Table of Contents    
ORIGINAL ARTICLE
Year : 2019  |  Volume : 10  |  Issue : 2  |  Page : 157-166  

Antioxidant and cytotoxic potential of leaf extracts of Costus igneus


PG and Research Centre in Biotechnology, MGR College, Hosur, Tamil Nadu, India

Date of Web Publication18-Jul-2019

Correspondence Address:
Balaji Paulraj
PG and Research Centre in Biotechnology, MGR College, Hosur - 635 130, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jnsbm.JNSBM_216_18

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   Abstract 


Background: Costus igneus leaves are traditionally used to treat diabetes and this plant is commonly known as insulin plant. Hypothesis: The purpose of this study was to evaluate the antioxidant and cytotoxic potential of different leaf extracts of C. igneus. Materials and Methods: The antioxidant potential of various leaf extracts of C. igneus was established by ferric reducing antioxidant power (FRAP) assay, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay, metal-chelating activity, phosphomolybdenum assay, superoxide radical scavenging activity, 2,2-diphenylpicrylhydrazyl (DPPH) assay, reducing power assay, and cytotoxic activity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on breast cancer cell line Michigan Cancer Foundation (MCF-7). Results: In FRAP assay, acetone extract showed higher activity of 276.31 mmol Fe (II) E/mg extract. In ABTS assay, hexane extract showed the highest scavenging activity of 12878.893 μM Trolox equivalent antioxidant capacity (TEAC)/g extract. Hot water extract registered higher metal ion-chelating ability with 2.94 mg ethylenediaminetetraacetic acid equivalent/g extract. Hexane extract has recorded the highest total antioxidant capacity of 65.00 mg AAE/mg in phosphomolybdenum assay. Superoxide radical scavenging activity was found maximum in acetone extract with 41.11%. In DPPH assay, acetone extract recorded total free radical scavenging capacity with IC50 value of 40.70 μg/ml. Hexane extract has recorded significant highest reducing power at concentration 100 μg/μl with maximum optical density of 0.177. Acetone extract of C. igneus leaf extract at concentration of 150 μg/ml showed the highest cytotoxic activity on cancer cell line with cell viability of 65.51%. Conclusion: The present study explored that C. igneus shows efficient antioxidant activity as well as cytotoxic activity and could act as safe and cost-effective with potential biological applications.

Keywords: 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), 2,2-diphenylpicrylhydrazyl, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, Costus igneus, ferric reducing antioxidant power, flavonoid, metal-chelating activity


How to cite this article:
Khanday WI, Wani NA, Paulraj B. Antioxidant and cytotoxic potential of leaf extracts of Costus igneus. J Nat Sc Biol Med 2019;10:157-66

How to cite this URL:
Khanday WI, Wani NA, Paulraj B. Antioxidant and cytotoxic potential of leaf extracts of Costus igneus. J Nat Sc Biol Med [serial online] 2019 [cited 2019 Oct 19];10:157-66. Available from: http://www.jnsbm.org/text.asp?2019/10/2/157/262954




   Introduction Top


Natural products are an excellent source of complex chemicals, possessing a wide variety of biological activities and having great potential therapeutic value.[1] The various disease are treated with medicinal plant extracts is one of the old practice people have adopted.[2],[3] The medicinal plants are useful for healing as well as for curing the human diseases because of the presence of phytochemicals.[4] Phytochemical compounds like alkaloids: flavonoids, coumarins, glycosides, polysaccharides, phenols, tannins, terpenes, and terpenoids are formed in plant during normal metabolic process. These phytochemical compounds possess protective or disease preventive properties. Antioxidant action is commonly well known in flavonoids.[5] Flavonoids have shown actions against diseases such as cancer, allergy, and microbes. The free radicals occur in the body during an imbalance between ROS and antioxidants.[6],[7],[8],[9] Considering the rich diversity and traditional knowledge, the world is looking toward India for developing the new natural, safe, herbal drugs to cure different diseases.[10] Natural antioxidants increase the antioxidant capacity of the plasma and reduce the risk of certain diseases such as heart and cancer.[11] It has been found that plants are having polyphenolic compounds such as flavonoids possess antioxidant activity.[12] Polyphenols have antioxidative properties which are due to their high reactivity as hydrogen donor or electron donor which stabilize and delocalize the unpaired electron.[13] Many of the diseases in our body arise from the imbalance between oxidants and antioxidants. Superoxide and other reactive oxygen species called as free radicals in physiological conditions are produced in our body by consumption of 1%–3% oxygen.[14],[15] The herbal products are also safer than synthetic formulation which may be hazardous and unsafe to human life.[16] The present investigation was designed to study the phytochemical profiling, in vitro antioxidant activity, and cytotoxic potential from acetone, hexane, and hot water extracts of the leaves of Costus igneus.


