In vitro Cytotoxicity Study on U87 Cells using Root Extracts of Plumbago Species and GC-MS Study

Panicker and Haridasan: In vitro Cytotoxicity Study on U87 Cells using Root Extracts of Plumbago Species and GC-MS Study

Authors

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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 leukemia1 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.3 Plumbagin induce apoptosis in human pancreatic cancer cells primarily through the mitochondria-related pathway which indicates that plumbagin can be potentially developed as a novel therapeutic agent against pancreatic cancer also.4

Gas 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.5

In vitro measurement of toxicity is purely a cellular pathway which can be measured by changes in cell survival or its metabolism.6 To test cell proliferation and cell survival; a simple calorimetric assay was developed by Mosmann7 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 bromide) (MTT) into an insoluble formazan precipitate by the mitochondrial succinate dehydrogenase enzyme which are active in living cells.8

MATERIALS AND METHODS

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- 65.2 kPa, 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% CO2 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 cm2 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 37oC for 72 h in 5% CO2 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 37oC in 5% CO2 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% (CTC50) values was generated from the dose-response curves (Figure 2) for each cell line.10

% Growth Inhibition=(Mean OD of individual test sampleMean OD of control)×100https://s3-us-west-2.amazonaws.com/jourdata/pj/pharmacognj-10-6s-71_g000.jpg
Figure 2

GC-MS Chromatogram of ethanolic extracts of P. zeylanica roots.

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Figure 2

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

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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).11 Lupeol isolated from Elaeodendron buchananii exhibited potent anticancer activity against LEUK-L1210 cells.12

Table 1

GC-MS study on the ethanolic extract of P. auriculata roots indicating 16 phytochemical constituents.

PeakR.TimeAreaArea%HeightHeight%NameBase m/z
15.543533040.15334020.59propane, 1,1-diethoxy-0.15
211.7812161400.601472372.58phenol, 2,4-bis(1,1-dimethylethyl)-0.60
313.151396113410.91194624934.121,4-naphthalenedione, 5-hydroxy-2-methyl-10.91
414.1371621170.45710091.25benzaldehyde, 4-ethoxy-0.45
516.7963770571.041926873.38hexadecanoic acid1.04
618.3259168602.53884761.55methyl commate c2.53
718.4515867691.621915873.361,3,12-nonadecatriene1.62
818.5007638452.101815633.187-tetradecenal, (z)-2.10
918.712349250.10199600.359,12-octadecadienoic acid (z,z)-0.10
1019.8677192671.981918233.36longifolenaldehyde1.98
1120.0821232674433.96116421720.41lupeol33.96
1221.7258731342.411038541.82methyl commate b2.41
1322.836872226024.0380361514.09nerolidyl acetate24.03
1425.32218075634.981277142.24stigmast-4-en-3-one4.98
1536.05833792069.313241735.68hexadecanoic acid, 9-octadecenyl ester, (z)-9.31
1636.29714009113.861159652.032,6,10-trimethyl,14-ethylene-14-pentadecne3.86
Table 2

GC-MS study on the ethanolic extract of P. zeylanica roots indicating 27 phytochemical constituents.

PeakR.TimeAreaArea%HeightHeight% NameBase m/z
15.545548790.19337840.75Propane, 1,1-diethoxy-59.05
211.7811772140.621220032.70Phenol, 2,4-bis(1,1-dimethylethyl)-191.15
313.14822649047.94107311323.751,4-Naphthalenedione, 5-hydroxy-2-methyl-188.05
414.1334065861.431683243.73Ethanone, 2-ethoxy-1-phenyl-121.10
514.780464060.16145860.3214,15,16-Trinorlabd-12-ene, 8,13-Epoxy-95.10
615.0681246570.44771031.71(-)-Drimenol109.10
715.217162970.0699860.223-Dodecanol59.05
815.333520530.18337540.751,3-Cyclopentadiene, 1,2,5,5-tetramethyl-107.15
915.63111374743.9960286713.342,4-Heptadiene, 2,6-dimethyl-109.15
1015.9384415161.552320185.141-Cycloheptene, 1,4-dimethyl-3-(2-methyl-1-propene-1-yl)-4-vinyl-107.10
1116.0921114690.39642661.42Alpha-guaien119.15
1216.478981230.34472891.05(3E)-5-Isopropylidene-2,7-dimethyl-6-oxa-1,3,7,10-UNDECATETRAENE109.15
1316.7963160031.111486613.29Hexadecanoic acid60.05
1416.9801489840.52766141.70Drimenin109.15
1517.1232066400.721360513.01Hexadecanoic acid, ethyl ester88.10
1617.659110150.04111530.256-Oxa-3,3,5-trimethyl-spiro[5,2]octa-5-ene219.10
1718.0121180020.41703321.56Retinol, acetate119.10
1818.076603760.21299330.665,8-Decadien-2-one, 5,9-dimethyl-, (E)-96.10
1918.450672350.24306080.68(5E)-5-Dodecen-1-ol #67.05
2018.5031440720.50539621.199-Octadecenoic acid (Z)-55.05
2118.7111781620.621003602.22ethanol, 2-(9,12-octadecadienyloxy)-, (z,z)-67.10
2218.7681674810.591107502.457-Tetradecenal, (Z)-55.05
2320.855196640.07104580.231-(2-Hydroxyethoxy)tridecane57.10
2434.5652050196471.86103574422.9211-Dodecen-1-ol difluoroacetate55.05
2535.10611012353.861092612.42Z,E-2,13-Octadecadien-1-ol55.05
2635.1673429501.20800141.7717-Oxolinoleic acid, methyl ester91.05
2739.3382139900.75351820.7822,23-Dibromostigmasterol acetate105.10

In 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 CTC50 (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.13 The side effects may be acute or chronic, self-limited, permanent, mild or potentially life threatening.14

Figure 1

GC-MS Chromatogram of ethanolic extracts of P. auriculata roots.

