Anti Cancer Potential of Cassia tora Linn. Extract

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Evaluation of antioxidant and anticancer potential of Cassia tora leaves / Asian Journal of Traditional Medicines, 2012, 7(6)

Regular articles

Evaluation of antioxidant and anticancer potential of Cassia tora leaves

Jinu Johna*, Archana Mehtab, Pradeep Mehtaba Inter University Instrumentation Centre, Mahatma Gandhi University, Kottayam, Kerala,

India;b School of Chemical and Biological Sciences, Dr. Hari Singh Gour University, Sagar, Madhya

Pradesh, India

Abstract

Cassia tora Linn. belongs to Leguminosae family has been traditionally reported to have medicinal properties. Reports on phyto-pharmacological evaluation of C. tora leaves for antioxidant and anticancer potential are rare. The aim of this study is to evaluate the anticancer potential, antioxidant activity and the total phenolic contents of C. tora leaf extracts. Methenolic extract and its ethyl acetate fraction were used for the analysis. Antioxidant assay was performed by in vitro models such as 2, 2-Diphenyl-1-picrylhydrazyl (DPPH), nitric oxide radical (NO.) scavenging and total reducing activity. The results were compared with that of standard compounds. Total phenolic content was measured by Folin-Ciocalteus reagent method using gallic acid as standard. Cytotoxicity of extracts was analyzed by sulforhodamine B (SRB) assay on cancer cell lines (MCF -7, SiHa, SK.N.SH, IMR-32, HT-29 and OVCAR-5). Ethyl acetate fraction showed highest percentage of phenolics (32.439%), relatively better antioxidant and cytotoxic property. Our results indicate that phenolic content may be responsible for the antioxidant activity of plant extracts and the observations were supportive to the traditional use of the plant as a nutraceutical leafy vegetable.

Key words: Cassia tora leaves; antioxidant potential; phenolic content; anticancer potential

1 Introduction

Herbal medicines play a major role in primary health care, mainly in the developing countries. Therapeutic potential of herbal drugs are attributed to the bioactive phytochemicals present in it. Plants are biosynthetic laboratories of a wide spectrum of chemicals of various physiological functions.

These phytochemicals are believed to have better compatibility with the human body and possess medicinal properties. Herbal drugs got a successful history as old as human civilization and today herbal medicines are coming back into prominence because of decreasing efficacy and serious side effects of the

modern medicines. Oxidation is necessary for energy production in all living systems. However it can produce free radicals, which can start chain reactions that may damage cells. Antioxidants terminate these chain reactions by removing radical intermediates, and inhibit other oxidation reactions by being oxidized themselves. Generally antioxidants of plant origin are of often

* Author to whom correspondence should be addressed. Address: Inter University Instrumentation Centre, Mahatma Gandhi University, Kottayam, Kerala, India; Tel.: +91-9446664201; Email: [email protected]

Received: 2012-09-29 Accepted: 2013-03-18

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reducing agents such as thiols or polyphenols. Recently there has been an increasing interest in free radicals in biological systems and their implied role as causative agents in the etiology of a variety of pathological physiologies as well as the search for phytochemicals with potential antioxidant activity [1]. Nature has immense source of different variety of medicinal plants, but only a small portion is phytochemically and pharmacologically evaluated or explored. Phyto-pharmacological evaluation of medicinal plants can provide potential bioactive lead compounds for the development of new drugs for several diseases like cancer. It is the need of the age to find out new sources of drugs due to the increased

demand and the development of resistance towards the existing chemo-therapeuticals. Phytochemicals shows anticancer effect may belong to various categories based on their chemical structures and the most commonly found active constituents include terpenes, alkaloids, coumarins, lignans, quinones, flavonoids, tannins, etc., some of them are alleged to

have antioxidant activity [2]. In 2002 plant derived natural products, paclitaxel and camptothecin were estimated to account for nearly one-third of the global anticancer market [3]. Cassia tora is a seasonal weed belongs to the Leguminosae family, traditionally reported to have medicinal properties, like laxative, antiperiodic, antihelmintic, ophthalmic and effective for leprosy, ringworm, flatulence, colic, dyspepsia, constipation,

cough, bronchitis, cardiac disorders, etc. In the Ayurvedic system of medicine Cassia tora has a great reputation in all kinds of skin diseases [4]. The leaves of Cassia tora are gently aperient. Both leaves and seeds constitute a valuable remedy in skin diseases, chiefly for ringworm and itch [5]. The leaves act like tincture of iodine and are useful against eczema [6]. This study was designed to evaluate the antioxidant potential and cytotoxic property of Cassia tora leaf extracts.

