1
SUPPLEMENTARY MATERIALS
Bioactive phenylpropanoid analogues from Piper betle L. var. haldia leaves.
Akhtar Atiya1*, Barij Nayan Sinha2 & Uma Ranjan Lal1*
1Division of Pharmacognosy and Phytochemistry, Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra-835215, Ranchi, Jharkhand, India
2Division of Medicinal Chemistry, Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra-835215, Ranchi, Jharkhand, India
Correspondence authors e-mail & contact number
[email protected]; [email protected]. Phone: +91-8986815195
[email protected]; [email protected]. Phone: +91-9006110464
2 Abstract
Phytochemical analyses of the chloroform extract of Piper betle L. var. birkoli, Piperaceae, leaves led to the isolation of two new phenylpropanoid analogues: bis-chavicol dodecanoyl ester (2) and bis-hydroxychavicol dodecanoyl ester (3), along with one known compound:
allyl-3-methoxy-4-hydroxybenzene (1) on the basis of spectroscopic data 1D (1H and 13C) and 2D (1H-1H COSY and HMBC) NMR, as well as ESI-MS, FT-IR, HR-ESI-MS and LC- ESI-MS. Compound 2 and 3 exhibited excellent antioxidant DPPH radical scavenging activity with IC50 values of 12.67 µg/mLand 1.08 µg/mL compared to ascorbic acid as a standard antioxidant drug with IC50 value of 6.60 µg/mL. Evaluation of cytotoxic activity against two human oral cancer cell lines (AW13516 and AW8507) showed significant effect with GI50 values of 19.61 and 23.01 µg/mL for compound 2 and 10.25 and 13.12 µg/mL for compound 3, compared to Doxorubicin® as a standard cytotoxic drug with GI50
value of <10 µg/mL.
Keywords: Piper betle; phenylpropanoid analogues; antioxidant; cytotoxic; SAR.
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Figure No Contents Page No
S1 400 MHz-1H-NMR Spectrum of Compound 1 in CDCl3. 10 S2 100 MHz-13C-NMR Spectrum of Compound 1 in CDCl3. 11
S3 LC-ESI-MS Spectrum of Compound 1. 12
S4 FT-IR Spectrum of Compound 1. 13
S5 400 MHz-1H-NMR Spectrum of Compound 2 in CDCl3. 14 S6 100 MHz-13C-NMR Spectrum of Compound 2 in CDCl3. 15
S7 ESI-MS Spectrum of Compound 2. 16
S8 LC-ESI-MS Spectrum of Compound 2. 17
S9 HR-ESI-MS Spectrum of Compound 2 (Positive mode). 18
S10 1H-1H COSY Spectrum of compound 2. 21
S11 HMBC Spectrum of compound 2. 22
S12 FT-IR Spectrum of Compound 2. 23
S13 400 MHz-1H-NMR Spectrum of Compound 3 in CDCl3. 24 S14 100 MHz-13C-NMR Spectrum of Compound 3 in CDCl3. 25
S15 ESI-MS Spectrum of Compound 3. 26
S16 LC-ESI-MS Spectrum of Compound 3. 27
S17 HR-ESI-MS Spectrum of Compound 3 (Positive mode). 28
S18 1H-1H COSY Spectrum of compound 3. 29
S19 HMBC Spectrum of compound 3. 30
S20 FT-IR Spectrum of Compound 3. 31
S21 Flow chart for the isolation of bioactive compounds 1-3. 32 S22 Gallic acid standard curve and absorbance (abs.) value of chloroform
extract of P. betle var. birkoli at 200 µg/mL for the determination of total phenolic content.
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S23 The basic proton peaks which were noticed in 1H NMR spectrum of compounds 2 and 3.
34 S24 Key 1H-1H COSY and HMBC correlation of isolated compounds 2 and
3.
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Table No Contents Page No
S1 Evaluation of antioxidant DPPH radical scavenging activity (IC50) of chloroform extract of P. betle var. birkoli leaf and isolated compounds (n=3; ± SD).
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S2 Evaluation of cytotoxic activity of isolated compounds against two human oral cancer cell lines (AW13516 and AW8507; n=3; ± SD).
