In vitro Cytotoxicity Study on U 87 Cells using Root Extracts of Plumbago Species and GC-MS Study

Plumbago is a genus of medicinal plants that are used in recent years to induce significant levels of apoptosis in various cancer cells. Purpose: The test substances that are obtained from Plumbago species are studied for their cytotoxicity against U87 cell line in a dose dependent manner and were first subjected to GC-MS study to know the bioactive constituents present in them. Methods: GC-MS was done using ethanol extracts of the roots of both the species. In vitro cytotoxicity of the roots of two species of Plumbago –P. zeylanica (sample I) and P. auriculata (sample II) were tested against U87 cell line. Test samples were taken at concentrations ranging from 400μg/ml to 3.12μg/ml to determine the percentage growth inhibition of both the test substances on U87 cell line. Results: GC-MS analysis on root extracts of P. zeylanica showed the presence of 27 phytochemical constituents and P. auriculata 16 in number .The test substances, Sample I and Sample II exhibited a cytotoxic CTC50 value of 88.07±4.4 and 23.11±0.9 respectively. Conclusion: P. zeylanica is more effective than P. auriculata in terms of its cytotoxicity as well in the number of useful bioactive compounds.


INTRODUCTION
Plumbagin, an active constituent found in various parts of the plant Plumbago was studied extensively against various cancer cell lines such as P388 lymphocytic leukemia 1 non-small cell lung cancer cell lines (A549, H262 and H460) 2 and apoptosis pathway in breast cancer and lung cancer as well.It was also found to inactivate the oncogenic transcription factor Forkhead Box M1 (FOXM1) signalling pathway in glioma cell. 3Plumbagin induce apoptosis in human pancreatic cancer cells primarily through the mitochondriarelated pathway which indicates that plumbagin can be potentially developed as a novel therapeutic agent against pancreatic cancer also. 4as Chromatography Mass Spectroscopy is a very compatible technique and most commonly used for the identification and quantification purpose.The unknown organic compounds in a complex mixture can be identified by matching the spectra with reference spectra. 5n vitro measurement of toxicity is purely a cellular pathway which can be measured by changes in cell survival or its metabolism. 6To test cell proliferation and cell survival; a simple calorimetric assay was developed by Mosmann 7 which was further modified for the measurement of chemosensitivity and cytotoxicity on human malignant cell lines.This assay is called MTT assay.The MTT assay involves the ability of viable cells to convert a soluble tetrazolium salt, (3-(4, 5 dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium

GC -MS STUDY
Preparation of the extract: The pulverized root powder was successively extracted with ethanol.The extracts were then concentrated under reduced pressure in a rotary evaporator.Extract: 1µl each of the ethanol extracts of the roots of P.zeylanica and P.auriculata was employed for GC -MS analysis. 5

Instruments and chromatographic conditions:
The analysis was performed on a Shimadzu GC-MS (Model Number: QP2010S) equipped with column consisting of Rxi-5Sil MS (30 meter length, 0.25 mm ID and 0.25 µm thickness).The instrument was operated at 70 eV in electron impact mode.Helium (99.995%) was used as carrier gas at a constant flow of 1ml/min and an injection volume of split ratio 50:0.Column Oven Temperature was at 80.0°C, Injection Temperature-260.00°C,Pressure-65.2kPa, Total Flow-54.0 mL/min, Linear Velocity: 36.8 cm/sec, Purge Flow: 3.0 mL/min.The total GC time was 45 minand the MS time was 40.00 min.The relative amounts of individual components of the total composition were expressed as percentage peak area relative to total peak area.
Identification of the compounds: Interpretation on mass spectrum of GC-MS was done using GCMS Software-GCMS Solutions and the libraries used were NIST 11 and WILEY 8.