2 Material and methods

2.1 Preparation of plant extract

Cassia tora leaves were collected from the University campus and has been authenticated by Dr. Pradeep Tiwari, herbarium in charge, Dr. Hari Singh Gour University, Sagar (Voucher specimen number: Bot/Her/3427G). Plant materials were shade dried, powdered and were extracted with 70% methanol at room temperature for 48 h by repeated shaking in an orbital shaker. The extract was then filtered, concentrated and divided into two parts. Half of the portion of this extract was then partitioned with ethyl acetate and water (3:1). Ethyl acetate layer was separated until it becomes completely colorless and then evaporated and dried at 60C to get ethyl acetate fraction. Another half portion of the extract was dried to get methanoloic extract.

2.2 Preliminary phytochemical analysis

The screening of phytochemical chemical constituents of extracts was carried by qualitative chemical methods and thin-layer chromatography. A small portion of the dry extract was subjected to tests for secondary metabolites such as tannins, flavonoids, steroids, glycosides, cardiac glycosides and saponins by characteristic colour reactions [7]. In order to find out the presence of alkaloids, about 0.5 g of the plant extract was dissolved in 5 mL of 1% HCl by heating and filtered. A small quantity of the filtrate was then treated with few drops of Dragendorff s reagent. Turbidity or precipitation was taken as indicative of the presence of alkaloid. The presence of tannins/ phenolics was detected by FeCl3 test and tannins were confirmed by gelatin precipitation test. A blue coloration resulting from the addition of ferric chloride reagent to the dilute water solution of extract indicated the presence of

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tannins. Gelatin solution (1%) containing sodium chloride was added to the extract. Formation of white precipitate confirms the presence of tannins.

For flavonoids, about 0.2 g of the extract was dissolved in 2 mL of methanol and heated; to this a few magnesium metal turnings were added followed by the addition of a few drops of concentrated HCl. The occurrence of a red or orange colouration was indicative of the flavonoids.

About 0.5 g of the extract was dissolved in 3 mL of chloroform and filtered. The filtrate was divided

into two portions. Concentrated H2SO4 was carefully added to one portion to form the lower layer. A reddish brown colour at the interface was taken as positive observation for terpenoids. The other portion of the filtrate was treated with few drops of

acetic anhydride, boiled and cooled. Concentrated sulphuric acid was then added slowly through the sides of the test tube. Formation of brown ring at the junction indicates the presence of phytosterols (Libermann Burchards test). Saponins were detected by the froth test. Extracts were diluted with distilled water and this was shaken in a test tube for 5 minutes. Formation of a persistent layer of foam indicates the presence of saponins. Presence of anthraquinone glycosides was tested by Borntragers Test. Extract was treated with ferric chloride solution and heated in boiling water bath for about 5 minutes. The mixture was then cooled and extracted with equal volumes of benzene. The benzene layer was then treated with ammonia solution and the formation of rose-pink colour in the ammoniacal layer considered as positive.

2.3 HPTLC analysis of extracts

HPTLC was performed on 1010 cm HPTLC plates coated with 0.25 mm layer of silica gel 60 F254 (Merck, Germany). The plates were washed with methanol and then activated at 110oC for 5 min. Samples were applied as 4 mm wide bands at 6

mm apart by using a Camag (Muttenz, Switzerland) Linomat V sample applicator equipped with 100 L

syringe. A constant application rate of 5 L/s

was used. Mobile phase was hexane: chloroform: methanol (2:2:1). After the development of the chromatogram, the plate was dried and the images were captured in photo-documentation chamber in white light and in UV (254 nm and 366 nm). At 365 nm fluorescent zones were detected, while at 254 nm

quenching zones were detected [8]. 2.4 Qualitative DPPH antioxidant assay

Qualitative antioxidant assay was performed by the standard TLC method [9].

