Free radicals contribute to more than one hundred disorders in human beings, including atherosclerosis, arthritis, ischemia, central nervous system disorders, gastritis, cancer and AIDS [6]. Free radicals due to environmental pollutants, radiations, Chemicals, toxins, deep fried and spicy foods, as well as physical stress, cause depletion of immune system, change in gene expression and induce abnormal proteins. Due to depletion of immune system, natural antioxidants in different maladies consuming antioxidants as free radical scavengers may be necessary [7].

An increasing interest in the search for natural replacements of synthetic antioxidants has led to the antioxidant evaluation of a number of plant sources. The neutraceutical trend towards doubling the impact of natural antioxidants that stabilize food and maximize health benefit presents distinct challenges in evaluating antioxidant activity. Thus, there is a renewing interest in phytomedicine during last decade, and nowadays many medicinal plant species are being screened for its pharmacological potential.

India is the major exporter of raw MAP’S (Medicinal and Aromatic Plants) and processed plant based drugs. About 45,000 plant species with medicinal properties have been assigned to several thousand for the turnover of herbal medicines in India. However, very few plant species have been thoroughly investigated for their medicinal properties.

Recently, there has been an upsurge of interest in the therapeutic potentials of medicinal plants as antioxidants in reducing such free radical induced tissue injury. Besides well-known and traditionally used natural antioxidants from tea, wine, fruits, vegetables, spices, some natural antioxidant are already exploited commercially either as antioxidant additives or a nutritional supplements [8]. Also, many other plant species have been investigated the search for novel antioxidants [9], but generally there is still a demand to find more information concerning the antioxidant activity of plants might be due to their phenolic compounds [10]. The number of reports on isolations of natural antioxidants, mainly of plant origin, has increased immensely during the last decade [11]. Phenolic compound are commonly produced in plants as a general line of their natural defence.

They have multiple applications in food, cosmetic and pharmaceutical industries [12]. The antioxidant capacity of phenolic compound is mainly due to their redox properties, which allow them to cut as reducing agents, hydrogen donor’s singlet oxygen quenchers or metal chelators. In addition to their roles as antioxidants, these compounds exhibit a wide spectrum of medicinal properties, such as anti-allergic, anti-inflammatory, anti-microbial, anti-thrombotic, cardio-protective and vasodilatory effect [13].

Cassia alata (belonging to the family Leguminosae) (Figure 1) has been reported to have various phytochemical activities. Pharmacological investigations performed so far on Cassia alata have revealed that this herb has several biological activities, such as antimicrobial [14,15], purgative [16], anti-inflammatory [17,18], analgesic [17,19,20], hypoglycaemic [17], and antitumor activities [21].

Figure 1: Cassia alata

Considering the growing demands of herbal medicines and those too natural antioxidants, the objective of the present investigation was to determine the antioxidant potential and immune stimulating property of the common ethno-medicinally important plant, Cassia alata.

Sample collection

The plant Cassia alata was collected from road side of Tarabag, the residential campus of Burdwan University, Burdwan, and West Bengal, India.

Determination of antioxidant property of Cassia alata

Leaf extracts of C. alata were used for determination of antioxidant properties of the plant. Estimation of different antioxidant compounds and assay of DPPH radical scavenging activity were studied. Methanolic extract of leaves was used for determination of antioxidant properties of this plant (Figure 2).

Figure 2: Inflorescence of C. alata

Preparation of alcoholic extract

Leaves dried at room temperature were taken and put in boiling methanol at a rate of 5-10 ml of methyl alcohol per g of tissue and allowed to boil for 5 to 10 minutes, in order to have extraction on a steam bath or hot water bath under a hood. It was then cooled in a pan of cold water, crushed thoroughly in a mortar and pestle for 5 to 10 minutes, and filtered through two layers of cheese cloth. The residue was again extracted with 2 to 3 ml of alcohol for 3 minutes in a hot water bath, cooled and passed through cheese-cloth. Both the extracts were mixed and the mixture was filtered through Whatman No. 1 filter paper. The alcohol from the extract was then evaporated till the volume was reduced by 80-90%.