In vitro Cytotoxicity Studies
Material used: The roots of two species of Plumbago -P.zeylanica (sample I) and P. auriculata (sample II) were shade dried-pulverized and extracted using ethanol.The extracts were allowed to vacuum evaporate and the residues deposited on the walls of the chamber was collected and used for the study.The in vitro cytotoxicity was performed for "Sample I and Sample II" on Human Glioblastoma cells to find toxic concentration of test substances.Test solution: For cytotoxicity studies, 100μl each of the test drug was separately suspended and volume was made up with Ham's F12 supplemented with 2% inactivated Fetal Bovine Serum to obtain a stock solution of 10% v/v concentration.This was sterilized by 0.22μ syringe filtration.Cytotoxicity studies were carried out using two fold serial dilutions from this.Cell line and Culture medium: U87 (Human Glioblastoma) cell line was procured from National Centre for Cell Sciences (NCCS), Pune, India.Stock cells were cultured in Ham's F12 supplemented with 10% inactivated Fetal Bovine Serum, Penicillin (100 IU/ml), streptomycin (100μg/ml) and Amphotericin B (5μg/ml) in a humidified atmosphere of 5% CO 2 at 37°C until confluent.The cells were dissociated with TPVG solution (0.2% Trypsin, 0.02% EDTA, 0.05% glucose in PBS).The stock cultures were grown in 25 cm 2 culture flasks and all experiments were carried out in 96 well micro titre plates (Tarsons India Pvt. Ltd., Kolkata, India).Cytotoxicity Studies: The monolayer cell culture was trypsinized and the cell count was adjusted to 100,000 cells/ml using Ham's F12 containing 10% FBS.To each well of the 96 well micro titre plates, 0.1 ml of the diluted cell suspension was added.After 24 h, when a partial monolayer was formed, the supernatant was flicked off, the monolayer washed once with medium and 100μl of different test concentrations of test drugs were added on to the partial monolayer in micro titre plates.The plates were then incubated at 37 o C for 72 h in 5% CO 2 atmosphere, and microscopic examination (Figure 2) was carried out and observations were noted every 24 h interval.After 72 h, the drug solutions in the wells were discarded and 50μl of MTT in PBS was added to each well.The plates were gently shaken and incubated for 3 h at 37 o C in 5% CO 2 atmosphere.The supernatant was removed and 100μl of propane was added and the plates were gently shaken to solubilise the formed formazan.The absorbance was measured using a micro plate reader at a wavelength of 540 nm. 9 The percentage growth inhibition was calculated using the standard formula and concentration of test drug needed to inhibit cell growth by 50% (CTC 50 ) values was generated from the dose-response curves (Figure 2) for each cell line. 10

% Growth Inhibition
Mean OD of individual test sample

RESULTS AND DISCUSSION
GC-MS study: P. zeylanica showed the presence of 27 phytochemical (Table 1) constituents where 1, 4-naphthalenedione, 5-hydroxy-2-methyl-(23.75%) was in the highest concentration.In P. auriculata (Table 2) 1, 4-naphthalenedione, 5-hydroxy-2-methy-(34.12%)was found in highest concentration followed by lupeol (20.41%).Plumbagin or 5-hydroxy-2-methyl-1,4-napthoquinone is derived naphthoquinone isolated from the roots of the Plumbago zeylanica L. (common name-Chitrak).It is anti-atherogenic, cardiotonic, hepatoprotective and neuroprotective agent.Topical application of non-toxic doses (100-500 nmol) of PL to skin elicits dose-dependent inhibition of ultraviolet radiation (UVR)induced development of squamous cell carcinomas (SCC). 11Lupeol isolated from Elaeodendron buchananii exhibited potent anticancer activity against LEUK-L1210 cells. 12n vitro Cytotoxicity Studies: Test substances "Sample I (P.zeylanica) and Sample II" (P.auriculata) were tested for in vitro cytotoxicity studies against Human Glioblastoma (U87) using MTT assay exposing the cells to different concentrations of test substances (Figure 1).The test   substances, Sample I and Sample II exhibited a CTC 50 (concentration of the sample tolerated by 50% of the cultures exposed) value of 88.07 ± 4.4 and 23.11 ± 0.9 respectively (Table 4).The antitumorous activity of the sample was recorded in a dose dependent manner.Drugs that target cancerous cells prevent its growth and division, become cytotoxic in nature targeting nucleic acids and their precursors that are rapidly synthesized during cell division. 13The side effects may be acute or chronic, self-limited, permanent, mild or potentially life threatening. 14

CONCLUSION
From the current studies it can be inferred that the ethanolic root extract of P. zeylanica contained a larger number of bioactive compounds (Table Table 3: Some of the bioactive compounds identified using ethanolic extracts of P. auriculata and P. zeylanica roots.3) compared to P. auriculata as revealed through the GC-MS studies.It has also been observed that the cytotoxicity is more in P. zeylanica on U87 (Human Glioblastoma) cell line than P. auriculata using MTT assay.Hence the potential of the roots of P. zeylanica can be further exploited for cancer therapy.

4 -
Ethoxybenzaldehyde is present in black tea.4-Ethoxybenzaldehyde is a flavouring agent.5Hexadecanoicacid Negative feedback on acetyl-CoA carboxylase (ACC) which prevents palmitate generation

Figure 1 :
Figure 1: Graph showing cytotoxic effect of test substances on U87 cell line.

Figure 2 :
Figure 2: Microscopic observation showing cytotoxicity of Sample I and Sample II on U87 cell line.

Table 3 : Cont'd.
Surfactants, lubricating oils, pharmaceuticals, in the formation of monolithic polymers and as a flavour enhancing food additive.In cosmetics, used as an emollient.It is also the precursor to dodecanal, an important fragrance.