   Materials and Methods Top


Collection and identification of plant material

The leaves of C. igneus were collected from the outskirts of Hosur, Krishnagiri district of Tamil Nadu and were identified and authenticated by Dr. M. Kumar, Assistant Professor, Department of Plant Biology and Plant Biotechnology, Madras Christian College, Chennai, Tamil Nadu, India. The collected leaf materials were cleaned, shade dried, and powdered for further extraction and analysis.

Preparation of extracts

The air-dried leaf powdered of C. igneus was extracted in Soxhlet extractor for 8–10 h, successively with acetone, hexane, and hot water.[17] The dried extract obtained with each solvent was weighed. The evaporated extracts thus obtained were dissolved in the respective solvents at the concentration of 1 mg/mL and used for further in vitro further studies.

Preliminary phytochemical screening

The acetone, hexane, and hot water leaf extracts of C. igneus were subjected to preliminary phytochemical screening of various plant constituents. 10 mg extract was diluted per 10 ml of the respective solvent to make the sample for preliminary tests.[18],[19]

Determination of total phenolics and tannins

Known concentration of acetone, hexane, and hot water extract of C. igneus was taken in test tubes and made up to the volume of 1 ml with distilled water. Then, 0.5 ml of Folin–Ciocalteu phenol reagent (1:1 with water) and 2.5 ml of sodium carbonate solution (20%) were added sequentially in each tube. Soon after vortexing the reaction mixture, the test tubes were placed in dark for 40 min, and the absorbance was recorded at 725 nm against the reagent blank. The analysis was performed in triplicate, and the results were expressed as the gallic acid equivalents (GAEs). The total phenolic content of acetone, hexane, and hot water extract of C. igneus was determined according to the method described by Siddhuraju and Becker.[20] Using the same extract, the tannins were estimated after treatment with polyvinylpolypyrrolidone (PVPP). One hundred milligrams of PVPP was weighed into a 100 mm × 12 mm test tube, and to this, 1.0 ml of distilled water and then 1.0 ml of tannin-containing phenolic extract were added. The content was vortexed and incubated at 4°C for 4 h. Then, the acetone, hexane, and hot water extract of C. igneus were centrifuged (3000 × g for 10 min at room temperature), and the supernatant was collected. This supernatant has only simple phenolics other than tannins (the tannins would have been precipitated along with the PVPP). The phenolic content of the supernatant was measured as mentioned above and expressed as the content of nontannin phenolics. From the above results, the tannin content of the sample was calculated as the difference between total phenolics and nontannin phenolics.

Tannin (%) = Total phenolics (%) – nontannin phenolics (%)

Determination of total flavonoids

The flavonoid content of the acetone, hexane, and hot water extract of C. igneus was determined by the use of a slightly modified colorimetric method described previously.[21] A 0.5 ml each extract of C. igneus was mixed with 2 ml of distilled water and subsequently with 0.15 ml of 5% NaNO2 solution. After 6 min, 0.15 ml of 10% AlCl3 was added and allowed to stand for 6 min, and then, 2 ml of 4% NaOH solution was added to the mixture. Immediately, distilled water was added to bring the final volume to 5 ml, and then, the mixture was thoroughly mixed and allowed to stand for another 15 min. Absorbance of the mixture was determined at 510 nm versus prepared water blank. Rutin was used as the standard compound for the quantification of total flavonoids. All the values were expressed as milligram of rutin equivalents (REs) per gram of extract.