Estimation of total phenols: Total phenol was estimated using Folin-Ciocalteu reagent [22]. For this purpose, alcoholic extract of plant tissue was used.

Reagent preparation: Folin Ciocalteu reagent was made by dissolving 100.0 mg of sodium tungstate and 25.0 gm of sodium molybdate in 700.0 ml of distilled water. 50.0 ml of 85% orthophosphoric acid and 100.0 ml of conc. HCl were added to it and boiled under reflux gently for about 10 hours and allowed to cool. 150.0 gm of lithium sulphate dissolved in 50.0 ml water and 4-5 drops of liquid bromine together were added to it gently. The mixture was boiled without condenser for about 15 mins to remove the excess bromine, cooled and diluted to 1000 ml with water and filtered. The reagent should be golden yellow in colour and stable for several months, and stored in amber coloured bottles. Just before use, the reagent was diluted with one volume of this stock solution with 2 volumes of water.

Estimation: For estimation of total phenol, 1 ml of alcoholic extract was taken in a graduated test tube and 1 ml of Folin Ciocalteu reagent was added to it, which was followed by the addition of 2 ml of 20% Na₂CO₃ solution. The tube was shaken and placed in a boiling water bath, for exactly 1 min., cooled under running tap water. The blue solution developed was diluted with distilled water up to 25 ml, and its absorbance was recorded at 650 nm in a spectrophotometer (Simadzu). Total phenol was calculated from a standard curve prepared from catechol. Blank set was made identically. Total phenol content was expressed in terms of (μg/ml) of extract.

Estimation of flavonoids: 5 ml of sample of the alcoholic extract of leaf was taken in a test tube containing 1.25 ml of distilled water [23]. Then 0.075 ml of 5% sodium nitrite solution was added in it and allowed to stand for 5 minutes. Subsequently, 0.15 ml of 10% Aluminium chloride (AlCl₃) was added to the mixture. After 6 minutes, 0.5 ml of 1 M sodium hydroxide was added, and the mixture was diluted with another 0.275 ml of distilled water. The absorbance was measured at 510 nm in a spectrophotometer (Simadzu). A standard curve was prepared from analytical grade Catechin. And the total flavonoids content in the leaf tissue of Cassia alata was calculated, and was expressed in terms of (μg/ml) of extract.

Estimation of Vitamin-C: Ascorbic acid was estimated following the method described by Oser [24]. 100 mg of fresh leaf tissue was homogenized with 5 ml of 6% trichloro acetic acid (TCA). After centrifugation at 5000 rpm for 5 min., the supernatant was collected and a pinch of activated charcoal (Norit) was added to it and filtered. The volume of the filtrate was made up to 100 ml with addition of distilled water. 4ml of supernatant was taken in a test tube and 2 ml of 2% 2,4 dinitrophenyl hydrazine and one drop of 10% thiourea solution (in 70% alcohol) were added to it. Dinitrophenyl hydrazine solution was prepared in another conical flask by adding 2 gm of dinitrophenyl hydrazine into 100 ml of 9(N) H₂SO₄ (concentrated). The mixture was then boiled for 15 min. in a water bath. After cooling at room temp., 5 ml of 80% H₂SO₄ was added in the mixture at 0°C. After waiting for 30 min, the absorbance of the solution was taken at 530 nm in a spectrophotometer (Simadzu). A standard curve was prepared from analytical grade ascorbic acid, and the Vitamin C content in the leaf tissue of Cassia alata was calculated and was expressed in terms of (μg/ ml) of extract.

