Cancer has been reported as the second leading cause of mortality worldwide, and is expected to surpass heart disease in the next few years [1
]. One recent report has revealed that cancer incidence is predicted to increase to as many as 22 million cases per year [2
]. Cancer is characterized by evasion of apoptosis or defective apoptosis mechanisms which in turn lead to uncontrollable development of cells [3
]. Most anticancer drugs aim to halt cancer cells from further proliferating and metastasizing to other parts of the body. In spite of great advances in anticancer research, current chemotherapy drugs have been reported to leave cancer survivors with lifelong side effects [4
]. Thus, investigation using a natural product with fewer side effects is crucial.
Antioxidants are the agents that have the ability to prevent, delay or eradicate oxidative stress of a molecule from atmospheric oxygen or reactive oxygen species [6
]. Antioxidants play an important role in the prevention of various chronic diseases, such as cardiovascular disease, neuronal disease, cataracts, and several types of cancer via the pathophysiology mechanism known as oxidative stress. In this context, there is an increasing interest to search for natural antioxidants present in functional herbs, fruits, and vegetables. Phytoconstituents such as phenolics, carotenoids, anthocyanins, and tocopherol have been found to exert chemo-preventive and cardio-protective effects [8
In this study, the natural products used for antioxidant and anticancer evaluation is porcupine bezoar (PB) as depicted in Figure 1
. PBs are concretions of undigested foreign materials that accumulate and calcify within the gastrointestinal tract of porcupines [9
]. The bezoar is a stone-like material and is traditionally believed to exert various medicinal benefits [10
]. The word bezoar itself has origins in Persian (pad = to expel, zahr = poison or antidote) [11
]. Bezoar was valued as gem and as a noble metal. A review on the importance of bezoar has revealed that the oriental bezoars found in monkeys and porcupines (Lapis bezoar orientalis) were considered the most expensive [12
]. Porcupine bezoars are generally composed of three concentric layers made up of vegetal matter [9
]. Traditionally, people have used bezoars to treat several deadly diseases such as cholera, plague, smallpox, and measles, and as an antidote for poisons of different origins [7
]. Due to traditional claims of medicinal benefits of porcupine bezoars and its rarity, traders tend to sell it at implausibly high prices. Moreover, there are undocumented traditional claims in several parts of Malaysia that porcupine bezoars can treat cancers and possess high antioxidant activity. Despite these undocumented traditional claims, there is limited information on the antioxidant and anticancer properties of porcupine bezoar. Therefore, the objective of this study was to characterize the PB chemical profile, investigate its antioxidant and anticancer properties to verify its traditional claim, and to explore its medicinal potential.
2. Materials and Methods
2.1. Chemicals and Reagents
Methanol, Folin–Ciocalteu phenol reagent, and gallic acid were purchased from Honeywell (Charlotte, NC, USA). Quercetin, 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH), trichloroacetic acid, and thin layer chromatography (TLC) plates were purchased from Sigma-Aldrich (St. Louis, MO, USA). Aluminium chloride hexahydrate (AlCl3·6H2O), sodium bicarbonate (Na2CO3), sodium phosphate dibasic (Na2HPO4), sodium phosphate monobasic (NaH2PO4), and ABTS [2,2′-azinobis (3-ethylbenzothiazoline)-6-sulfonic acid] diammonium salt were purchased from Roche, Basel, Switzerland. Human melanoma (A375) and normal human dermal fibroblast (NHDF) were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were grown in complete growth medium (CGM) which was made of Dulbecco’s modified Eagle medium (DMEM) (Nacalei Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (Nacalei Tesque, Kyoto, Japan) and 1% penicillin–streptomycin (Nacalei Tesque, Kyoto, Japan). Phosphate-buffered saline (PBS) (Gibco, Waltham, MA, USA) was used for cell washing. For analyzing apoptosis and cell cycle, Nexin reagent (Merck Millipore, Burlington, MA, USA) and cell cycle reagent (Merck Millipore, Burlington, MA, USA) were used, respectively. For the mRNA expression analysis, InnuPREP DNA/RNA mini kit (Analytik Jena, Jena, Germany), SensiFAST cDNA synthesis kit (BIOLINE, Memphis, TN, USA), SensiFAST SYBR® No-ROX Kit (BIOLINE, Memphis, TN, USA), and primer (Integrated DNA technologies, Singapore) were used. 5-Fluorouracil (5-FU) (Sigma-Aldrich, St. Louis, MO, USA), a standard anticancer drug, was used as a positive control.
