EVALUATION OF ANTI – CANCER ACTIVITY OF BARK OF CRATAEVA NURVALA BUCH. HAM AGAINST THREE CELL LINES

EVALUATION OF ANTI - CANCER ACTIVITY OF BARK OF CRATAEVA NURVALA BUCH. HAM AGAINST THREE CELL LINES

N.S. Wagh^* and N. J. Gaikwad

Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, Amravati road, Nagpur- 440033, Maharashtra, India.

ABSTRACT: The present study was aimed to evaluate in-vitro anti-cancer activity of ethanolic extract of Crataeva nurvala Buch. Ham. The method used for determining the anticancer activity was the MTT bio-assay using A549(Human Lung Carcinoma) cell line, Hela (Human Cervix)cell line and MDA-MB(Human Adenocarcinoma, Mammary Gland)cell line. The ethanolic extract of Crataeva nurvala bark at different concentration (10µg, 20 µg and 30 µg) was used as test for this bioassay. Vincristine30µg/ml (anticancer drug) was used as reference standard and blank as control. Optical density and % cell lysis was estimated at the end of the study. Extract showed promising anticancer effect. % Cell lysis was found to be in the range nearer to the reference standard for all the cell lines and optical density at 492nm is (0.575to 1.191) almost equal to that of reference standard i.e. 1.151 and always more than blank.  The IC₅₀ values were found to be less than 10μg against A549 cell line, the activities against HELA cell line were found to be 13μg and IC₅₀ values were found to be 20μg against MDA-MB cell lines. These finding support that Crataeva nurvala bark extract was found to have a good anti-cancer activity.

Crataeva nurvala, MTT assay, A549 cell lines, Hela cell lines, MDA-MB cell lines, Anti-cancer activity

INTRODUCTION: Cancer is a group of diseases characterized by abnormal, excessive, uncoordinated, autonomously controlled and purposeless proliferation of cells due to local tissue invasion and distant metastasis ¹. Cancer is the second to the cardiovascular disease in causes of mortality in world. More than 1.2 million cases of cancer are diagnosed annually and more than 500,000 lives in United State each year. According to Kidwai Memorial Institute of Oncology, the crude incidence of cancer in India is about 1000 per 100,000 populations i.e. approximately one third the rate of developed countries and continuously increasing per day due to stressful life style ².

Inrecentyears,theincreaseinthenumberofcancer cases hasmotivatedthegrowthofcancerresearch.A large number ofnaturalproductsanddietarycomponentshavebeen evaluated aspotentialchemo preventiveagents,andherbalremedies usedintraditionalfolkmedicineprovidealargelyunexplored sourceofpotentialnoveldrugs³.Chemotherapy is an effectivetreatment against various types of cancer either singlyor in combination with surgery and/or radiotherapy.In spite of a number of new anti-cancer drugs the prevalence of cancer has been increased now-a-days.

However, chemotherapeutic effects of most of thedrugs showed limited efficacies due to thedevelopment of various side effects⁴.It hasbecome a major cause of death and there is an urgent need forits control. The most important drawback of the current cancertherapeutic practices such as chemotherapy and radiationtherapy is the suppression of immune system. Plants have beena prime source of natural products for the treatment of variousdiseases and they are highly effective conventional drugs for thetreatment of many forms of cancer^5-9.A traditional and folklore medicine plays an important role in the health services around the globe. About three quarters of the world populationrelies on the plants and plant extracts for healthcare. India has an extensive forest cover, enriched with plant diversity. Several plants have been used folklore medicine¹⁰.

Crataeva nurvala was yet not scientifically reported having anti-cancer activity. Howeverplant is frequently preferred in the treatment of Urinary disorders, Prostate enlargement,Bladder sensitivityand Kidney stones. Plant also showedAnti-arthritic, Hepatoprotective, and Cardio-protective actions¹¹.Lupeol is a naturally occurring triterpene isolated from Crataeva nurvala stem bark,various In-vitro and Preclinical animal studies suggested that lupeol has a potential to act as an Anti-inflammatory, Anti-microbial, Anti-protozoal, Anti-proliferative, Anti-invasive, Anti-angiogenic and cholesterol lowering agent. It is noteworthy that lupeol has been reported to selectively target diseased and unhealthy human cells, while sparing normal and healthy cells^12-13. The objective of the present work is to evaluate the anti-cancer activity of the ethanolic extract of Crataeva nurvalabark.

MATERIALS AND METHODS:

Plant Material: Crataeva nurvala Buch-Ham (Family: Capparidaceae) commonly known as Varuna, is an evergreen tree indigenous to India ¹⁴. It is a medium sized branched deciduous plant distributed throughout the river banks of southern India and other tropical, sub-tropical countries of the world, wild or cultivated¹⁵. It requires dry, hot climate and shady places to grow effectively. Vedic literatures described its potentiality as blood purifier and to maintain homeostasis.