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1. Experimental 1.1 Instruments
Melting points were determined using an OPTIMELT (Automated melting point system, Stanford Research Systems, USA). The mass spectra were recorded on WATERS-Q-T of Premier HAB213 using the (ESI-MS) electro spray Ionization technique. LC-ESI-MS spectra were achieved by using Agilent 6400 Series Triple Quadrupole. 1D NMR experiments were acquired in CDCl3 as solvent at VNMRS-400 spectrometer, observing 1H and 13C at 400 MHz and 100 MHz, respectively. In the proton NMR spectra the coupling constants (J) are expressed in hertz (Hz). Chemical shifts (denoted as δ) of NMR are reported in parts per million (ppm) units relative to tetra methyl silane (TMS). 2D NMR spectra were acquired in CDCl3, at VNMRS-400 MHz spectrophotometer, observing 1H-1H COSY and HMBC spectrum, respectively. HR-ESI-MS experiments were recorded on WATERS-SYNAPT G2 HDMS spectrometer and FT-IR spectra were acquired on FT-IR-8400S-Shimadzu spectrophotometer with KBr pellets.
1.2 Chemicals
All chemicals and solvents were of analytical grade (AR grade) and were purchased from S.D.
fine chemicals Ltd., and Sigma Aldrich. Isolated compounds were monitored by Thin-layer chromatography (TLC) was performed on TLC plates (Merck, Mumbai, India) precoated with silica gel 60 GF254 thickness 0.2 mm and activate at 110 °C for 1 hour. The spots were detected in either Iodine/UV light (254 and 365 nm). For column chromatography, silica gel 60 (# 230- 240 mesh, Merck, Mumbai, India) was used.
5 1.3 Plant material
The leaves of P. betle var. birkoli were collected from certified organic growers in the state of Odisha district Nayagarh, during the month of October-November 2011. The age of P. betle plant was 2-3 years old. Plant leaves were collected from upper part of the plant specially branches. The voucher specimens were identified by a taxonomist, Dr. K. Karthigeyan (Scientist C) at the Central National Herbarium, Botanic Garden Howrah Kolkata, India. The voucher specimen (CNH/10B/2011/Tech.II/502) was deposited in the Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra-835215, Ranchi, Jharkhand, India for future reference.
1.4 Extraction procedure
The air-dried plant leaves of P. betle var. birkoli (800 g) were powdered coarsely and were extracted with petroleum ether and chloroform in soxhlet extractor (not more than ten siphoning (Harborne 1993). The obtained chloroform extract was filtered and the filtrate was evaporated to dryness under reduced pressure at 40 °C to obtain a dark brown green viscous mass with yield 32.71 g. The obtained extract keeps it in dried, tightly caped amber coloured bottle for further analysis.
1.5 Isolation of bioactive compounds
The chloroform extract (10 g) was dissolved in chloroform (150 mL) and adsorbed on silica gel (approximately 30 g, # 60-120 mesh) for column chromatography. The slurry was dried and chromatographed over column (diameter 43 cm length x 3.5 cm width) packed with silica gel.
The column was eluted successively with petroleum ether, mixtures of petroleum ether and chloroform (9:1, 3:1, 1:1, and 1:3 v/v), pure chloroform and finally mixture of chloroform and methanol (99:1, 97:3, 95:5, 92:8, 9:1, 3:1, 1:1, and 1:3 v/v) (Figure S21). Various fractions were collected separately and matched by TLC to check homogeneity. The same fractions having same retention factor (Rf) values were incorporate together.
1-allyl-3-methoxy-4-hydroxybenzene or eugenol (1)
Pale yellowish semi solid mass, soluble in petroleum ether, n-hexane; MF: C10H12O2; MW:
164.08; Yield 21.0 mg; yield (%) 0.21. 1H NMR (400 MHz, CDCl3, δ, ppm): 6.78 (1H, d, J = 2.1 Hz, Ar H-2), 6.80 (1H, dd, J = 2.1, 7.9 Hz, Ar H-6), 6.64 (1H, d = 7.9 Hz, Ar H-5), 5.91 (2H, m,
=CH2-γ), 5.12 (1H, d, J = 10.1 Hz, -CH-β), 3.82 (3H, s, OCH3-3), 3.36 (2H, d, J = 7.8 Hz, -CH2-
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α); 13C NMR (100 MHz, CDCl3, δ, ppm): 133.40 (C-1), 145.50 (C-3), 144.93 (C-4), 119.80 (C- 6), 137.62 (C-β), 115.48 (C-5), 114.86 (C-2), 110.67 (C-γ), 55.98 (C-3), 39.56 (C-α); LC-ESI- MS (m/z): 163 [M-H] −, 149 [M-Me]–.
