INTRODUCTION
Traditionally, medicinal plants have found applications in the formulation and production of modern drugs. They are used in the treatment of severe life-threatening ailments including cancer especially in developing countries.1 Over the years, cancer patients have relied on surgery, radiotherapy, and chemical derived drugs for their treatment, which further damage patients’ health and increase their suffering. Hence, there is a need to search for and focus on medicinal plants that are used traditionally in treating tumor-related ailments. One such plant, Cnidoscolus aconitifolius (Mill.) I.M.Johnst., has been used as a constituent of herbal preparations for the treatment of cancer patients by Nigerian herbalists.
C. aconitifolius (Euphorbiaceae) originates from southeast Mexico and Guatemala and the southwestern part of Nigeria.2 It is locally known as Iyana Ipaja.3 In Nigerian traditional health practices, it is also referred to as “Hospital too far” due to its rapid healing properties against certain health conditions.4 In ethnomedicine, it has been reported to have a blood boosting effect in both pregnant women and young anemic children,5 and as an antidote for alcoholism, insomnia, and scorpion bites. Previously, the cytotoxic and antiproliferative activity of the methanol extracts of C. aconitifolius leaves, stem, and root barks against tadpoles of Ranicep ranninus and radicle length of Sorghum bicolor has been reported.6 The cytotoxicity of ethanol extract of its leaf against the brine shrimp has also been noted.7 However, there is presently no report on the anticancer activity of this plant. Therefore, the present study was aimed at validating the antitumor ethnomedicinal uses of C. aconitifolius using human breast and lung cancer cell lines.
MATERIALS AND METHODS
Chemicals
Dimethyl sulfoxide (DMSO), fetal bovine serum, gentamicin sulfate, L-glutamine penicillin streptomycin solution, Roswell Park Memorial Institute-1640 medium (RPMI-1640), sulforhodamine B (SRB), trichloroacetic acid (TCA), tris base, trypan blue, and trypsin-EDTA were purchased from Sigma (St. Louis, MO, USA). Acetic acid (Lab Scan, Ireland), amphotericin B (Formepharma, Pakistan), and doxorubicin (ICN, USA) were also obtained.
Consumables
The cell culture boats, flasks (25 and 75 cm2), centrifuge tubes (15 and 50 mL), culture plates (96-well, transparent with flat bottom), and serological pipettes (1, 5, and 10 mL) were purchased from Falcon BD (USA). The microcentrifuge tubes (2 mL) were purchased from Kartel (Italy).
Equipment
The equipment included an analytical balance, milligram balance, pH meter (Precisia, Switzerland), centrifuge, CO2 incubator (Kendro Lab Products, Germany), safety cabinet class 2 (Heraeus Germany), microscope: inverted TS-100 (Nikon, Japan), multiwell microplate reader (Stat fax 2100, Awareness Technology, USA), multiwell plate shaker (PMS 1000, Grant Instruments, UK), ultrasonic bath (MXB6, Grant Instruments), and Neubauer’s chamber (0.1 mm and 0.0025 mm2, HBG, Germany).
Collection and processing of the plant
The leaves of C. aconitifolius (Mill.) I.M.Johnst. were collected in February 2014 at Sabongida-Ora in Edo State, Nigeria, and the identity of the plant was confirmed by Dr. Shasanya Olufemi, a plant taxonomist. It was preserved at the Forest Research Institute of Nigeria, Ibadan, with herbarium specimen number FHI 109574. The plant material was air dried in the laboratory for 5 days at room temperature, followed by oven drying at 40°C, and subsequently ground to powder form and stored in an air-tight container.
Extraction of plant materials
About 2.5 kg of each plant part was exhaustively extracted in methanol (95%) using a Soxhlet apparatus, and dried using a rotary evaporator maintained at 40°C.
Preliminary phytochemical screening
Phytochemical screenings of the extracts (leaves stem and root barks) were carried out using standard methods previously described.8
Sulforhodamine-B assay
The growth inhibitory activities of methanol extract of C. aconitifolius and its fractions were tested using human cancer cell lines [breast (MCF-7) and lung cancer (NCI-H460)].9
The stock solutions of plant extracts and fractions were prepared as 40 mg/mL in DMSO. However, doxorubicin (1 mM) was prepared in distilled water. On the experimental day, respective dilutions were prepared in RPMI-1640 containing gentamicin (50 µg/mL).
Monolayer trypsinization, cell viability determination, and cell counting from a confluent flask (75 cm2) were carried out. Cells (10,000 cells/well/100 µL for MCF-7 and NCI-H460) were seeded for monolayer formation and incubated in a CO2 incubator at 37°C for 24 h. Various concentrations of methanol extracts of C. aconitifolius (1, 10, 50, 100, 200, and 250 µg/mL) and fractions (1, 25, 50, 75, and 100) were added (100 µL/well) in appropriate wells, followed by incubation for 48 h. Appropriate controls and blanks (drug and extract) were also prepared. At the end of 48 h, time zero-1 (Tz1 plate) and time zero-2 (Tz2 plate) plates were fixed with gentle addition of 50% w/v cold TCA (50 µL/well) before and after the addition of extract and fractions in experimental plates. These were left at room temperature for 30 min, washed (3×), and dried overnight. After 48 h, the experimental plates were also fixed in a similar manner.