2.2. Porcupine Bezoar Extract Preparation
The investigation using Hystrix brachyura
bezoar was performed with the permission from the Malaysian government authority viz. Department of Wildlife and National Parks, Malaysia, for academic and research purposes (JPHL&TN (IP): 100-34/1.24 Jld 8). The PB (9.26 g) was crushed into a powdered form using mortar and pestle. Subsequently, 1 g of powdered PB was extracted using deionized water with 1:20 ratio for 30 min using ultrasonication method to get PB aqueous extract [15
]. The supernatant was filtered and freeze-dried obtained PB aqueous extract in powdered form (250 mg, 25%). The dry powdered form of PB aqueous extract was then subjected to chemical profile characterization, bioautography antioxidant assay, and anticancer activity evaluation. For anticancer activity, PB aqueous extract was prepared into stock solution (2.0 mg/mL) and further diluted with CGM or DMEM accordingly. On the other hand, the positive control (5-FU) was prepared by dissolving in DMSO, before making it into stock solution (1.0 mg/mL) with CGM and diluted further accordingly.
2.3. Fourier-Transform Infrared Spectroscopy Analysis Of Porcupine Bezoar
A Fourier-transform infrared (FTIR) spectrometer (Perkin Elmer Inc., Waltham, MA, USA) was used for the functional groups analysis. Prior to FTIR analysis, the PB aqueous extract was thoroughly freeze-dried to ensure an absence of water molecule. A small mass of PB aqueous extract was placed on the diamond crystal and the spectra were collected in wave number region of 400–4000 cm−1 at a resolution of 4 cm−1. The data were processed using PerkinElmer Spectrum (version 10.03.09) software (PerkinElmer, Waltham, MA, USA).
2.4. Gas Chromatography Mass Spectroscopy (GCMS) Profile Analysis of Porcupine Bezoar
The aim of this assay was to determine the chemical profile of PB aqueous extract. The PB aqueous extract was derivatized as described previously [16
]. GCMS-TQ8030 linked to a GCMS-QP 2010 Plus Shimadzu gas chromatograph equipped with a capillary column DB-5 (0.25 μm thickness × 0.25 mm diameter × 30 mm length) was used for compound identification. Helium (1.0 mL/min) was used as the carrier gas (Shimadzu, Japan). The instrument was operated in electron impact mode at ionization voltage (70 eV), injector temperature (250 °C), and detector temperature (280 °C). The starting oven temperature was set at 50 °C and held for 5 min. This temperature was then programmed to 150 °C at 5 °C per min and held for 5 min. Finally, the temperature was programmed to 300 °C for 5 min at a rate of 5 °C per min. The compound identification from the spectral data was based on the available mass spectral records in National Institute of Standards and Technology database.
2.5. Total Phenol Content (TPC)
Folin–Ciocalteu’s method was modified and adapted to the 96-well plate assay as described by previously for the quantification of total phenolic content [17
]. Mixtures of 25% (w/v) Folin–Ciocalteu’s reagent and 75% (w/v) sodium carbonate were prepared. Then, 50 μL of both mixtures were mixed with 10 μL of sample (5 mg/mL) and incubated for 45 min at room temperature. Absorbance was set at 765 nm using a Tecan microplate reader (Infinite®
200 PRO, Männedorf, Switzerland) against blank. All assays were carried out at least in triplicate. The results were expressed as microgram gallic acid (GAE) equivalent per gram dry weight basis of the fresh sample (mg GAE/g dw basis).