2-(γ′-dodecanoyl chavicol)-hydroxy chavicol or bis-chavicol dodecanoyl ester (2)
Brownish solid mass, soluble in chloroform and methanol; MF: C30H40O4; MW: 464.29; Yield 21.44 mg; yield (%) 0.27; mp (°C) 62-68; 1H NMR (400 MHz, CDCl3, δ, ppm): 7.36 (1H, s, Ar H-3), 7.26 (1H, d, J = 3.1 Hz, Ar H-4ʹ), 7.12 (1H, d, J = 7.2 Hz, Ar H-6ʹ), 6.86 (1H, dd, J = 3.1, 7.2 Hz, Ar H-5ʹ), 6.80 (1H, m, Ar H-6), 6.73 (1H, d, J = 3.2, Hz, Ar H-2ʹ), 5.86 (1H, m, -CH-βʹ), 5.78 (1H, m, -CH-β), 5.18 (2H, m, =CH2-γ), 5.10 (1H, m, -CH-γʹ), 3.26 (2H, dd, J = 2.0, 6.1 Hz, -CH2- α), 3.24 (2H, dd, J = 2.1, 6.2 Hz, -CH2-αʹ), 2.04 (2H, m,-CH2-2ʹʹ), 1.62-1.20 (2H, m, 8 ×- CH2, H-3ʹʹ-10ʹʹ), 1.16 (2H, m, -CH2-11ʹʹ), 0.88 (3H, t, J = 6.2 Hz,-CH3-12ʹʹ); 13 C NMR (100 MHz, CDCl3, δ, ppm): 169.38 (C-1ʹʹ), 147.05 (C-3ʹ), 146.66 (C-5), 145.07 (C-4), 139.24 (C-β), 136.82 (C-βʹ), 127.01 (C-1), 124.42 (C-2), 124.42 (C-γʹ), 123.94 (C-2ʹ), 122.30 (C-1ʹ), 122.12 (C-4ʹ), 121.02 (C-5ʹ), 117.76 (C-6), 117.71 (C-6ʹ), 116.11 (C-3), 115.59 (C-γ), 39.14 (C-α), 34.82 (C-αʹ), 33.63 (C-2ʹʹ), 31.87 (C-3ʹʹ), 31.38 (C-4ʹʹ), 29.54 (C-5ʹʹ), 29.38 (C-6ʹʹ), 29.31 (C-7ʹʹ), 29.19 (C-8ʹʹ), 29.02 (C-9ʹʹ), 28.87 (C-10ʹʹ), 24.66 (C-11ʹʹ), 14.06 (C-12ʹʹ); HR-ESI-MS and ESI- MS (m/z): 465.2608 and 465.0 [M+H]+ .
2-[γ′-(α′, β′-dihydro-α′, γ′-3′-trihydroxy-4′-dodecanoyl) chavicol]-hydroxy chavicol or bis- hydroxychavicol dodecanoyl ester (3)
Brownish solid mass, soluble in chloroform and methanol; MF: C30H42O7; MW: 514.29; Yield 92.04 mg; yield (%) 0.92; mp (°C) 75-78; 1H NMR (400 MHz, CDCl3, δ, ppm): 7.56 (1H, d, J = 8.1 Hz, Ar H-5ʹ), 7.38 (1H, s, Ar H-3), 7.12 (1H, dd, J = 2.1, 8.1 Hz, Ar H-6ʹ), 6.80 (1H, d, J = 2.8, Hz, Ar H-2ʹ), 6.71 (1H, s, Ar H-6), 5.96 (1H, m, -CH-β), 5.04 (2H, m, -CH2-γ), 4.32 (1H, t, d, J = 6.1 Hz, -OH-αʹ), 4.21 (1H, t, d = 6.1 Hz, -OH-γʹ), 3.24 (2H, dd, J = 1.2, 6.4 Hz, -CH2-α), 2.38 (2H, m, -CH2-βʹ), 2.02 (2H, m, -CH2-2ʹʹ), 1.52-1.20 (2H, m, 8 × -CH2, H-3ʹʹ-10ʹʹ), 1.18 (2H, m, -CH2-11ʹʹ), 0.86 (3H, t, J = 6.1 Hz, -CH3-12ʹʹ); 13 C NMR (100 MHz, CDCl3, δ, ppm): 167.81 (C-1ʹʹ), 147.49 (C-3ʹ), 147.15 (C-4ʹ), 143.56 (C-4), 141.78 (C-5), 138.52 (C-β), 132.98 (C-1), 132.37 (C-2), 130.89 (C-1ʹ), 130.86 (C-6), 119.01 (C-6ʹ), 118.99 (C-3), 115.59 (C-γ), 115.45 (C- 5ʹ), 115.23 (C-2ʹ), 68.17 (C-γʹ), 65.58 (C-αʹ), 37.05 (C-α), 34.82 (C-βʹ), 34.48 (C-2ʹʹ), 31.87 (C- 3ʹʹ), 30.51 (C-4ʹʹ), 30.30 (C-5ʹʹ), 30.14 (C-6ʹʹ), 29.98 (C-7ʹʹ), 29.64 (C-8ʹʹ), 29.19 (C-9ʹʹ), 29.02
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(C-10ʹʹ), 22.64 (C-11ʹʹ), 14.06 (C-12ʹʹ). HR-ESI-MS and ESI-MS (m/z): 515.2506 and 515.1 [M+H]+.