The dried fixed plates were stained with 100 µL of sulforhodamine solution (0.4% wt/vol prepared in 1% acetic acid) for 10 min, followed by washing (5×) with 1% acetic acid to remove excess stain, and air-dried. Finally, 100 µL of tris base solution (pH 10.2, 10 mM) was added to solubilize protein bound stain and absorbance was recorded at 545 nm using a microplate reader. All the experiments were conducted in triplicate. The results of the extracts and fractions were presented as GI50, TGI, and LC50 (µg/mL) values.
Fractionation of methanol extract of the leaf of C. aconitifolius
About 25.0 g of the crude methanol extract was re-dissolved in methanol–water (1:1) and partitioned exhaustively with chloroform (400 mL×4) volumes in a separating funnel. The chloroform layer (lower) was collected first, followed by the aqueous fraction. This was repeated until a clear lower layer was obtained. The aqueous and the chloroform fractions were concentrated to dryness on a rotary evaporator and their respective yields noted.
RESULTS
The 2.5 kg of the powdered leaves, stem, and roots of C. aconitifolius yielded 87.65, 71.10, and 68.43 g of the methanol extracts, corresponding to 3.5%, 2.8%, and 2.7%, respectively.
Phytochemical screening of extract of the leaves, stem, and roots of C. aconitifolius showed the presence of saponins, tannins, terpenes, and flavonoids in varying intensities. However, alkaloids and anthraquinones were absent (Table 1).
Results of the effect of the methanol extracts on MCF-7 and NCI-H460 cell lines
The methanol extract of leaves produced growth inhibitory and cytotoxic effects on MCF-7 to varying extents. At a concentration of 50 µg/mL, the extract had +63.08±3.63% growth inhibition, which became more cytotoxic at 200 and 250 µg/mL as -14.70±0.76, and -26.25±2.18% were recorded. The GI50 and TGI were recorded as 26.67±3.33 and 95 µg/mL while the LC50 was greater than 250 µg/mL (Table 2). At the maximum concentration of 250 µg/mL, stem and root barks extracts of C. aconitifolius showed no significant activities against breast cancer cell lines.
Extract of C. aconitifolius at 50-100 µg/mL exhibited cell growth inhibitory effects on the lung cells unlike the breast cancer cells. Significant growth inhibition ranging between ~28% and 77% was recorded in a concentration-dependent manner against the human lung cancer cell line (NCI-H460). A GI50 value of ~59.67 µg/mL was recorded, while LC50 and TGI were greater than 250 µg/mL (Table 3).
Effects of the aqueous and chloroform fractions of C. aconitifolius against breast (MCF-7) and lung cancer (NCI-H460) cell lines
The chloroform fraction at 25 µg/mL displayed significant growth inhibition of ~48.62% against MCF-7 cells, which became cytotoxic at 50-100 µg/mL in a concentration-dependent manner with GI50, LC50, and TGI of 22.50, 68.75, and 43.75 µg/mL, respectively (Table 4).
Similar effects were also observed with the chloroform fraction against NCI-H460 cells, giving GI50 and TGI of 35.40 and 55.8 µg/mL with LC50˃100 µg/mL. The aqueous fraction did not show any activity at 100 µg/mL on either cell line (Table 5).
DISCUSSION
Among all human diseases, cancer remains the most deadly and life-threatening pathological condition.10 The global burden of this disease has continued to surge due to the adoption of high level of cancer-inducing lifestyles such as smoking, eating of a westernized diet, and physical inactivity.11
According to global cancer statistics, breast and lung cancers are the most frequently diagnosed cancers in females and males, respectively.12 The commonly employed treatment includes chemotherapy, radiotherapy, and in some cases surgery, which also exhibit series of side effects among patients.13 Due to this, research into ethnomedicinal plants with antitumor properties as an alternative medicine at the early stage of the disease has become necessary.
Phytochemical screening of extracts revealed the presence of various phytochemicals including tannins and flavonoids, which are known to possess free radical scavenging activity, hence preventing the development of diseases. The higher activity demonstrated by the leaf extract over the stem and root extracts could be due to the abundance of one or more secondary metabolites such as terpenes and phenolic compounds, which are known for their antifree radical potentials and inhibition of carcinogenesis.14,15
Our previous work demonstrated that the extracts from the leaf, stem, and root barks of C. aconitifolius displayed cytotoxic and antiproliferative effects on guinea corn radicle length and tadpoles in vitro. The present study further validates the previous results with the leaf extract being most active against breast and lung cancer cells. This could happen due to variation in the chemical constituents of the different morphological parts of the plant occasioned by translocation.
The leaf extract exhibited a concentration-dependent effect with higher growth inhibitory effect against breast cancer by ~2× than that of lung cancer cells. Partitioning of the extract was observed to increase the activity compared to the crude as the chloroform fraction at 100 µg/mL produces cytotoxicities of -72±9.1% and -22±11% as well as GI50 of 22.5 and 35 µg/mL against MCF-7 and NCI-H460 cells, respectively. This variation in sensitivities of the crude extracts and fraction could be as a result of the interference of some molecular process at some stages in the cell division processes, e.g., the G2/M phase and the induction of some apoptotic process such as mitochondrial transmembrane depolarization.14
The results obtained have validated the traditional uses of this plant in the treatment of cancer. However, further work towards the isolation of the constituents responsible for the observed activities is required.
ACKNOWLEDGEMENTS
The authors are thankful to NAM S&T center for providing a six-month grant to conduct this research as well as the director of the International Center for Chemical and Biological Sciences (ICCBS), University of Karachi, Pakistan, Prof. Muhammad Iqbal Choudhary, for providing the necessary facilities to carry out this work.
Conflicts of interest: No conflict of interest was declared by the authors.