The
Traditional medicines for human diseases have been widely used in many parts of the world. Herbal plants are usually the primary source of medicine in many developing countries. Natural product compounds from plants provide biologically active compounds, many of which have been developed as new lead chemicals for pharmaceuticals [
The stem bark of
All the apparatus used for cell culture were sterilized and decontaminated using Hirayama HICLAVE HVE-50. Cell culture handling was carried out in an ESCO Class II BSC Biosafety Cabinet. The healthy cells were spun down, adhered together, and separated from unhealthy and dead cells by using Thermo Scientific Sorvall ST 16R centrifuge machine. All cultures were incubated in 5% CO2 humidified incubator at 37°C (ESCO Celculture CO2 Incubator with model number CCL-170B-8). Cell stocks were placed in a −86°C ultralow temperature freezer (Scancool SCL 50 P) and preserved in a liquid nitrogen tank (Taylor-Wharton LS300).
The aerial parts of the plant samples were extracted using the conventional extraction method in which solvent was added and removed in batches. The dried and milled samples were soaked in nonpolar (hexane), medium polar (dichloromethane, or ethyl acetate) and polar (methanol) solvents in a polarity increasing order. The solvent was decanted and replaced with new solvent after 48 hours.
The air-dried and powdered
Preliminary screening tests for cytotoxic activity against a panel of human cancer cell lines on the crude extracts and quercetin (standard) were undertaken by using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra zolium bromide) assay. The nine tested cancer cell lines were Raji (human B lymphocyte), SNU-1 (human gastric carcinoma), K562 (human erythroleukemia cells), LS-174T (human colorectal adenocarcinoma), HeLa (human cervical cells), SK-MEL-28 (human malignant melanoma cells), NCI-H23 (human lung adenocarcinoma), IMR-32 (human neuroblastoma), and Hep-G2 (human hepatocellular liver carcinoma). The Raji, SNU-1, K562, HeLa, Hep-G2, and NCI-H23 cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), while the LS-174T, SK-MEL-28, and IMR-32 were maintained in MEM supplemented with 10% FBS. All the cell lines were cultured in 75 cm2 T-flask and maintained at 37°C in 5% CO2 humidified incubator. The MTT assay is based on the protocol illustrated by Mosmann [
The plate was then incubated for 72 hours at 37°C in 5% CO2 humidifier incubator. After 72 hours, 20
The absorbance of each well was determined using a microplate reader at 550 nm. The average absorbance of each crude extract was calculated, and the average value was used to determine the percentage of cell viability by using the following formula:
A graph of percentage of cell viability versus concentration was plotted for each extract. The half maximal inhibitory concentration (IC50) values were obtained from the plotted graph. Further dilutions will only be performed on the extracts with IC50 values less than 3.13
The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay was used to evaluate the antioxidant property of the sample. The assay was performed in 96-well flat bottom plates. The DPPH solution was prepared in a stock solution of 5 mg/2 mL in ethanol (EtOH) and wrapped with aluminium foil. The varying concentrations of 3.13, 6.25, 12.50, 25.00, 50.00, and 100.00
A graph of percentage of total radical scavenging activity versus concentration was plotted for each extracts. The half maximal effective concentration (EC50) values were obtained from the plotted graphs. Three independent experiments were conducted to ensure the precision of the results. Ascorbic acid (vitamin C) was used as the standard drug.
The TPC analysis is to analyze the total phenolic content present in the extract. The TPC analyses were carried out in 24-well flat bottom plates. The extract was initially oxidized by Folic-Ciocalteu reagent (FC) and subsequently neutralized by sodium bicarbonate, NaHCO3. Gallic acid was used as the standard in the experiment.
A standard curve was constructed using the equation
An aliquot of 100
Nutrient agar (NA) and Mueller Hinton agar (MHA) were prepared according to the manufacturer’s instructions. The medium was cooled to 50°C after autoclaving, before approximately 25.0 to 30.0 mL of the medium was poured into 15 × 100 mm plastic petri dishes at a uniform depth of 4 mm. Each type of microorganism (from the stock culture) was streaked onto a noninhibitory NA plate to obtain isolated colonies. After an overnight incubation (16–20 hours) at 37°C, one single colony was selected and inoculated with a loop, transferred into 10.0 mL of sterile nutrient broth (NB), and incubated in a shaking incubator at 37°C for 16–20 hours. The density of bacteria was standardized using McFarland 0.5 turbidity standard to 1 × 108 coliform units (cfu)/mL by diluting each suspension in 10 mL of sterile distilled water to the appropriate density.