Ferric reducing antioxidant power assay

The antioxidant capacity of acetone, hexane, and hot water extract of C. igneus was estimated according to the procedure described by Pulido R et al.[22] The acetone, hexane, and hot water extracts of C. igneus and reagent blank were incubated at 37°C for 30 min in a water bath. The final dilution of different leaf extracts of C. igneus in the reaction mixture was 1/34. The ferric reducing antioxidant power (FRAP) reagent contained 2.5 mL of 20 mM TPTZ solution in 40 mM HCl, 2.5 mL of 20 mM FeCl3.6H2O, and 25 mL of 0.3 M acetate buffer (pH 3.6) as described by Siddhuraju and Becker, 2003. The absorbance readings were taken immediately at 595 nm, using a spectrophotometer. The results were compared with the commercially available antioxidants such as butylated hydroxytoluene (BHT) and rutin. Methanolic solutions of known Fe (II) concentration, ranging from 100 to 2000 μM (FeSO4.7H2O), were used for the preparation of the calibration curve. The FRAP value is expressed as μmol Fe (II) equivalent/g extract.

Antioxidant activity by ABTS •+ assay

The total antioxidant activity of acetone, hexane, and hot water extract of C. igneus was measured by 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation decolorization assay according to the method.[23] The stock solution of the different leaf extracts of C. igneus was diluted such that after introduction of 10 μl aliquots into the assay, they produced between 20% and 80% inhibition of the blank absorbance. After the addition of 1 ml of diluted ABTS solution to 10 μl of different leaf extracts of C. igneus or Trolox standards (final concentration 0–15 μM) in ethanol, absorbance was measured at 30°C exactly 30 min after the initial mixing. Appropriate solvent blanks were also run in each assay. Triplicate determinations were made at each dilution of the standard, and the percentage inhibition was calculated of the blank absorbance at 734 nm and then was plotted as a function of Trolox concentration. The unit of Trolox equivalent antioxidant capacity (TEAC) is defined as the concentration of Trolox having equivalent antioxidant activity expressed as μM/g sample extract on dry matter.

Metal-chelating activity

The metal-chelating activity of ferrous ions by acetone, hexane, and hot water extract of C. igneus was estimated by the method given by Dinis TC et al.[24] Initially, about 100 μl different leaf extracts of C. igneus was added to 50 μl solution of 2 mM FeCl2. The reaction was initiated by the addition of 200 μl of 5 mM ferrozine, and the mixture was shaken vigorously and left standing at room temperature for 10 min. The mixture was vigorously shaken and left to stand at room temperature for 10 min. The absorbance of the solution was thereafter measured at 562 nm. The analysis was performed in triplicate, and the results were expressed as ethylenediaminetetraacetic acid (EDTA) equivalent/g of the extract. The metal-chelating capacity of the different leaf extracts of C. igneus was evaluated using the following equation:

Metal-chelating capacity (%) = ([A0 − A1]/A0] ×100, where A0 is the absorbance of the control and A1 is the absorbance of the sample extract/standard. The chelating activity of acetone, hexane, and hot water extract of C. igneus was evaluated using EDTA as standard, and results were expressed as mg EDTA equivalent/g extract.

Phosphomolybdenum assay

The antioxidant activity of acetone, hexane, and hot water extract of C. igneus was evaluated by the green phosphomolybdenum complex formation according to the method.[25] A 100 μL of different leaf extracts of C. igneus solution (in 1 mM dimethyl sulfoxide) was combined with 1 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate) in a vial. The vials were capped and incubated in a water bath at 95°C for 90 min. After the different leaf extracts of C. igneus had cooled to room temperature, the absorbance of the mixture was measured at 695 nm against a blank. The results reported are mean values expressed as grams of ascorbic acid equivalents/100 g extract.

Superoxide radical (O2•−) scavenging activity

The assay was based on the capacity of acetone, hexane, and hot water extract of C. igneus to inhibit formazan formation by scavenging the O2•− generated in riboflavin-light-nitro blue tetrazolium (NBT) system.[26] Each 3 ml reaction mixture contained 50 mM sodium phosphate buffer (pH 7.6), 20 μg riboflavin, 12 mM EDTA, 0.1 mg NBT, and 1 ml of different leaf extracts of C. igneus solution (50–250 μg/ml). Reaction was started by illuminating the reaction mixture with different concentrations of different leaf extracts of C. igneus for 90 s immediately; after illumination, the absorbance was measured at 590 nm. Identical tubes with reaction mixture kept in the dark served as blanks. The percentage inhibition of superoxide anion generation was calculated using the following formula: % of inhibition = ([A0 – A1]/A0) ×100, where A0 is the absorbance of the control and A1 the absorbance of acetone, hexane, and hot water extract of C. igneus/standard. The results were compared with the commercially available antioxidants such as rutin and BHT.

