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SHORT COMMUNICATION
Year : 2015  |  Volume : 4  |  Issue : 4  |  Page : 267-270

Cytotoxicity activities of chloroform extract of Cichorium intybus seed against HCT-15 and Vero cell line


Department of Pharmacology, NGSPMS, College of Pharmacy, Brahma Valley Educational Campus, Nashik, Maharashtra, India

Date of Web Publication20-Oct-2015

Correspondence Address:
Prashant Y Mali
Department of Pharmacology, NGSPMS, College of Pharmacy, Anjaneri, Trimbak Road, Nashik - 422 213, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2278-344X.167647

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  Abstract 

Background: Cichorium intybus L., (Asteraceae) is well-known as a coffee substitute but is also widely used medicinally to treat various ailments ranging from wounds to diabetes. Other plant parts are also used for liver and cancer disorder. Objective: The objective was to study the cytotoxic potential of chloroform extract of C. intybus seed against HCT-15 and Vero (normal) cell line. Materials and Methods: Fourier transform infrared spectroscopy (FTIR) analysis of the extract was performed. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay was used for evaluation of the cytotoxic potential of chloroform extract of C. intybus seed. Doxorubicin was considered as standard reference drug. The concentrations 1000–0.05 μg/ml was used in the experiment. Result and Discussion: FTIR spectrum showed 1025.363, 1083.126, 1291.366, 1389.144, and 1569.294 peaks/centers in the wavelength region of 4,000.00–650.00 cm−1. The chloroform extract of C. intybus seed and doxorubicin was showed 1411.37 μg/ml and 460.13 μg/ml 50% cell growth inhibition (IC50) against the HCT-15 cell line. Both extract and doxorubicin were safe against the Vero (normal) cell line. Conclusion: It can be concluded that the chloroform extract of C. intybus seed was not efficient against the HCT-15 cell line at the concentrations used in the experiment. Furthermore, there is no need to explore the said studies by in vivo models.

Keywords: Cytotoxicity, Cichorium intybus, HCT-15, and Vero cell line


How to cite this article:
Mali PY. Cytotoxicity activities of chloroform extract of Cichorium intybus seed against HCT-15 and Vero cell line. Int J Health Allied Sci 2015;4:267-70

How to cite this URL:
Mali PY. Cytotoxicity activities of chloroform extract of Cichorium intybus seed against HCT-15 and Vero cell line. Int J Health Allied Sci [serial online] 2015 [cited 2020 Aug 8];4:267-70. Available from: http://www.ijhas.in/text.asp?2015/4/4/267/167647


  Introduction Top


Carcinoma of the breast, colon, and prostate are highly prevalent malignancies in the Western nations and accounts for approximately half of the total cancer-related deaths among men and women.[1] The limited success of clinical therapies including radiation, chemotherapy, immunomodulation, and surgery in treating cancer, as evident by the high morbidity and mortality rates, indicates that there is an imperative need for new cancer management. Chemoprevention involves the use of pharmacological, dietary bio-factors, phytochemicals, and even whole plant extracts to prevent, arrest, or reverse the cellular and molecular processes of carcinogenesis due to its multiple intervention strategies.[2]

Cichorium intybus L., (Asteraceae) seeds are one of the main ingredients of jigrine a commercial product of India used for the treatment of various diseases of the liver.[3] Prior to the wars in Afghanistan, folkloric reports described the use of aqueous root extracts as a light-sensitive plant remedy for malaria. This indigenous knowledge has since been confirmed, and the antimalarial compounds of C. intybus roots have been identified as the light-sensitive sesquiterpene lactones lactucin and lactucopicrin.[4] The flowers of the chicory plant are used as an herbal treatment of everyday ailments such as a tonic and appetite stimulant and as a treatment of gallstones, gastroenteritis, sinus problems, cuts, and bruises.[5] Other plant parts are also used for liver disorders, cancers, namely, aerial parts in Bosnia and Herzegovina,[6] and roots in Serbia and India.[7],[8] Chicoric acid has been identified as the major compound in methanolic extracts of chicory which includes Cichorioside-B, Artesin, Cichoriolide, and Cichorioside, etc.[9],[10]C. intybus has reported as antimalarial,[11] hepatoprotective,[12],[13],[14] gastroprotective,[15] and anti-inflammatory,[16] etc., for its pharmacological actions. Toxicological data on C. intybus is currently limited. Therefore, the present study was an effort to investigate cytotoxicity study of C. intybus seed against HCT-15 and Vero cell line.


  Materials and Methods Top


Preparation of extract

The seeds of C. intybus were purchased from local market of Jalgaon, Maharashtra, and were authenticated by a taxonomist, Department of Botany, Dr. AGD Bendale Mahila Mahavidyalaya, Jalgaon, Maharashtra, India. The seeds were then powdered and extracted with chloroform using percolation extraction method. The completely dried extract was preserved in an airtight container under refrigeration for further use.

Fourier transform infrared spectroscopy analysis of Cichorium intybus seed extract

The Fourier transform infrared spectroscopy (FTIR) (Agilent Cary 630, Diamond ATR) analysis of chloroform extract of C. intybus seed was performed.

Procurement and culturing of cell lines

HCT-15 and Vero cell lines were procured from National Centre for Cell Science, Pune. Stock cells of these cell lines were cultured in Dulbecoos Modified Eagels Medium, supplemented with 10% fetal bovine serum. Along with media cells were also supplemented with 5% HBSS, penicillin, streptomycin, and amphotericin – B, in a humidified atmosphere of 5% CO2 at 37°C until confluence reached. The cells were dissociated with 0.2% trypsin, 0.02% ethylenediaminetetraacetic acid in phosphate buffer saline solution. The stock cultures were grown initially in 25 cm 2 tissue culture flasks, then in 75 cm 2, and finally in 150 cm 2 tissue culture flask and all cytotoxicity experiments were carried out in 96 micro titer well plates.