1.6 Total phenolic content
Total amount of phenolic contents were measured with Folin–Ciocalteu reagent, slightly modified using the method of (Baba & Malik 2015). Gallic acid was used as standard (in a range of 25 to 200 µg/mL) and the absorbance of samples were read in triplicate at 765 nm in a spectrophotometer. Briefly, 200 μL of each test sample (1 mg/mL) were made up to 3 mL with distilled water, add 1 mL 1/10 dilution of Folin-Ciocalteau’s reagent and after 3 min, 2 mL of sodium carbonate (Na2CO3) 20%, w/v was added. The contents were mixed thoroughly and incubated at 45 °C for 15 minutes. Total phenolic content was expressed in micrograms equivalents of gallic acid (GAE) per gram of dry weight of plant material (µg GAE/g dry weight). The equation obtained for the calibration curve of gallic acid was Y = 0.007x + 0.027 (R2 = 0.9999). The standard curve for gallic acid is shown in figure S22.
1.7 Evaluation of antioxidant activity by DPPH radical scavenging method
Free radical scavenging activity was measured by 1, 1- diphenyl-2-picryl hydrazyl (DPPH) (Shekhar & Anju 2014). In brief, 1 mL of 0.1 mM solution of DPPH was added to 1 mL of different extracts in ethanol at different concentration (10, 20, 30, 40, 50 µg/mL). The mixture was shaken vigorously and allowed to stand in a dark at room temperature for 30 minutes. The absorbance was measured at 517 nm by using spectrophotometer. Reference standard compound being used was ascorbic acid and experiment was done in triplicate. The IC50 value of the sample (the concentration of sample required to inhibit 50% of the DPPH free radical), was calculated using Log dose inhibition curve. Lower absorbance of the reaction mixture indicated higher free radical activity. Calibration curve for the DPPH method as generated the absorbance of the reaction medium at 517 nm. The percentage (%) DPPH scavenging effect was calculated by using following equation:
Percentage inhibition or DPPH scavenging effect (%) = Whereas
A0 = absorbance of the control reaction;
A1 = absorbance in the presence of test and standard sample.
8 1.8 In vitro cytotoxic activity
As reported previously (Houghton et al. 2007). Cell culture for the anticancer activities of each test samples were conducted at Advanced Center for Treatment, Research and Education in Cancer (ACTREC), Mumbai where the two human cell lines (AW13516 and AW8507, respectively) were maintained in ideal laboratory conditions. Cells lines were harvested in suitable media supplemented with 10% fetal bovine serum, 1 mM L-glutamine, and 1%
Penicillin-Streptomycin in an adherent tissue culture microtiter plate at 37 °C with 5.0% CO2. 96-wells microtiter plate were seeded with 5 × 103 cells per well and incubated again at 37 °C with 5% CO2, 95% air and 100 % relative humidity for 24 hours prior to addition of experimental test samples. When the seeded plates achieved confluency, the cells were treated with different percentage of test samples in different concentrations.