Its bark is hot, bitter at first and then sweets harp taste, easy to digest, Stomachic, Laxative, Anthelmintic, Expectorant and Anti-pyretic. Plant of Crataeva nurvala collected from the forest region nearby Amravati, Maharashtra, India. The collected plant was authenticated from Department of Botany, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur; the voucher specimen has been deposited in the office (specimen No.9802)

FIGURE 1: AUTHENTICATED HERBERIUM OF CRATAEVA NURVALA FROM RASHTRASANT TUKADOJI MAHARAJ NAGPUR UNIVERSITY, NAGPUR

 Preparation of Extract: Dried coarsely powdered bark of Crataeva nurvala (500 g) were defatted with petroleum ether at 50⁰ - 60⁰ C for 72 h. using Soxhlet apparatus. The marc left subsequently extracted with ethanol (95%, 60-70^oC) for 72 h. The crude brown residue mass of extract were concentrated, stored and preserved (2-8^oC). The Percentage yield of extract (5 % w/w) was found on dry wet basis.¹⁶.

Maintenance of Cell culture: The method used was the MTT assay for evaluation. The A549 cell lines, Hela cell lines and MDA-MB cell lines were maintained at The Maratha Mandal Dental College, Belgaum, Karnataka. Cell lines were grown in Minimal Essential Medium(MEM) supplemented with 4.5 g/L Glucose, 2 ml L-Glutamine and 5% Fetal Bovine Serum (FBS) (growth medium) at 37 ⁰C in 5% CO₂ incubator.

MTT Assay^17-18:

Materials and Equipment: 5mg/ml MTT (3-{4, 5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromine solution in PBS. Sterilized by filtration, Stored in dark at 4°C, Stable a month, 96-well plates, Multichannel  pipettes, Micro-plate reader equipped with a 490 nm filter.

Experimental procedure: After treatment of cells with the xenobiotics, the cell monolayers were getting washed with warm PBS using a multichannel pipette.100 µL of culture medium containing MTT solution (10:1) was added to each well. It was recommended to have some wells in the plate without cells but incubated with MTT solution in order to have blanks of the readings. Cells were kept for 3h in the cell incubator. Incubation medium was carefully removed.100µL of Dimethyl sulphoxide was added and the plate is gently shaken to re-suspend formed for reason and waited until a homogenized color a formed.

Reading of results and calculations: Absorbance was measured at 490 nm of wells containing cells and blanks. The mean of the absorbance of wells was calculated with the all treatments after subtracting of blank absorbance. The results were normalized considering control wells as 100% (maximum absorbance obtained), expressing then the results as percentage of controls. The O.D. of control was taken to be negative. Increase in O.D. indicates increased cell lysis. The O.D. increases due to increased turbidity which was due to increased cell lysis. Three concentrations (10, 20 and 30µl) of extract was used. The IC₅₀ value and % lysis cell at every concentration was also calculated. Three cell lines were used for performing anticancer activity i.e A549, HELA, MDA-MB.

The percentage inhibition of cancer cell lines was calculated by the following equation

100-(At-Ab) / (Ac-Ab) × 100

Where   At: Absorbance of test,

Ab: Absorbance of blank,

Ac: Absorbance of control.

 RESULT: The effect of ethanolic extracts of Crataeva nurvala (EtCn) was studied by using MTT assay.

Against A549-Human lung carcinoma cell line: The IC₅₀ value of Crataeva nurvala against A549- Human lung carcinoma cell line was found to be 10μg and the results are mentioned in Table 1 and Figure 2 and Figure 3.

TABLE 1: IN-VITRO CYTOTOXICITY EFFECT AGAINSTA549 CELL LINES

Where, O.D. –Optical Density

FIGURE 2: MTT BIOASSY MICRO PLATES FOR THE CELL LINE A549 - HUMAN LUNG CARCINOMA AT VARIOUS CONCENTRATIONS

FIGURE 3: GRAPHICAL PRESENTATION OF OPTICAL DENSITY AND % CELL LYSIS AGAINST THE CELL LINE A549 - HUMAN LUNG CARCINOMA AT VARIOUS CONCENTRATIONS

Against HELA-Human cervix carcinoma cell line: The IC₅₀ value of Crataeva nurvala against HELA cell line was found to be 13μg and the

results are mentioned in Table 2. and Figure 4 and Figure 5

TABLE 2: IN-VITRO CYTOTOXICITY EFFECT AGAINST HELA CELL LINE

Where, O.D. –Optical Density

FIGURE 4: MTT BIOASSY MICROPLATE FOR THE CELL LINE HELA - HUMAN CERVIX CARCINOMA AT VARIOUS CONCENTRATIONS

FIGURE 5: GRAPHICAL PRESENTATIONOF OPTICAL DENSITY AND % CELL LYSIS AGAINST THE CELL LINE HELA - HUMAN CERVIX CARCINOMA AT VARIOUS CONCENTRATIONS

Against MDA-MB-Human Adenocarcinoma, Mammary Gland Carcinoma cell line:

The IC₅₀ value of Crataeva nurvala against MDA-MB cell

line was found to be 20μg and the results are mentioned in Table 3 and Figure 6 and Figure 7.