Test samples were

spotted on a TLC plate, air dried and then the plates were sprayed with 0.004% methanolic DPPH (2, 2-Diphenyl-1-picrylhydrazyl, HIMEDIA) solution using an atomizer. The positive activity was detected by the pale yellow spots on a reddish purple background due to the decolorization of DPPH by the antioxidant. Ascorbic acid (HIMEDIA) was used as the positive control.

2.5 Quantitative antioxidant assay

2.5.1 DPPH radical scavenging activity

The free radical scavenging activity was measured in terms of hydrogen donating or radical scavenging ability, using the stable radical, DPPH [10]. The scavenging reaction between (DPPH) and an antioxidant (H-A) can be written as: (DPPH) + (HA) DPPHH + (A)

(Purple) (Yellow) The degree of discoloration indicates the scavenging potential of the antioxidant compounds in the extracts in terms of hydrogen donating ability. 2, 2-Diphenyl-1-picrylhydrazyl solution (0.1 mM)was prepared in methanol and 1.0 mL of this solution was added to 3.0 mL of control (without the

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test compound, but with an equivalent amount of methanol), while test solutions and standard ascorbic acid (HIMEDIA) at different concentrations (10-200 g/mL). Thirty minutes later, the absorbance

was measured at 517 nm. Samples were tested in triplicate. Percentage inhibition was calculated by comparing the absorbance values of control and test samples. % Inhibition of DPPH= A(c)-A(s) 100 A(c) Where A(c) = absorbance of control, A(s) = absorbance of sample. IC50 value is determined as the concentration of extract corresponding to 50% inhibition graphically.

2.5.2 Nitric oxide scavenging activity

Nitric oxide scavenging potential was measured by the method proposed by Rajeshwer et al., [11]. Nitric oxide, generated from sodium nitroprusside (SNP) in aqueous solution at physiological pH, interacts with oxygen to produce nitrite ions which were measured by Griess reaction. The reaction mixture (3 mL) containing sodium nitroprusside (10 mM) in phosphate buffered saline (PBS) was mixed with extract and standard (10 to 500 g/mL) separately and were incubated at 25C for 150 min. After incubation, 0.5 mL of the reaction mixture was removed and mixed with 0.5 mL of Griess reagent (1% sulfanilamide, 2% H3PO4 and 0.1% naphthylethylene diamine dihydrochloride). The absorbance of the chromophore formed was evaluated at 546 nm. Percentage of NO scavenged and IC50 values were determined. Curcumin (HIMEDIA) was used as the positive control.

2.5.3 Determination of reducing power

The reducing power of the crude extracts was determined on the basis of the reaction of Fe3+- Fe2+ in presence of the extracts [12]. Extracts at different

concentrations (10 to 500 g/mL) were mixed with an equal volume of 0.2 M phosphate buffer (pH 6.6) and 1% potassium ferricyanide. The mixture was incubated at 50C for 20 min, after this an equal volume of 1% trichloroacetic acid (TCA) was added to the mixture and centrifuged at 5,000 g for 10 min. The upper layer of the solution was mixed with distilled water and 0.1% FeCl3 with a ratio of 1:1:2, the absorbance was measured at 700 nm.

2.5.4 Estimation of total phenolics

Phenolic estimation was performed by Folin - Ciocalteus phenol reagent method [13]. 0.4 mL aliquot of the plant extracts (100 g/mL) was transferred into a test tube containing 0.8 mL of the 10% Folin-Ciocalteu phenol reagent (Sigma). After 3 min, 1.6 mL of the 10% sodium carbonate solution was added. The contents were mixed well and left to stand at room temperature for 2 h. Absorbance measurements were recorded at 750 nm (Systronics 118). Gallic acid (HIMEDIA) was used as standard. Each experiment was triplicated; the observations were reported as mean SD and expressed as g equivalent of gallic acid/100 g sample.