Evaluation of anti-cancer activity by sulforhodamine-B assay (SRB), the test samples were solubilized in appropriate solvent at 400-fold and stored frozen prior to use. At the time of drug addition, an aliquot of frozen test concentrate was defrost and diluted to 10 times the desired final maximum test concentration with complete medium containing test article at a concentration of 100, 200, 400 and 800 µg/mL. Aliquots of 10 µL of these different dilutions were added to the appropriate microtiter wells already containing 90 µL of cell suspension, resulting in the required final test sample concentrations of 10-80 µg/mL. For each of the experiments a known anticancer drug was used as a positive control.
For the endpoint measurement, after samples addition, plates were incubated at standard conditions for 48 hours. Thereafter, 100 µL of 10% cold trichloro-acetic acid (TCA) was gently added to each well of the plates and incubated for 50 minutes at 4 °C to fix the cells attached to the bottom of the wells. After fixation, the supernatant was discarded; the plates were washed five times with distilled water in order to remove excessive fixative and dead cells and kept overnight at 4 °C. Once the plate was dried, 100 µL of SRB dye (0.4%, w/v in 1% acetic acid) was added to each of the wells, and plates were incubated for 25 minutes at room temperature.
After staining, unbound dye was recovered and the residual dye was removed by washing four times with 1% acetic acid. Viable cells take up SRB dye and stain pink. The plates were allowed to air dried. Bound stain was subsequently eluted with 100 µL of 10 mM Trizma base [tris (hydroxymethyl) amino methane (pH 10.5)]. Plates were kept over mechanical shaker for 5-10 minutes for complete mixing of bound dye with tris base. Blank wells contained medium but no
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cells and the control wells contained cells but no test samples. The absorbance was read on an Elisa Plate Reader at a wavelength of 540 nm with 690 nm reference wavelength. Percent growth was calculated on a plate-by-plate basis for test wells relative to control wells.
Percent Growth was expressed as the ratio of average absorbance of the test wells to the average absorbance of the control walls × 100. Using the six absorbance measurements [time zero (Tz), control growth (C), and test growth in the presence of test sample at the four concentration levels (Ti)]; the percentage growth was calculated at each of the sample concentration levels.
Percentage growth inhibition (GI50) was calculated as: [(Ti-Tz)/(C-Tz)] × 100. TGI = Drug concentration resulting in total growth inhibition (TGI) will calculated from Ti=Tz and LC50 = Concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning indicating a net loss of 50% cells following treatment is calculated from [(Ti-Tz)/Tz × 100 = -50.
Statistical analysis
All the experiments were performed in triplicates and the data were reported as the mean ± SD.
The results obtained were analyzed using one-way ANOVA method using Graph Pad Prism version 5.0. A difference was considered statistically significant if p<0.005.
References
Baba SA, Malik SA. 2015. Determination of total phenolic and flavonoid content, antimicrobial and antioxidant activity of a root extract of Arisaema jacquemontii Blume, J Taibah Univ Sci. 9: 449-454.
Harborne AJ. 1993. Phytochemical methods: a complete guide to modern techniques of plant, 2nd edn. Chapman and Hall, London; pp 288.
Houghton P, Fang R, Techatanawat I, Steventon G, Hylands PJ, Lee CC. 2007. The sulphorhodamine (SRB) assay and other approaches to testing plant extracts and derived compounds for activities related to reputed anticancer activity, Methods. 42: 377-387.
Shekhar TC, Anju G. 2014. Antioxidant activity by DPPH radical scavenging method of Ageratum conyzoides Linn. Leaves, Am J Ethnomed. 1: 244-249.
10 Figures
Figure S1. 400 MHz-1H-NMR Spectrum of Compound 1 in CDCl3.
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Figure S2. 100 MHz-13C-NMR Spectrum of Compound 1 in CDCl3.
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Figure S3. LC-ESI-MS Spectrum of Compound 1.
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Figure S4. FT-IR Spectrum of Compound 1.
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Figure S5. 400 MHz-1H-NMR Spectrum of Compound 2 in CDCl3.
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Figure S6. 100 MHz-13C-NMR Spectrum of Compound 2 in CDCl3.
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Figure S7. ESI-MS Spectrum of Compound 2.
OH OH
O O
Molecular weight: 464.2926
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Figure S8. LC-ESI-MS Spectrum of Compound 2.
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Figure S9. HR-ESI-MS Spectrum of Compound 2 (Positive mode).