2.6. Total Flavonoid Content (TFC)
Total flavonoid content was determined through modified method using 96-well plate as described earlier [17
]. An aliquot of (10 μL) of sample (5 mg/mL) was mixed with 90 μL of 2% (w/v) aluminum chloride and incubated for 10 min at room temperature. The absorbance was set at 415 nm and the mixture was read using microplate reader (Tecan Infinite®
200 PRO) against blank. All tests were performed in triplicate. The data were expressed as microgram quercetin (QE) equivalent per gram dry weight basis of fresh sample (mg QE/g dw basis).
2.7. Rapid Screening of Antioxidant by Dot-Blot Assay
2.7.1. DPPH Dot-Blot Assay
Sixteen grids of TLC plates were separately loaded with sample (PB aqueous extract) and standard (ascorbic acid) in decreasing concentrations (5–0.153 mg/mL) via two-fold serial dilution. The plate was left to completely dry at room temperature. Both plates were dipped for 10 s in 0.05% of DPPH in methanol. Excess solution was removed with tissue paper and the plate was air dried. After 30 min result was taken. Stained silica layer revealed a purple background with white/yellow spots at the location where radical-scavenger capacity presented. The intensity of the white/yellow color depends upon the amount and nature of radical scavenger present in the sample [18
2.7.2. ABTS+ Dot-Blot Assay
The 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation (ABTS+
) is a stable free radical frequently used for estimating the total antioxidant capacity of natural products. ABTS+
solution was prepared by mixing 7 mM ABTS stock solution with 2.45 mM potassium persulfate with ratio 1:1 (v/v
) leaving the mixture in dark at room temperature for 16 h [19
]. Dried TLC plates with pre-loaded sample (PB aqueous extract) and standard (ascorbic acid) were dipped in ABTS+
solution exhibited green background with white or pink spot indicated the presence of radical scavengers in PB aqueous extract [20
2.7.3. β-Carotene Dot-Blot Assay
Pre-loaded TLC plates with sample (PB aqueous extract) and standard (quercetin) were prepared and left to dry completely. The TLC plates were dipped in 0.05% solution of β-carotene in chloroform and exposed to sunlight for 2–3 hours or until the yellow background bleached [20
]. Active compound will remain in yellow with white background.
2.8. Cell Culture Maintenance
Human malignant melanoma cells (A375) were maintained in complete growth medium (CGM) supplemented with 89% of Dulbecco’s modified eagle medium (DMEM), 10% fetal bovine serum and 1% of penicillin streptomycin antibiotic at 37 °C and 5% CO2 humidified atmosphere.
2.9. Cytotoxicity Assay of Porcupine Bezoar on Cancer Cell and Normal Cell
This assay was performed to determine the median inhibition concentration on cancer cell of A375. Cell viability to determine 50% inhibitory concentration (IC50) and proliferation assay were evaluated using Promega CellTiter 96® AQueous Non-Radioactive Cell Proliferation (Promega, Madison, WI, US) assay as recommended by the manufacturer. In determining IC50, cells were seeded at 4.0 × 102 cells/mL and treated with PB extract with serial concentrations of 3.9–1000 µg/mL in a 96-well plate before incubation for 72 h. Then, 10 µL of MTS reagent were added in each well and incubated for 3 h before measuring at 490 nm using a microplate reader. The IC50 values for PB aqueous extract and 5-FU were calculated using Graphpad Prism 6. Further test for the proliferation assay over time (24, 48, 72 and 95 h) used untreated (UT) cells as negative control and 5-fluorouracil as positive control. Additionally, the IC50 of PB aqueous extract was evaluated on NHDF with UT and 5-FU as controls.
2.10. Cell Cycle Arrest Assay
Cell cycle assay was performed to determine whether PB aqueous extract has the ability to induce cell arrest in A375 cells and through which phase. Grown cells were treated with PB aqueous extract at IC50 for 72 h. All cells from the well were collected and prepared in triplicate. The cells were fixed with cold ethanol for 2 h and stained with cell cycle kit according to the manufacturer’s instructions. Cells were analyzed using guava flow cytometer (Luminex, Austin, TX, USA).