Estimation of Vitamin-A: Vitamin A was estimated by method of Carr and Price [25]. 100 mg of fresh leaf tissue sample was weighed and ground well using mortar and pestle. To this, 1 to 2 ml chloroform was added and then centrifuged, supernatant was collected, and it was made up to 50 ml with distilled water. This served as test solution was taken in series of test tubes and made up to 3 ml with chloroform. Blank was also setup with 3 ml of chloroform, 0.2 ml of test solution was taken and made up to 3 ml with chloroform. To all the test tubes, 2 ml of antimony trichloride was added. The blue colour developed was read at 620 nm within 15 seconds.

Standard vitamin A solution: About 40 to 50 mg of a vitamin A concentrate, which comes in gelatin capsules, Control KO. PC-3 from Distillation Products, Inc., were diluted in chloroform to give a concentration of vitamin A (as the alcohol) from 2 to 5 micrograms per ml of solution. Standard curve was drawn. Vitamin-content in the leaf tissue of Cassia alata was calculated, and was expressed in terms of (μg/ ml) of extract.

Estimation of total carotenoids: Extraction and estimation of total carotenoids was done following the method of Thimmaiah [26]. 1 gm of the fresh leaf tissue sample was homogenized with peroxide free ether, until it was free from pigments, filtered and the filtrates were pooled and partitioned with equal quantity of peroxide free ether. The ether phase which contains the carotenoids was evaporated and the residue was dissolved in 1 ml of ethanol, and absorbance was measured at 450 nm. The carotenoids present in the extract were calculated from the A₄₅₀ values using the following formula:

C=D×V×F/2500

Where, C=Total amount of carotenoids (mg)

D=Absorbance at 450 nm

V=Volume of the original extract in ml

f=Dilution factor and

2500=Average extinction coefficient of the pigments

Estimation of anthraquinone: Sample preparation: Cassia alata leaf powder (100 mg) was extracted with methanol (20 ml) under reflux conditions for 1 hr. The extract was then filtered and concentrated. The sample was adjusted to 5 ml with methanol [27].

Conformity test for anthraquinone: Anthraquinone, which is the major component of Cassia alata, is identified by this procedure [28]. Borntrager’s reaction was used to detect anthraquinone in the extract. HCl (2 N) was added to the sample, and the mixture was heated on a hot water bath for 15 min, then cooled and filtered. The filtrate was mixed well with chloroform layer and was separated with it. The mixture was shaked with 10% KOH solution, the aqueous layer become pink-red, which confirms the presence of anthraquinone in the sample (Figure 3).

Figure 3: Strongly positive reaction for Anthraquinone: Solution becomes red to pink red.

Method of estimation of anthraquinone in the extract: Crude extract was taken and mixed with 30 ml water, weighed and refluxed for 15 min. The aqueous mixture allowed to cool, weighed and adjusted with H₂O to the original weight. The sample was centrifuged at 4000 rpm for 10 min. 10 ml of supernatant was taken with 20 ml of 10.5% FeCl₃.6H₂O and refluxed for 2 min where aglycones and glycosides are produced. These were extracted with 25 ml×3 ml of ether. The ether layer and the aqueous layer formed separately. The ether layer was washed separately with water (15 ml) for 2 times and adjusted to 100 ml with ether. 25 ml from the ether layer was taken and evaporated to dryness. The residue was dissolved with 10 ml of 0.5% Magnesium acetate in methanol and absorbance was taken at 515 nm, and calculated the percentage (%) of total anthraquinones and expressed in terms of (μg/ml). Standard curve was prepared from sodium anthraquinone-2- sulfonate (analytical grade, SIGMA-ALDRICH).