Antiradical activity using DPPH method

The antioxidant activity of acetone, hexane, and hot water extract of C. igneus was determined in terms of hydrogen donating or radical scavenging ability using the stable radical 2,2-diphenylpicrylhydrazyl (DPPH ), according to the method.[27] Different leaf extracts of C. igneus at various concentrations were taken, and the volume was adjusted to 100 μl with methanol. 5 ml of 0.1 mM methanolic solution of DPPH was added and shaken vigorously. The tubes were allowed to stand for 20 min at 27°C. The absorbance of the sample was measured at 517 nm. Radical scavenging activity of the samples was expressed as IC50. Concentration of different leaf extracts of C. igneus necessary to decrease initial concentration of DPPH by 50% (IC50) under the experimental condition was determined. Therefore, lower value of IC50 indicates a higher antioxidant activity. BHT and rutin were used as standard antioxidants in DPPH assay.

Reducing power

Various concentrations of acetone, hexane, and hot water extract of C. igneus (dissolved in appropriate solvents) were taken in 1 ml of phosphate buffer, and 5 ml of 0.2 M phosphate buffer (pH 6.6) was added. To this, 5 ml of 1% potassium ferricyanide solution was added and the mixture was incubated at 50°C for 20 min. After the incubation, 5 ml of 10% trichloroacetic acid was added. The content was then centrifuged at 1000 rpm for 10 min. The upper layer of the supernatant (5 ml) was mixed with 5 ml of distilled water and 0.5 ml of 0.1% ferric chloride. The absorbance of the reaction mixture was read spectroscopically at 700 nm. The reducing power of acetone, hexane, and hot water extract of C. igneus was determined by the method reported by SiddhurajuP et al.[28]

Cytotoxic potential of different leaf extracts of Costus igneus on Michigan Cancer Foundation-7 breast cancer cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

Cell culture and maintenance

Breast cancer cell lines (Michigan Cancer Foundation [MCF]-7) was obtained from the National Cell Culture Science, Pune, India. The cells were cultured in a cell culture flask using Dulbecco's Modified Eagles Medium/Nutrient Mixture F-12 HAM supplemented with 10% fetal bovine serum, 15 mM HEPES, NaHCO3, pyridoxine, and L-glutamine. The cells were maintained in a 5% CO2 incubator with 97% humidity at 37°C.

3--(4,5--dimethylthiazol--2--yl)--2,5--diphenyltetrazolium bromide assay procedure

Cytotoxicity assay was carried out according to the 3--(4,5--dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method as described by Mosmann T.[29] Breast cancer cell line (MCF-7) cells (5 × 103 cells/well) were seeded to 96-well microtiter plates (NUN96ft-Nunclon-96 Flat transparent) and incubated at 37°C in humidified atmosphere with 5% CO2 for 24 h. After 24 hrs of incubation, 50 μg/ml, 100 μg/ml, and 150 μg/ml acetone, hexane, and hot water leaf extracts of C. igneus were added in serum-free medium, and 0.2% (v/v) DMSO was kept as negative control. After 24 h of treatment, 50 μl of MTT reagent (2 μg/ml) was added to each well. The plates were incubated at 37°C in humidified atmosphere with 5% CO2 for 4 h. Medium was removed and 200 μl DMSO was added. The absorbance at 585 nm was measured using a Model 680 Microplate Reader (Shimadzu UV-1800). Percentage of cell viability was calculated according to the following equation:

Percentage of cell viability = (OD of treated cells/OD of control cells) ×100.

Crystal violet assay

MCF-7 cells have a polygonal morphology with irregular dimensions, and they have a habit of clumping into large aggregates. One simple method to detect maintained adherence of cells is the staining of attached cells with crystal violet dye, which binds to proteins and DNA. Cells that undergo cell death lose their adherence and are subsequently lost from the population of cells, reducing the amount of crystal violet staining in a culture.[30] The MCF-7 cells were treated with hexane, acetone, and hot water leaf extracts of C. igneus for 24 h and then washed twice with PBS for 3 min. The treated cells were then stained with 0.5 ml crystal violet dye for 5 min. After washing with PBS for three times, cells were observed under a fluorescent microscope by ×400 magnification.