The antibacterial assay was performed using disc diffusion (Kirby-Bauer) method as described by Aksoy et al. The standardized bacterial suspensions were swabbed on MHA surface using sterile cotton wool. For initial screening, 1 mg of DCM : MeOH extract was loaded onto each Whatman No. 1 filter paper discs (ø, 6 mm) and were impregnated. The positive (streptomycin 10
According to the National Cancer Institute (NCI), a crude extract can be considered active if it possesses an IC50 value of less than 20
On the other hand, the hexane and dichloromethane extracts of
Additionally, the hexane extract of
In conclusion, only nonpolar to semipolar extracts contribute to the cytotoxic effects on the panel of human cancer cell lines except for the polar extract of
IC50 values of a panel of human cancer cell lines treated with extracts of the three
Crude Extracts | Cell lines with IC50 values ( | ||||||||
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Raji | SNU-1 | K562 | LS-174T | SK-MEL-28 | IMR-32 | HeLa | Hep-G2 | NCI-H23 | |
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Hexane | — |
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— |
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— |
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DCM | — |
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— | — |
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— | — |
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EA | — | — | — | — | — | — | — | — |
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MeOH |
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— | — | — | — | — | — | — | — |
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Hexane | — |
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DCM | — |
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— |
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EA | — | — | — | — | — | — | — | — | — |
MeOH | — | — | — | — | — | — | — | — | — |
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Hexane | — |
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— | — | — | — | — |
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EA | — | — | — | — | — | — | — | — | — |
MeOH | — | — | — | — | — | — | — | — | — |
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Quercetin |
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— |
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**Each data represents the mean of three independent experiments.
IC50 values of the extracts of (a)
All the extracts of
Several semipolar to polar extracts possess medium to strong scavenging activity against DPPH radical where the methanol (MeOH) extracts of
The EC50 values for the extracts of three
EC50 values of crude extracts towards DPPH free radicals.
Compounds | EC50 values ( | |
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Crude extracts |
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Hexane | — | |
Dichloromethane |
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Ethyl acetate |
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Methanol |
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Hexane | — | |
Dichloromethane |
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Ethyl acetate | — | |
Methanol |
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Hexane | — | |
Ethyl acetate |
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Methanol | — | |
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Standard drug | Ascorbic acid |
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EC50 values of crude extracts of the three
The total phenolic content (TPC) analysis was carried out on all the crude extracts by using Folin-Ciocalteu method. The TPC values give information on the phenolic contents of the crude extracts and their antioxidant activities. The methanolic extracts of
Total phenolic contents of crude extracts in GAE.
Plant species | Crude extracts | Total phenolic content ( |
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Hexane |
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Dichloromethane |
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Ethyl acetate |
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Methanol |
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Hexane |
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Dichloromethane |
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Ethyl acetate |
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Methanol |
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Hexane |
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Ethyl acetate |
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Methanol |
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Total phenolic content of crude extracts of the three
The extracts of
Antibacterial activity of crude extracts from
Crude extract (1 mg) | 1 | 2 | 3 | 4 |
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Hex | 8.33 | — | — | — |
DCM | — | — | — | — |
EA | — | — | — | — |
MeOH | 7.00 | — | 8.00 | 8.67 |
Streptomycin (10 |
18.70 | 23.00 | 17.0 | not tested |
Vancomycin (30 |
not tested | not tested | not tested | 21.00 |
**Each value represents the inhibition zone (mm).
***1-
Weights of the extracts obtained from the three
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Hex (g) | 15.6 | 49.6 | 5.5 |
DCM (g) | 21.2 | 19.5 | — |
EA (g) | 15.8 | 16.7 | 61.0 |
MeOH (g) | 80.5 | 62.2 | 120.5 |
Both DPPH radical assay and total phenolic content indicate that semipolar to polar extracts had high antioxidant properties, while non-polar extracts exhibited good cytotoxic activity. The pharmacognosy investigation showed adverse effects of crude extracts suggesting that the three
The authors wish to thank Associate Professor Dr. Rusea Go for the identification of plants. Financial support from UPM under the RUGS research fund is gratefully acknowledged.