Assay of DPPH free radical-scavenging activity: The antioxidant activity was determined according to the DPPH radical scavenging assay. The effect of the methanolic extracts of leaf, flower and pods of C. alata on 1, 1-diphenyl-2-picrylhydrazyl (DPPH, Fluka Chemie) was estimated following the method, as described by Brand-Williams et al. [29]. Samples were diluted in methanol prior to the analysis (1 mg/ ml). The DPPH solution was added to the diluted samples, thoroughly mixed, then left for 30 min for the reaction to occur. After that, the absorbance of the samples was measured at 515 nm using a UV−Vis spectrophotometer (Simadzu). The absorbance of DPPH solution in methanol, without any antioxidant (control), was also measured. The percentage of DPPH radical scavenging activity was calculated by using the following equation:

DPPH scavenging (%)=[(Acontrol−Asample)/Acontrol]×100

Where, a sample is the absorbance of the sample after the time necessary to reach the plateau (30 min), and a control is the absorbance of DPPH. Extract concentrations providing IC₅₀ inhibition values (defined as the concentration of the compounds that was able to inhibit 50% of the total DPPH radicals) were calculated from graph plotting using nonlinear regression, and expressed in microgram material equivalents per milliliter for sample extracts. Butylated hydroxytoluene (BHT) was used as a positive control. A lower value of IC₅₀ indicates a higher antioxidant activity and vice versa.

Study of immune stimulating activity of C. alata

For assay of immune modulating/stimulating activity of C. alata, aqueous extract of leaves of C. alata was prepared following standard protocol, and was injected to adult rabbit with the help of a hypodermic syringe at a dosage of 10 ml aqueous extract per kg of body weight. Control group of rabbit were maintained which received no of leaf extract of C. alata. Estimation of haemoglobin, total count, differential count, count of peritoneal macrophages was done at an interval of 7 days up to 21 days.

Estimation of haemoglobin

Haemoglobin content was estimated following acid haematin method, in which haemoglobin gets converted into acid haematin by the action of hydrochloric acid, and was measured using haemoglobinometer. Content of haemoglobin was expressed as g/100 ml blood.

Total count of Leucocytes (TC)

It was done by haemocytometer method. For calculating total leucocyte content, blood was diluted with WBC diluting fluid, which removes the red cells by haemolysis, and also accentuates the nuclei of the white cells. Counting was done under a microscope, and by knowing the volume of the fluid examined and the dilution of the blood, the number of white cells per cu mm in undiluted whole blood was calculated.

Count of leucocytes (DC or granulocyte-agranulocyte ratio)

It was done following the Leishman stain method. Blood sample smear was prepared and observed under microscope. Approximately, 100 leucocytes were counted and from that observation, the ratio of granulocyte and agranulocyte was calculated.

Count of peritoneal macrophages

Count of peritoneal macrophages was done following the method of Hudson and Hay [30].

It is evidenced from the results that the plant Cassia alata possesses strong antioxidant activity, as it contains good quantity of phenols ( 3.1 ± 0.67 μg/g ), Vitamin-C (1.98 ± 0.47 μg/g), Vitamin- A (1.2 ± 0.22 μg/g), flavonoids (2.2 ± 0.51 μg/g ), carotenoids (0.35 ± 0.50 μg/g) and anthraquinone (1.57 ± 0.36 μg/g) (Table 1).

Table 1: Antioxidant compounds present in the leaf tissue of Cassia alata.

In addition, it has strong DPPH radical scavenging activity (IC₅₀=54 ± 2.20), in contrast to the DPPH radical scavenging activity of a synthetic antioxidant compound, BHT (IC₅₀=72 ± 1.18) (Table 2).

Table 2: DPPH Radical Scavenging activity of C. alata as compared to the synthetic antioxidant compound-BHT.

Administration of aqueous leaf extract to the rabbits showed a noticeable increase in blood haemoglobin content, as well as in RBC count. There appeared a steep rising in the total count of leucocytes, with concomitant increasing in granulocyte: agranullocyte ratio. It is also evident from the results that the rabbit treated with the leaf extracts of Cassia alata resulted into a tremendous increase in the total number of peritoneal macrophages (Table 3). Thus, it is apparent from the results that aqueous leaf extract of Cassia alata plant has strong immune-modulating or immune-stimulating potency. During the course of experiment, abnormal behavior of the experimental animals was not shown. Though there appeared an increase in eating habit, but there seemed to be no significant enhancement in their body weight.