   Results Top


Preliminary analysis of phytochemicals in different leaf extracts of Costus igneus

Qualitative tests for detecting the presence of various types of bioactive compounds were carried out in hot water, acetone, and hexane extracts of C. igneus. All the three extracts responded positively for alkaloid, flavonoid, carbohydrate, glycosides, saponins, tannin, phytosterol, and phenolics which have been depicted in [Table 1]. The test for phytochemical analysis inferred it was a good source of multiple phytoconstituents. Immediate production of orange precipitate and yellowish buff precipitate was observed after the addition of Dragendroff's and Mayer's reagent, respectively, in the hot water extract, indicating the highest amount of alkaloids presence followed by acetone extract and hexane extract. Flavonoid presence was recorded highest in acetone extract with the immediate disappearance of intense yellow color in the presence of dilute acid followed by hot water extract and hexane extracts. Carbohydrates were recorded the highest in acetone extract with the formation of a brown ring at the junction of two liquids on addition of alcoholic alpha-naphthol solution and concentrated H2 SO4. Following acetone extract carbohydrates were observed in hot water extract and hexane extract. Acetone extract of C. igneus recorded maximum of glycosides with the change of pink to red color in the extract after the addition of Legal's and Borntrager's reagent followed by hexane and hot water extracts. Saponin analysis showed that hot water extract contains highest saponins with the foam test and leads acetate test followed by acetone and hexane extracts. Tannin test revealed that hot water extracts recorded highest followed by acetone and hexane extract. Phytosterol was found highest in acetone extract followed by hexane and hot water extracts. Preliminary analysis of phenols revealed that all the three extracts recorded the presence of phenols by the formation of blue color while added with Folin–Ciocalteu reagent.
Table 1: Phytochemical test of different leaf extracts of Costus igneus

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Determination of total phenolic content in different leaf extracts of Costus igneus

Total phenolic content of different leaf extracts of C. igneus is shown in [Table 2]. Among the different extracts analyzed, hot water extract has recorded the highest phenolic content with the value of 169.08 mg GAE/1 g extract. However, it was followed by acetone extracts (168.74 mg GAE/1 g extract) and hexane extracts (105.57 mg GAE/1 g extract).
Table 2: Total phenolics contents in different extracts of Costus igneus leaves

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Determination of total tannin content in different leaf extracts of Costus igneus

The tannin content analysis results are shown in [Table 3]. The acetone leaf extract of C. igneus was found to contain highest tannin content of 86.66 mg GAE/1 g at 100 μg concentration followed by hot water leaf extract. It was found to be 58.90 mg GAE/g at 100 μg concentration, and hexane extracts of C. igneus leaves at 100 μg concentration were found to contain 45.71 mg GAE/1 g.
Table 3: Total tannin contents in different extracts of Costus igneus leaves

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Determination of total flavonoids content in different leaf extracts of Costus igneus

The estimation of total flavonoid contents of the different leaf extracts of C. igneus carried during this experiment is shown in [Table 4], and it was observed that all the extracts contain total flavonoid content. Hexane extract was found to contain maximum total flavonoid content with 535.48 mg RE/1 g extract, followed by acetone (481.11 mg RE/1 g extract) and hot water extract (59.07 mg RE/1 g extract).
Table 4: Total flavonoids contents in different extracts of Costus igneus leaves

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Ferric reducing antioxidant power assay in different leaf extracts of Costus igneus

Acetone extract showed higher FRAP activity of 276.31 mmol Fe (II) E/mg extract followed by hexane extract which recorded 146.19 mmol Fe (II) E/mg extract and hot water extract (127.14 mmol Fe (II) E/mg extract) as shown in [Table 5].
Table 5: Ferric reducing antioxidant power assay in different extracts of Costus igneus leaves

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ABTS •+ scavenging activity in different leaf extracts of Costus igneus

The capacity of ABTS •+ scavenging activity of different leaf extracts of C. igneus was tested as given in [Table 6]. The results obtained clearly imply that all different leaf extracts of C. igneus inhibit or scavenge ABTS radical cation. Among the different leaf extracts of C. igneus, hexane extract showed the highest scavenging activity of 12878.893 μM TEAC/g extract followed by acetone extract (12696.08 μM TEAC/g extract) and hot water extract (12656.18 μM TEAC/g extract). Triplicate determination was made at each dilution of the standard, and the percentage inhibition was calculated of the blank absorbance at 734 nm and then was plotted as a function of TEAC. The results were compared with the commercially available antioxidants such as BHT and rutin.
Table 6: ABTS scavenging activity in different extracts of Costus igneus leaves