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Figure 1

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

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Table 4

Cytotoxic properties of test drugs against U87 cell line.

Sl. No.Name of Test compoundTest Conc. (µg/ml)% CytotoxicityCTC50 (µg/ml)
1Sample I40070.13±1.188.07±4.4
20058.87±1.6
10051.77±1.1
5044.92±1.3
2533.10±3.0
12.518.83±1.21
6.2515.92±1.12
3.126.15±1.65
2Sample II40074.39±0.623.11±0.9
20071.16±0.9
10061.86±0.8
5058.08±0.9
2552.48±1.6
12.536.80±1.5
6.2532.07±1.2
3.1213.40±1.0

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 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.

Table 3

Some of the bioactive compounds identified using ethanolic extracts of P. auriculata and P. zeylanica roots.

Sl. noCompound nameStructureFunction
1Propane, 1,1-diethoxy-https://s3-us-west-2.amazonaws.com/jourdata/pj/pharmacognj-10-6s-71_g004.jpgFood additives -Flavouring Agents
2Phenol, 2,4-bis(1,1-dimethylethyl)-https://s3-us-west-2.amazonaws.com/jourdata/pj/pharmacognj-10-6s-71_g005.jpgSolvent for cleaning or degreasing
31,4-Naphthalenedione, 5-hydroxy-2-methyl-https://s3-us-west-2.amazonaws.com/jourdata/pj/pharmacognj-10-6s-71_g006.jpgantimicrobial
antimalarial,
anti-inflammatory,
anticarcinogenic,
cardiotonic,
immunosuppressive,
antifertility action,
neuroprotective,
anti-atherosclerosis effects
4Benzaldehyde, 4-ethoxy-https://s3-us-west-2.amazonaws.com/jourdata/pj/pharmacognj-10-6s-71_g007.jpg4-Ethoxybenzaldehyde is present in black tea. 4-Ethoxybenzaldehyde is a flavouring agent.
5Hexadecanoic acidhttps://s3-us-west-2.amazonaws.com/jourdata/pj/pharmacognj-10-6s-71_g008.jpgNegative feedback on acetyl-CoA carboxylase (ACC) which prevents palmitate generation.
6Methyl commate chttps://s3-us-west-2.amazonaws.com/jourdata/pj/pharmacognj-10-6s-71_g009.jpgAntibacterial, anti-inflammatory
77-Tetradecenal, (z)-https://s3-us-west-2.amazonaws.com/jourdata/pj/pharmacognj-10-6s-71_g0010.jpgAgrochemical Category-
Attractant
89,12-Octadecadienoic acid (z,z)-https://s3-us-west-2.amazonaws.com/jourdata/pj/pharmacognj-10-6s-71_g0011.jpgFood additives - Flavoring Agent
9Longifolenaldehydehttps://s3-us-west-2.amazonaws.com/jourdata/pj/pharmacognj-10-6s-71_g0012.jpganticancer and antibacterial activities
10Lupeolhttps://s3-us-west-2.amazonaws.com/jourdata/pj/pharmacognj-10-6s-71_g0013.jpgantiprotozoal, antimicrobial, anti-inflammatory, antitumor and chemopreventive properties
11Methyl commate bhttps://s3-us-west-2.amazonaws.com/jourdata/pj/pharmacognj-10-6s-71_g0014.jpgTriterpenes glycoside
12Nerolidyl acetatehttps://s3-us-west-2.amazonaws.com/jourdata/pj/pharmacognj-10-6s-71_g0015.jpgFlavour and fragrance agent
13Stigmast-4-en-3-onehttps://s3-us-west-2.amazonaws.com/jourdata/pj/pharmacognj-10-6s-71_g0016.jpgHypoglycaemic effect
1414,15,16-trinorlabd-12-ene, 8,13-epoxy-https://s3-us-west-2.amazonaws.com/jourdata/pj/pharmacognj-10-6s-71_g0017.jpgUsed in perfumery
153-Dodecanolhttps://s3-us-west-2.amazonaws.com/jourdata/pj/pharmacognj-10-6s-71_g0018.jpgSurfactants, 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.
16Alpha-guaienhttps://s3-us-west-2.amazonaws.com/jourdata/pj/pharmacognj-10-6s-71_g0019.jpgfragrance and flavouring industries
17Hexadecanoic acidhttps://s3-us-west-2.amazonaws.com/jourdata/pj/pharmacognj-10-6s-71_g0020.jpgsoaps, cosmetics, and industrial mold release agents
18Hexadecanoic acid, ethyl esterhttps://s3-us-west-2.amazonaws.com/jourdata/pj/pharmacognj-10-6s-71_g0021.jpghair- and skin-conditioning agent
19Retinol acetatehttps://s3-us-west-2.amazonaws.com/jourdata/pj/pharmacognj-10-6s-71_g0022.jpgAnti neoplastic and chemo preventive activities
209-octadecenoic acid (z)-https://s3-us-west-2.amazonaws.com/jourdata/pj/pharmacognj-10-6s-71_g0023.jpgInsect pheromone

ACKNOWLEDGEMENT

The authors wish to thank Department of Botany, Post Graduate and Research Centre, St. Joseph’s College -Bangalore for its valuable support in carrying out the studies.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

ABBREVIATIONS

GC-MS

Gas Chromatography Mass Spectroscopy

SCC

Squamous cell carcinomas

U87

Human Glioblastoma cell line

CTC50

Concentration of the sample tolerated by 50% of the cultures exposed

UVR

Ultraviolet radiation.

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