3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay

The method described by Mossmann with some modifications was used.[17],[18],[19],[20] Briefly, cell lines in exponential growth phase were washed, trypsinized and resuspended in complete culture media. Cells were seeded at a concentration of 2 × 104 cells/well in 96 well micro titer plate and incubated for 24 h during which partial monolayer forms. The cells were then exposed to various concentrations of the test compounds and standard drug doxorubicin, (i.e., concentration 1000–0.05 µg/ml). Control wells were received only maintenance medium. The plate was incubated at 37°C in humidified incubator with 5% CO2 and 75% relative humidity for a period of 24 h. At the end of 24 h, 10 µl 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide labeling mixture was added and incubates for 4 h. The absorbance was measured using microplate ELISA reader at wavelength 590 nm. The percentage cell growth inhibition or percentage cytotoxicity was calculated by following formula,



where, AT = Absorbance of treated cells (drug); AB = Absorbance of blank (only media); and AC = Absorbance of control (untreated).

% cell growth inhibition/% cytotoxicity = 100 − % cell survival.

Statistical analysis

All the values of percent cell growth inhibition was replicated of three independent observations (n = 3). The 50% cell growth inhibition (IC50) was determined by interpolating concentration (X-axis) versus % cell inhibition (Y-axis) by linear regression equation using Microsoft Excel, 2007, Microsoft Corporation, USA application.


  Results Top


Fourier transform infrared spectroscopy analysis of C. intybus seed extract

The spectral analysis of chloroform extract of C. intybus seed were performed using FTIR spectrophotometer at the wavelength region of 4,000.00–650.00 cm 1 and resolution 4 cm 1 [Figure 1] and [Table 1]. It has showed five peaks/centers that correspond to 1025 (C–N stretching), 1300–1000 (C–O stretching) alcohol and phenols, 1310–1250 (aromatic C–O stretching) esters, 1300–1100 (aliphatic and anhydrides C–O stretching) esters, 1220–1020 (aliphatic C–N stretching) amines, 1360–1250 (aromatic C–N stretching) amines, 1300–1270 (nitrate NO2 symmetric stretching) various nitrogen-containing compounds, 1300–1000 (C–F stretching) organic halogen compounds, and 1390–1290 (SO2 asymmetric stretching) sulfur group compounds might be present in the extract.
Figure 1: Fourier transform infrared spectroscopy spectrum is showing peaks/centers in chloroform extract of Cichorium intybus seed

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Table 1: FTIR spectrophotometer parameters and peak ranges of chloroform extract of C. intybus seed

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3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay

The chloroform extract of C. intybus seed and doxorubicin was showed 1411.37 µg/ml and 460.13 µg/ml 50% cell growth inhibition (IC50) against the HCT-15 cell line [Figure 2], [Figure 3] and [Table 2]. The IC50 range of chloroform extract of C. intybus seed was more than the concentrations used in the experiment. Therefore, the said extract was less efficient against HCT-15 cell line. The 50% cell growth inhibition (IC50) of chloroform extract of C. intybus seed, and doxorubicin was 1160.65 µg/ml and 2392.71 µg/ml against Vero (normal) cell line [Figure 4], [Figure 5] and [Table 2]. Both the chloroform extract of C. intybus seed and doxorubicin was safe with respect to the concentrations used against Vero (normal) cell line.
Figure 2: % cell inhibition of chloroform extract of Cichorium intybus seed against HCT-15 cell line

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Figure 3: % cell inhibition of doxorubicin against HCT-15 cell line

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Figure 4: % cell inhibition of chloroform extract of Cichorium intybus seed against vero (normal) cell line

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Figure 5: % cell inhibition of doxorubicin against vero (normal) cell line

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Table 2: % cell inhibition of chloroform extract of C. intybus seed against HCT-15 and vero cell line

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  Discussion and Conclusion Top


Approximately, 60% of the anticancer drugs currently have been isolated from natural products from the plants.[21] Based on the FTIR spectrum of chloroform extract of C. intybus seed, further there will be a need to isolate, elucidate, identify, and confirm the phytoconstituents present in the extract by using mass, nuclear magnetic resonance, and Gas chromatography-mass spectrometry, etc., techniques. At this time, more than 3000 plants worldwide have been reported to possess anticancer properties.[21] In the US, National Cancer Institute Plant Screening Programme, a crude extract is generally considered to have in vitro cytotoxic activity if the IC50 value in carcinoma cells, following incubation between 48 h and 72 h, is <20 μg/ml, while it is <04 μg/ml for pure compounds.[22] Based on these criteria, chloroform extract of C. intybus seed was not showing its IC50 value <20 μg/ml due to because of non-extraction of cytotoxic bioactive constituents into chloroform solvent which are not cytotoxic to HCT-15 cell line. This extract was safe to Vero (normal) cell line. It can be concluded that chloroform extract of C. intybus seed was not efficient against the HCT-15 cell line at the concentrations used in the experiment. Hence, further there is no need to explore the said studies by in vivo models.


  Acknowledgment Top


The author would like to thankful to Dr. Vipul P. Patel, Department of Pharmaceutical Biotechnology, S.K. Patel College of Pharmaceutical Education and Research, Ganpat University, Mehsana, Gujarat and Dr. Ramprakash S. Gupta, Department of Zoology, Dr. AGD Bendale Mahila Mahavidyalaya, Jalgaon, Maharashtra, India for providing the necessary facilities to the tenure of investigation.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2]



 

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