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Metal-chelating activity in different leaf extracts of Costus igneus

In metal-chelating activity, among the different leaf extracts, the hot water extract registered higher metal ion-chelating ability with 2.94 mg EDTAE/g extract followed by hexane extract (2.86 mg EDTAE/g extract) and acetone extract (2.14 mg EDTAE/g extract) as depicted in [Table 7]. The absorbance of the solution was thereafter measured at 562 nm. The analysis was performed in triplicate, and the results were expressed as mg EDTA equivalent per gram of the extract.
Table 7: Metal-chelating activity in different extracts of Costus igneus leaves

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Phosphomolybdenum assay in different leaf extracts of Costus igneus

The reducing ability of different extracts of C. igneus leaf expressed in terms of ascorbic acid equivalents is presented in [Table 8]. All the extracts tested in the present study recorded significant antioxidant activity. Among the different extracts, hexane extract has recorded highest total antioxidant capacity of 65.00 mg AAE/mg of plant extract followed by acetone extract (56.66 AAE/mg of plant extract) and hot water extract (53.33 AAE/mg of plant extract). The results were compared with the commercially available antioxidants such as BHT and rutin.
Table 8: Phosphomolybdenum activity in different extracts of Costus igneus leaves

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Superoxide radical scavenging activity in different leaf extracts of Costus igneus

The scavenging capacity of superoxide radicals by the different leaf extracts of C. igneus is shown in [Table 9]. Among the different extracts, highest superoxide radical scavenging activity was found in acetone extract with 41.11% followed by hexane extract which showed the superoxide radical scavenging activity of 38.36% and hot water extract with 35.35%. After illumination, the absorbance was measured at 590 nm. The results were compared with the commercially available antioxidants such as BHT and rutin.
Table 9: Superoxide radical scavenging activity in different extracts of Costus igneus leaves

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Free radical scavenging activity using 2,2- diphenylpicrylhydrazyl in different leaf extracts of Costus igneus

DPPH radical scavenging activity of different leaf extracts of C. igneus is shown in [Table 10]. The free radical scavenging activity of different leaf extracts was determined, among which acetone extract recorded total free radical scavenging capacity with IC50 value of 40.70 μg/ml followed by hot water extract with IC50 value of 42.30 μg/ml and hexane extract with IC50 value of 48.33 μg/ml which is comparable activity to the reference standards BHT (IC5045.56 μg/ml) and rutin (IC5024.71 μg/ml). Radical scavenging activity of the leaf extracts was expressed as IC50 which is the concentration of different leaf extracts required to inhibit 50% of DPPH˙ concentration. BHT and rutin were used as positive control.
Table 10: 2,2-diphenylpicrylhydrazyl scavenging activity in different extracts of Costus igneus leaves

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Reducing the power of Costus igneus

Among the different leaf extracts of C. igneus, hexane extract has recorded significant highest reducing power at concentration 100 μg/μl with maximum optical density of 0.177, whereas hot water and acetone extracts showed 0.166 and 0.092, respectively, as given in [Table 11]. The standard reference BHT and rutin had significantly highest activity of 1.516 and 0.531, respectively. The absorbance of the reaction mixture was read spectroscopically at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power.
Table 11: Reducing power in different leaf extracts of Costus igneus

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Cytotoxic potential of different leaf extracts of Costus igneus on Michigan Cancer Foundation-7 breast cancer cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

The cytotoxic activity of different leaf extracts of C. igneus on MCF-7 breast cancer cell line is depicted in [Table 12]. Among three different hexane, acetone, and hot water extracts studied for cytotoxic activity against breast cancer cell line (MCF-7), acetone extract at concentration of 150 μg/ml showed highest cytotoxic activity on cancer cell line with cell viability of 65.51% (65.41 ± 0.08) as compared to positive control doxorubicin (53.85 ± 0.16). The morphological changes of MCF-7 are shown of both treated and untreated cells which are shown in [Figure 1].
Table 12: Cytotoxic activity of different leaf extracts of Costus igneus on Michigan Cancer Foundation-7 breast cancer cell line

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Figure 1: Morphological changes of Michigan Cancer Foundation-7-treated cells observed under a fluorescent microscope (×400). (a) Untreated cells (b) Treated cells