OH OH
O O
Molecular weight: 464.2926
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Figure S9 (a). HR-ESI-MS Spectrum of Compound 2 (Positive mode; zoom from m/z 100 to m/z 470).
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Figure S9 (b). HR-ESI-MS Spectrum of Compound 2 (zoom from m/z 297 to 318 m/z).
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Figure S10. 1H-1H COSY Spectrum of compound 2.
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Figure S11. HMBC Spectrum of compound 2.
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Figure S12. FT-IR Spectrum of Compound 2.
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Figure S13. 400 MHz-1H-NMR Spectrum of Compound 3 in CDCl3.
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Figure S14. 100 MHz-13C-NMR Spectrum of Compound 3 in CDCl3, δ, ppm.
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Figure S15. ESI-MS Spectrum of Compound 3.
Molecular weight: 514.29
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Figure S16. LC-ESI-MS Spectrum of Compound 3.
Molecular weight: 514.29
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Figure S17. HR-ESI-MS Spectrum of Compound 3 (Positive mode).
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Figure S18. 1H-1H COSY Spectrum of compound 3.
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Figure S19. HMBC Spectrum of compound 3.
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Figure S20. FT-IR Spectrum of Compound 3.
500 750
1000 1250
1500 1750
2000 2500
3000 3500
4000
1/cm 20
30 40 50 60 70 80 90
%T
2924.18 2853.78 1733.10 1493.92 1458.23 1363.72 1277.88 1187.23 1122.61 1081.14 969.26 894.04 818.81 721.40
b3
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Figure S21. Flow chart for the isolation of bioactive compounds 1-3.
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20 40 60 80 100 120 140 160 180 200 220
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
µg/mL B
(1.011 abs. = 140.57 µg)
Absorbane (765 nm)
Concentration (µg/mL)
20 40 60 80 100 120 140 160 180 200 220
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 A
Y=0.007x+0.027 R2= 0.999
Absorbance (765 nm)
Concentration (µg/mL)
Figure S22. A, Gallic acid standard curve and B, absorbance (abs.) value of chloroform extract of P. betle var. birkoli at 200 µg/mL for the determination of total phenolic content.
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Figure S23. The basic proton peaks which were noticed in 1H NMR spectrum of compounds 2 and 3.
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Figure S24. Key 1H-1H COSY and HMBC correlation of isolated compounds 2 and 3.
36 Tables
Table S1. Evaluation of antioxidant DPPH radical scavenging activity (IC50) of chloroform extract of P. betle var. birkoli leaf and isolated compounds (n=3; ± SD).
Samples DPPH free radical scavenging (µg/mL)
Compound 1 14.09 ± 0.12
Compound 2 12.67 ± 0.11
Compound 3 1.08 ± 0.03
Extract 17.58 ± 0.14
Standard (Ascorbic acid) 6.60 ± 0.02
IC50 = concentration in µg/mL capable of reducing DPPH by 50%. Results were presented as the mean ± S.D, compared with Ascorbic acid, positive control.
Table S2. Evaluation of cytotoxic activity of isolated compounds against two human oral cancer cell lines (AW13516 and AW8507; n=3; ± SD).
Samples AW13516 AW8507
Drug concentrations (µg/mL)
LC50 TGI GI50 LC50 TGI GI50
Comp 1 67.41 ± 4.04 38.42 ± 3.20 29.01 ± 1.10 74.11 ± 2.04 46.63 ± 3.12 28.31 ± 1.21 Comp 2 39.22 ± 2.10 27.31 ± 1.10 19.61 ± 2.20 64.03 ± 4.04 45.04 ± 2.02 23.01 ± 2.01 Comp 3 41.60 ± 3.74 22.12 ± 3.12 10.25 ± 1.12 60.05 ± 5.02 31.21 ± 1.13 13.12 ± 1.10
ADR 36.70 ± 1.20 ˂10 ˂10 36.60 ± 1.23 ˂10 ˂10
LC50 is defined as concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning indicating a net loss of 50%
cells; TGI = Drug concentration resulting in total growth inhibition and GI50=Drug concentration resulting in a 50% reduction in the net protein increase. Results were presented as the mean ± S.D, compared with ADR=Adriamycin (Doxorubicin®, positive control drug). Values with different superscript letters within the same column are significantly different (p<0.05).