2.6 In vitro anticancer study

The in vitro viability test has been performed on cell lines [MCF -7 (breast cancer cell line), SiHa (derived from carcinoma of uterus), SK.N.SH (Human neuroblastoma cell line), IMR-32 (Human Neuroblastoma cell line), HT-29 (Human colon adenocarcinoma cell line) and OVCAR-5 (ovarian cell line)] cultured under 37C, 5% CO2 and 95% relative humidity in DMEM medium containing 2 mM L-glutamine, penicillin (100 IU/mL), streptomycin (100 g/mL) and 10% FBS. 3,000

cells were added per well (in a 96 wells-plate). After 24 hours, plant extract (100 g/mL) prepared by dilution with medium containing 50 g/mL of

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gentamycin from stock solution in 50% aqueous DMSO were added, and cells were cultured for 180 min. Effect of extract on the cell growth was measured by sulforhodamine B colorimetric assay. The results were presented as percentage inhibition of cell proliferation in comparison to the control [14].

2.7 Statistical analysis

Statistical analyses were performed using a one-way analysis of variance (ANOVA). The level of confidence required for significance was selected at

P < 0.05.

3 Results and discussion

3.1 Preliminary phytochemical analysis

Methenolic extract of C. tora leaves gave a yield of 5.3% (w/w) of the dried plant material, had a dark brown colour with a sticky appearance, while the percentage yield of ethyl acetate fraction was 1.2. Preliminary phytochemical analysis of extracts has shown the presences of tannins, flavonoids, anthraquinons, terpenoids and glycosides. Ethyl acetate mainly showed the presence of flavonoids and tannins (Table 1). Chromatographic analysis of ethyl acetate fraction showed about twelve components. Most of the components were colored and shown fluorescence at 366 nm and quenching of fluorescence was observed at 254 nm. HPTLC studies showed the presence of flavonoid, quercetin

and anthraquinone glycosides in the ethyl acetate fraction.

3.2 Qualitative antioxidant assay

TLC-DPPH method is one of the widely used models for testing preliminary radical scavenging activity a plant extract. Both the extracts were

found to be active. Ethyl acetate fraction showed relatively better activity when compared to that of crude methenolic extract with a yellow-white spots on purple background similar to that of vitamin C.

3.3 DPPH radical scavenging activity

Methanolic extract (MCT) and ethyl acetate fraction (EACT) showed concentration depended decolourization of DPPH (Fig. 1). The ethyl acetate extract showed potent antioxidant activity by DPPH radical quenching. The ethyl acetate extract strongly scavenged DPPH radical with an IC50 value 30.50 g/mL, which is almost comparable to that of ascorbic acid, 19.80 g/mL while that of methanol

Note: (+: present : absent)

Table 1. Phytochemical analysis of extracts of Cassia tora leaves.

Methanol Ethyl acetate

1. Alkaloids- -

(Draggendorffs & Mayers)

2. Coumarins+ +

(Alch. KOH)

3. Steroids+ -

(Liebermanns test)

4.Terpinoids- -

(Salkowski test)

5. Flavonoids+ +

(Shibatas reaction)

6. Saponins + -

(Froth test)

7. Tannin + +

(5% FeCl3)

8. Glycosides + +

(Fehlings test)

9. Cardiac Glycosides+ -

(Keller-Kiliani test)

10.Anthraquinone+ +

(Borntragers test)

11. Amino acids (Ninhydrin test) + -

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extract was 46.20 g/mL.

3.4 Nitric oxide scavenging activity

Both the extract showed concentration dependent elevation in NO scavenging activity up to 500 g/mL (Fig. 2). In the present study curcumin was

used as the reference NO radical scavenger. Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates NO, which interacts with oxygen to produce a nitrite ion. Scavenging activity inhibited nitrite formation by competing with oxygen to react with NO, which lead to the reduction of nitrite concentration in the assay media. Nitrite ions were measured by Griess reaction. Antioxidant potential and nitrite formation maintains an inverse relationship.

3.5 Reducing power of the extracts

Antioxidant activity can be explained as inactivation of oxidants by reductants like redox reactions in which one reaction species (oxidant) is reduced at the expense of the oxidation of another antioxidant. The extract caused significant elevation of reducing power potential in a dose depended manner (Fig. 3). The redox homeostasis is very important for the equilibrium among cancer development, apoptosis, regeneration and necrosis.