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Crystal violet assay

The crystal violet assay involves simultaneous cell lysis and the released nuclei to stain purple in a hypertonic solution. Cells that undergo cell death lose their adherence and are subsequently lost from the population of cells which are shown in [Figure 2].
Figure 2: The treated cells stained with 0.5 ml crystal violet dye cells were observed under a fluorescent microscope by × 400. (a) Untreated cells (b) Treated cells

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   Discussion Top


The reports of previous studies carried by Palanivel et al., 2013,[31] observed that phytochemical screening of ethanolic extract of C. igneus showed the presence of alkaloids, phytosterols, saponins, glycosides, carbohydrates, tannins, and phenolic compounds. In the present study, different leaf extracts of C. igneus underwent preliminary phytochemical screening, and the preliminary phytochemical tests indicated the presence of alkaloids, glycosides, saponins, tannins, phytosterols, phenolics, and carbohydrates.

Study on determination of total phenolic content and total antioxidant activity in locally consumed foodstuffs in Moodabidri, Karnataka, India, by Samshuddin et al., 2015,[32] while on screening of foodstuffs revealed total phenolic content ranged from 0.38 to 18.40 mg GAE/g of dry weight of the sample. The total antioxidant activity of analyzed samples varied from 2.75 to 48.94 mg AAE/g of dry weight of the sample. The high total phenolic content of curry leaves (18.40 mg GAE/g) contributed to its high antioxidant activity (48.94 mg AAE/g). C. igneus in the present study for total phenolic content estimation revealed that hot water extract has recorded the highest phenolic content with the value of 169.08 mg GAE/1 g extract. However, it was followed by acetone extract (168.74 mg GAE/1 g extract) and hexane extracts (105.57 mg GAE/1 g extract).

Previous studies recorded the total tannin content of the water, methanol, and 95% ethanol extracts of T. chebula which varied from 33.9% to 40.3%/mg extract.[33] The present study describes the tannin content analysis which was expressed as the GAEs and showed that the acetone leaf extract of C. igneus was found to contain highest tannin content of 86.66 mg GAE/1 g at 100 μg concentration followed by hot water leaf extract. It was found to be 58.90 mg GAE/g at 100 μg concentration, and hexane extracts of C. igneus leaf at 100 μg concentration were found to contain 45.71 mg GAE/1 g.

Flavonoid are referred as nature's biological modifiers due to their ability to respond to allergens, viruses and carcinogens. The major flavanol in the litchi fruit pericarp is reported to be procyanidin B4, procyanidin B2, and epicatechin, while cyanindin-3-rutinside, cyanidin-3-glucoside, quercetin-3-rutinosde, and quercetin-3-glucoside are identified as the important anthocyanins.[34] In the present study, the estimation of total flavonoid contents of the different leaf extracts of C. igneus revealed that all the extracts contain total flavonoid content. Hexane extract was found to contain maximum total flavonoid content with 535.48 mg RE/1 g extract followed by acetone (481.11 mg RE/1 g extract) and hot water extract (59.07 mg RE/1 g extract).

During the metabolic process, oxidation is normal phenomenon, while some of highly reactive oxygen components such as hydrogen peroxide and hypochlorous acid and various free radicals are generated.[35],[36],[37],[38] During the FRAP assay among all extracts, acetone extract showed higher FRAP activity of 276.31 mmol Fe (II) E/mg extract followed by hexane extract which recorded 146.19 mmol Fe (II) E/mg extract and hot water extract (127.14 mmol Fe (II) E/mg extract). The absorbance readings were taken immediately at 595 nm, using a spectrophotometer.

ABTS on oxidation by potassium persulfate releases ABTS+ measures antioxidant effect by electron-donating antioxidants (scavengers of aqueous phase radicals) and chain-breaking antioxidants (scavengers of lipid peroxyl radicals).[39] ABTS •+ also involves an electron transfer process.[40] The results obtained in the present study clearly imply that all different leaf C. igneus extracts inhibit or scavenge ABTS radical cation. Among the different leaf extracts of C. igneus, hexane extract showed the highest scavenging activity of 12878.893 μM TEAC/g extract followed by acetone extract (12696.08 μM TEAC/g extract) and hot water extract (12656.18 μM TEAC/g extract).