2.11. Apoptosis Assay
Apoptosis Annexin V/7AAD assay was performed to determine the apoptosis distribution. A375 cells were treated with PB aqueous extract at IC50 for 72 h. All cells from the well were harvested and prepared in triplicate. For apoptosis analyses, cells were stained using Nexin reagent and analyzed using guava flow cytometer following the manufacturer instruction. Distribution of early and late apoptotic cells after exposure with PB aqueous extract was reported in a dot plot graph.
2.12. Real Time Quantitative PCR (RT-qPCR) Analysis
The aim of this assay was to quantify the mRNA expression of mRNA of targeted primers related to apoptosis, cell cycle arrest and metastasis. Total RNA was extracted from PB aqueous extract-treated or untreated A375 cells after 72 h using RNA extraction kit according to the manufacturer’s instructions. The RNA’s quality and integrity were assessed before quantification process. The complementary DNA (cDNA) was prepared using cDNA synthesis kit according to manufacturer’s instructions. Subsequently, 200 ng of RNA template were synthesized into cDNA and later diluted in the experiments. The primers were chosen from the National Centre for Biotechnology Information (NCBI) database with a melting temperature (Tm) of 59–65 °C. Amplicon size was 70–150 bases. Forward and reverse primers spanning exon–exon junctions were selected to avoid amplification of genome sequences. PCR amplifications were performed using SYBR green in CFX96 Touch™ Real-Time PCR (Bio-Rad, Hercules, CA, USA). Annealing temperature was optimized using gradient temperature setting. Melting curves were analyzed to ensure amplification specificity and null primer-dimer formation. Primer PCR efficiency was evaluated using serial dilutions of cDNA sample (1:10, 1:100, 1:1,000, 1:10,000, and 1:100,000) in CFX Manager Software. The amplification efficiency (E) and correlation coefficients (R2) of the standard curve ranged from 93.0% to 114.7% and 0.984 to 0.998, respectively. Details information about the primers are attached in supplementary Table S1
. The amplifications were done by following the conditions viz., 2 min at 50 °C, 10 min at 95 °C, and 45 cycles of 15 s at 95 °C and 1 min at 60 °C. Results were analyzed using CFX Manager Software based on the threshold cycle (CT) values. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin (ACTB) were used as reference genes. The genes used, and the nucleotide sequences are summarized in Table 1
2.13. Statistical Analysis
All values were presented as mean ± SD of triplicate from three different experiments (n = 3). A one-way analysis of variance (ANOVA) was performed using the Prism statistical software package (GraphPad Software, San Diego, CA, USA). This study used the Dunnet test as follow-up test of ANOVA which compared PB aqueous extract and 5-FU with UT. Differences between PB aqueous extract treated and UT were considered statistically significant at p < 0.01.
Porcupine bezoars have been used for past centuries for its medicinal benefits, however the information about its properties are still limited. Hence, the current study was performed to investigate the chemical characterization of PB, and evaluate its antioxidant activity and anticancer activity on melanoma cells. In the current study, the extraction method of PB was carried out using sonication procedure. As mentioned in [21
], the sonication mechanism diffuses the solid area of the sample into the solvent. By forming cavitation bubbles that assist to break down the plant cells and increase the pores of the cell wall, the diffusion process is improved, leading to enhanced mass transfer into the solvent [22
]. The time factor also is very important when using the sonication technique to extract the phenolic compounds. A longer extraction time allows more contact time for the cavitation bubbles to break more plant cells, resulting an increase of the extracted of phenolic compounds [23
Phenolic compounds are powerful antioxidants and act in a structure-dependent manner; they can scavenge reactive oxygen species (ROS) and chelate transition metals which play vital roles in the initiation of deleterious free radical reactions [24
]. Clearly, the total phenolic content is considered as an important indication of antioxidant properties of natural product extracts. Crude extracts of fruits, vegetables, and other natural product materials are rich in phenolic content. TFC was recorded as having the lowest antioxidant activity and the reason is due to the solvent chosen; this is in agreement with a previous study which reported that antioxidant activity of flavonoids depends on the structure and substitution pattern of hydroxyl groups [8
]. Porcupines are herbivores and their dietary intake is from tree barks, seeds, twigs, and grasses [25
]. This explains the reason for PB to have a low flavonoid content, as mostly flavonoid content can be found mainly in fruits, vegetables, leaves and is rarely found in bark or twigs [27
]. Some of our initial data using different PB revealed that PBs have similar finding in this study where the phenolics content is low and absence of flavonoid content [28
]. However, another study using three different types of PB (black, powdered, and grassy) reported black and powdered PB have significant higher amount of phenolic content compared to grassy PB [29
]. Additionally, the GCMS profile also revealed main constituents are amino acids found in animal, which further clarified why PB extract have low phenolic and flavonoid content.