3.6 Total phenolic content

Ethyl acetate fraction showed relatively higher phenolic content (32.44%) compared to that of crude methanolic extract (26.19%). The phenolic contents of the extracts were tested using the Folin-Ciocalteu reagent as gallic acid equivalents.

3.7 Antiproliferative potential

The antiproliferative activities of C. tora leaf extracts varied with the type of cancer cell lines analyzed. They influenced the cell growth depending

on cell line. Ethyl acetate fraction of the extract showed better antiproliferative effect. It showed maximum activity against MCF-7, SiHa then OVCAR-5 and least against IMR-32 (Figs. 4 & 5). The study suggests that the higher antioxidant

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Fig. 1. Scavenging of DPPH radical by C. tora leaf extracts and ascorbic acid.

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Fig. 2. Nitric oxide scavenging by C. tora leaf extracts.

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Fig. 3: Reducing power of Cassia tora leaf extracts Fig. 3. Reducing power of Cassia tora leaf extracts.

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and cytotoxic property of ethyl acetate fraction may attribute to its high phenolic content. There are previous reports of direct relationship between antioxidant activity and total phenolic content in herbs, vegetables and fruits [15, 16]. Phenolics in herbs might prevent cancer through antioxidant action and/ or the modulation of several protein functions. They may inhibit carcinogenesis by affecting the molecular events in the initiation, promotion, and progression stages [17]. They modulated the secretion of protein kinases in tumor cell proliferation and induced the expression of anticarcinogenic enzymes or inhibited induction of cancer-promoting enzymes [18]. Moreover, some tannin molecules (e.g., tea polyphenols) have anticancer or anticarcinogenic or antimutagenic activity, might be related to their antioxidative properties [19]. Polyphenolics like kaempferol are major contributor to the antioxidant capacity of Cassia alata, while emodin, an anthraquinone glycoside, is reported to be an active antioxidant compound in C. tora seeds [20].

In conclusion, the ethyl acetate-soluble fraction from 80% methanolic extract exhibited the highest radical scavenging and relatively better cytotoxicity towards the cancer cell lines tested which is attributed to the phytochemicals present in the plant.

The present study shows a significant correlation between phenolic content and antioxidant property therefore the consumption of these leafy vegetables is good for health.

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MCF -7 SiHa SK.N.SH IMR-32 HT-29 OVCAR-5

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Ethyl acetate

Methanol

Fig. 4: Anti-proliferative effect of extracts of cassia tora leaves

Fig. 5: Antiproliferative effect of C. tora leaf extracts: EACT & MCT extracts shows inhibition on cell proliferation by cytoplasmic shrinkage and loss of normal nuclear architecture, became detached and found floating in the medium compared to the control. *EACT- Ethyl acetate extract, MCT- Methenolic extract

Fig. 4. Anti-proliferative effect of extracts of cassia tora lea-ves.

Fig. 5. Antiproliferative effect of C. tora leaf extracts: EACT & MCT extracts shows inhibition on cell proliferation by cy-toplasmic shrinkage and loss of normal nuclear architecture, became detached and found floating in the medium compared to the control. *EACT- Ethyl acetate extract, MCT- Metheno-lic extract. **MCF -7 (breast cancer cell line), SiHa (derived from carcinoma of uterus), SK.N.SH (Human neuroblastoma cell line), IMR-32 (Human Neuroblastoma cell line), HT-29 (Human colon adenocarcinoma cell line) and OVCAR-5 (ovari-an cell line).

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Fig. 4: Anti-proliferative effect of extracts of cassia tora leaves

Fig. 5: Antiproliferative effect of C. tora leaf extracts: EACT & MCT extracts shows inhibition on cell proliferation by cytoplasmic shrinkage and loss of normal nuclear architecture, became detached and found floating in the medium compared to the control. *EACT- Ethyl acetate extract, MCT- Methenolic extract

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Acknowledgments

The authors are grateful to Head, Department of Botany, Dr. H. S. Gour University, Sagar as well as Director, Hislop school of Biotechnology, Nagpur for their technical assistance and support to carry out this investigation.

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Anti Cancer Potential of Cassia tora Linn. Extract