TABLE 3: IN-VITRO CYTOTOXICITY EFFECT AGAINST MDA-MB CELL LINE

Where, O.D. – Optical Density

FIGURE 6: MTT BIOASSY MICROPLATE FOR THE CELL LINE MDA-MB - HUMAN ADENO CARCINOMA, MAMMARY GLAND CARCINOMA AT VARIOUS CONCENTRATIONS

FIGURE 7. GRAPHICAL PRESENTATION OF OPTICAL DENSITY AND % CELL LYSIS AGAINST THE CELL LINE MDA-MB - HUMAN ADENO CARCINOMA, MAMMARY GLAND CARCINOMA AT VARIOUS CONCENTRATIONS.

DISCUSSION: Table 1 recorded the anticancer effect of Crataeva nurvala using MTT assay represented by optical density and % cell Lysis against different cell lines. Also the inhibitory concentration (IC₅₀) of each extract is recorded to determine the potency. The cell lines used is A549- Human lung carcinoma, the extract recorded cell lysis upto 100% at highest conc. It also recorded good value of the inhibitory concentration i.e.IC₅₀ (i.e.10 µg) indicating the potential of the ethanolic extract.  Data of cell line-A549 indicate that optical density of the samples plate was increase with increase in the concentration which was more than the value for the control at lowest concentration and at the highest concentration it reached near the value of the standard drug, indicating that extracts possesses the anticancer activity.  Graphical representation of the data is given in the Figure 3 and Images of micro plates represented the effect of the drug on the cell in comparison to the control and the standard. Number of dead cells in the plate can be seen easily in Figure 2.

Table 2 recorded anticancer effect of the extract using cell line HELA- Human Cervix Carcinoma, the extract recorded cell lysis upto 100% at highest conc. It also recorded good value of the inhibitory concentration i.e.IC₅₀ (i.e.13µg) indicating the potential of the ethanolic extract.  Data of cell line-HELA indicate that optical density of the samples plate was increase in the same manner that of the A549 which was more than the value for the control at lowest concentration and at the highest concentration it reached near the value of the standard drug, indicating that extracts possesses the anticancer activity.  Graphical representation of the data is given in the Figure 5 and Images of microplates represented the effect of the drug on the cell in comparison to the control and the standard. Number of dead cells in the plate can be seen easily in Figure 4.

Table 3 recorded anticancer effect of the extract using cell line MDA-MB- Human adenocarcinoma, mammary gland carcinoma, and the extract recorded cell lysis upto 75% at highest conc. It also recorded good value of the inhibitory concentration IC₅₀ (i.e.20µg) indicating the potential of the ethanolic extract. Data of cell line-MDA-MB indicate that optical density of the samples plate was also increased with the increase in the concentration.  Graphical representation of the data is given in the Figure 7 and Images of microplates represented the effect of the drug on the cell in comparison to the control and the standard. Number of dead cells in the plate can be seen easily in Figure 6.

 CONCLUSION: In the present investigation the different concentration of the ethanolic extracts of Crataeva nurvalawith three different cell lines, Vincristine as standard anticancer drug were used. The parameter like % Cell Lysis, Optical Density and IC₅₀ value were evaluated for the anticancer activity.  The result of Cell line- A549 - Human lung carcinomashowed that optical density of t samples plate was increase with increase in the concentration which was more than the value for the control at lowest concentration and at the highest concentration it reached near the value of the standard drug, indicating that extract posses the anticancer activity. The parameter of % Cell lysis value for the test sample was found to be always more than that of the control i.e. ranging from 70 to 100 % and equal to the standard, which support the data for anticancer potential of the extract. The IC₅₀ value of 10µG is indicative of good anticancer potential of the extract.

In order to conform reproducibility of the result, more work is needed with the cell line Hela - Human cervix and cell line MDA-MB - Human adenocarcinoma, mammary gland. The Results shown in Table 2, 3 and Figure 4, 5, 6, 7 respectively were found to be identical and indicative of anticancer potential of the extract.

ACKNOWLEDGEMENTS: Author wants to thank Mr. Bhosle, Retired Forest officer, who guided for collecting the plant which is very rare in India and also like to thank the Dr. K. G. Bhat, Head of Department, Department of Microbiology, Maratha Mandal's NGH Institute of Dental Sciences and Research Centre, Belgaum, Karnataka, India, for his valuable guidance and support to carry the Anti-cancer studies.

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    How to cite this article:

    WaghNS and Gaikwad NJ:Evaluation of anti - Cancer Activity of Bark of Crataeva Nurvala Buch. Ham against three Cell Lines. Int J Pharm Sci Res2014; 5(11): 4851-57.doi: 10.13040/IJPSR.0975-8232.5 (11).4851-57.

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