The study reports carried by Amin et al., 2013,[41] on Costus pictus leaves observed the high Fe2+-chelating effect of Costus pictus leaves. In the present study of metal-chelating activity, among the different leaf extracts, the hot water extract registered higher metal ion-chelating ability with 2.94 mg EDTAE/g extract followed by hexane extract (2.86 mg EDTAE/g extract) and acetone extract (2.14 mg EDTAE/g extract). The absorbance of the solution was thereafter measured at 562 nm.

The antioxidant activity by phosphomolybdenum assay was found highest in aqueous extract of C. cajan.[42] In the present study, all the different leaf extracts of C. igneus recorded significant antioxidant activity. Among three different leaf extracts of C. igneus, hexane extract of C. igneus has recorded the higher total antioxidant capacity with value of 65.00 mg AAE/mg of plant extract followed by acetone extract (56.66 AAE/mg of plant extract) and hot water extract (53.33 AAE/mg of plant extract). The results of reducing ability of different leaf extracts C. igneus were expressed in terms of ascorbic acid equivalents.

Report of previous study by Malairaj et al., 2012[43] observed that chloroform fraction of bark of Costus pictus exhibits excellent superoxide anion scavenging activity lower than that of ascorbic acid. In the present study among the different extracts, the highest superoxide radical scavenging activity was found in acetone extract with 41.11% followed by hexane extract which showed the superoxide radical scavenging activity of 38.36% and hot water extract with 35.35%.

In vitro study of the alcoholic extract of leaves of C. igneus showed moderate antioxidant activity.[44] The antioxidant activities of methanol, aqueous, ethanol, and ethyl acetate extracts of leaves and rhizome were assessed using different models such as DPPH. Leaves and rhizomes of insulin plant showed antioxidant activity of about 89.5 and 90.0% when compared with standard BHT (85%) at a concentration of 400 μg/ml. Results obtained revealed that methanolic extracts of both leaves and rhizomes of C. igneus possess higher antioxidant activity when compared with other extracts.[45] Radical scavenging activity of different leaf extracts of C. igneus in the present study showed among all extracts, acetone extract recorded total free radical scavenging capacity with IC50 value of 40.70 μg/ml followed by hot water extract with IC50 value of 42.30 μg/ml and hexane extract with IC50 value of 48.33 μg/ml which is comparable activity to the reference standards BHT (IC5045.56 μg/ml) and rutin (IC5024.71 μg/ml). In the previous study of evaluation of aqueous and methanolic leaf extract of Epipremnum aureum reported by Sherikar and Mahanthesh, 2015,[46] for the reducing power assay recorded as concentration of sample increases, absorbance also increases. Higher absorbance for the reaction mixture indicated greater reducing power. Among the different leaf extracts of C. igneus hexane extract have recorded significant highest reducing power at concentration 100 μg/μl with maximum optical density of 0.177, whereas hot water and acetone extracts showed 0.166 and 0.092, respectively.

Balijepalli et al., 2010,[47] investigated the ethyl acetate extract from Grewia asiatica roots as antiproliferative agents on human breast cancer cells (MCF-7) which were due to the presence of lignins and flavonoids. Merlin et al., 2010,[48] have investigated that the chloroform extract of aerial parts of G. asiatica possesses potential anticancer activity in caspase three deficient breast cancer cell line MCF-7. Among three different hexane, acetone, and hot water leaf extracts of C. igneus studied for anticancer activity against breast cancer cell line (MCF-7), acetone extract of C. igneus at concentration of 150 μg/ml showed highest cytotoxic activity on cancer cell line with cell viability of 65.51% (65.41 ± 0.08) as compared to positive control doxorubicin (53.85 ± 0.16).


   Conclusion Top


Bioactive screening revealed that all the three extracts are good source of phytochemicals such as alkaloid, flavonoid, carbohydrate, glycosides, saponins, tannin, phytosterol, and phenolics. In vitro antioxidants assays, acetone extract of C. igneus showed higher FRAP activity, superoxide radical scavenging activity, and DPPH scavenging. Hexane extract of C. igneus showed the highest potential in ABTS scavenging activity and antioxidant capacity in phosphomolybdenum assay and also recorded significant highest reducing power with maximum optical density, whereas hot water extract showed the highest metal ion-chelating ability. Acetone extract of C. igneus has shown highest cytotoxic activity on cancer cell line (MCF-7) as compared to positive control doxorubicin. The present study thus explored that C. igneus shows efficient antioxidant activity as well as cytotoxic potential and can act safe and cost-effective in biological applications.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10], [Table 11], [Table 12]



 

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