Table 3: Assay of immune stimulating property of Cassia alata.

An increase in these blood components is very significant, as they are essentially important parameters of body’s defense system. Present study had revealed that the leaf extract of Cassia alata plant under consideration had significant impact on stimulation of host’s immune system. It has been well established that macrophages play vital role for immune stimulation, tumor cell lysis and inhibition of tumor growth [31,32].

Cassia alata is one of the most important species of the genus Cassia, which is rich in anthraquinones and polyphenols. The leaves of C. alata have been qualitatively analyzed for the presence of primarily five pharmacologically active anthraquinones: rhein, aloeemodin, chrysophanol, emodin and physcion, as well as the flavonoid, kaempferol [18]. The flavonoid, kaempferol has been reported to have anticancer properties [33].

Idu et al. [34] observed that preliminary phytochemical analysis of Cassia alata showed the presence of phenols, tannins, anthraquinones, saponins and flavonoids. Odunbaku and Lusanya [35] also confirmed that this plant also had alkaloids and cardenolides.

Sharma et al. [36] also reported in their study that Preliminary phytochemical screening of alcoholic extract revealed the presence of anthraquinone, phenolic compounds; saponin, glycosides, and while aqueous extract showed presence of glycosides and Phenolic compounds.

These anthraquinone derivatives are well known to exhibit a variety of biological activities, such as antibacterial and antifungal [37], antitumor [38], antioxidant [16], cytotoxic and hypoglycaemic activities [16,39]. Other chemicals contained in the plant include saponin, which acts as a laxative and expels intestinal parasites. Rhein and chrysophanol are also known to be present in the roots, in addition to two other quinone pigments [40,41].

Among the compounds obtained from the leaf extract of the plant Cassia alata, phenols and polyphenols have gained utmost importance due to their large array of bioactivities, including free radical scavenging, metal chelating, immune modulating activities, as well as modulation of enzyme activities, inhibition of lipid oxidation, etc. [42-44]. The term polyphenol refers to a complex group of compounds that contain in their structure an aromatic ring bearing one or more hydroxyl groups, which comprise simple phenols, such as phenolic acids and other derivatives of phenols, as well as some complex structures like flavones, flavonoids, anthocyanins, anthraquinones, etc.

Vitamin C (ascorbic acid) is an important component of our nutrients, and has been proved to exert a protective role against various stress related diseases, such as stroke, heart problems, cancer, and many neurodegenerative diseases [45]. And carotenoid, particularly, β-carotene is a vital antioxidant defense.

Presence of significant amount of phenols, anthraquinones, flavonoids, carotenoids, Vitamin-C and Vitamin-A in the methanolic leaf extract of C. alata indicates strong antioxidant properties of this plant.

Pieme et al. [46] investigated the acute to sub-acute toxicities of hydro-ethanolic extract of leaves of Cassia alata on Swiss mice and Wistar albino rat. However, the present study exhibited no cytotoxic effects of the aqueous leaf extract of Cassia alata. Cassia alata showed some protective effects on hepatocytes and improved liver architecture [30]. These results showed that the use of the extract of Cassia alata is safe and explained the extensive utilization of the plant in traditional medicine. In the light of this, Chukeatirote et al. [47] stated that since ancient time, plant and animal products have been used for treatment of diseases and disorders, and that, plants in particular, have been used to treat infections due to its antimicrobial properties.

Today a lot of antioxidant products are available in the market. Though a burning topic of the day, plants need to be evaluated for their antioxidant potential, which will definitely be the right step towards the benefit of the mankind, keeping in view the present study, may certainly be a positive step towards green chemistry in the field of modern drug discovery, and this novel plant Cassia alata may extensively be used in therapeutic medicines as a resource of natural antioxidants and immune stimulating agent. However, further research is needed for its safety level and judicious use.

I wish to acknowledge UGC RFMS Scheme for financial support. I am also thankful to Mrs. Pampa Pal, Pubali Dev and my family for their help and inspiration throughout the work.