Food antioxidants play an important role in the promotion and maintenance of health as the antioxidant capacity is able to reduce the risk for chronic diseases such as cancer and heart disease [30
]. ABTS antioxidant assay observed reaction kinetics of specific enzymes to act as electron donor to form the ABTS•+. Meanwhile, DPPH is a stable free radical and it receives an electron or hydrogen radical to become a stable diamagnetic molecule which is widely used to investigate radical scavenging activity. The ABTS and DPPH assays show PB aqueous extract had strong hydrogen-donating capacity. The results are in agreement with the phenol contents determined for each sample. Antioxidant agents act as reducing agents and antioxidants by the hydrogen-donating property of their hydroxyl groups [31
]. The method is influenced by the solvent and the pH of the reactions.
Therefore, it can be concluded here that the extraction of plants is related to its polarity of extraction solvent used. A previous study has reported that pentadecyl acrylate has DPPH and ABTS antioxidant activity [32
]. Another study described ursodeoxycholic acid as having high capacity to scavenge hydroxyl and lipid peroxidation antioxidant assays [33
]. Another study conducted antioxidant evaluation using three different types of bezoar namely black, grassy and powdered PB revealed that all three types of PB have different antioxidant capacity in 2, 2-diphenyl-1-picrylhydrazyl free radical scavenging activity assay (DPPH), ferric reducing power assay (FRAP), intracellular reactive oxygen species scavenging activity assay (ROS), and reactive nitric oxide scavenging activity assay [29
]. However, the grassy PB displayed the lowest antioxidant activity.
The cytotoxic assay revealed low concentrations for PB aqueous extract to inhibit 50% of A375 cell growth at 72 h. The cytotoxic study also suggested that PB exhibited cytotoxic and cytostatic capacity in a dose- and time-dependent manner. However, the flow cytometry analysis revealed PB did not cause cell cycle arrest but exhibited apoptosis. Annexin V/7AAD assay evaluated the cells and categorized them into a quadrant base in the presence or absence of phosphatidylserine and 7-AAD, which helped in differentiating early, late apoptotic and necrotic cells [34
]. The PB aqueous extract was revealed to induce apoptosis when compared to UT cells and 5-FU treated cells. The underlying mechanism of apoptosis induction revealed PB aqueous extract exhibited apoptosis via mitochondria apoptosis pathway. The intrinsic pathway is initiated within cells involving the Bcl2 families such as pro-apoptosis (Bax
) and anti-apoptosis (Bcl2
) genes to regulate the promotion or inhibition of apoptosis [35
]. PB aqueous extract also found to up-regulate the expressions of cytochrome C
, caspase 9
, and caspase 3
. The finding was found to be in line with the intrinsic pathway in which internal stimuli induce bax to activate cytochrome C release into cytosol and activate caspase cascade through caspase 9 and caspase 3 [37
]. The GCMS analysis revealed two compounds detected from PB aqueous extract possessed anticancer activity, namely stearic acid and ursodeoxycholic acid. Stearic acid is an 18-carbon chain of fatty acid found primarily in animal derivatives and plant fat [39
]. Stearic acid has earlier been reported to induce apoptosis in MDA-MB-231 through mitochondria pathway by reducing mitochondria membrane potential, which leads to the release of cytochrome c into cytosol [40
]. Ursodeoxycholic acid described to exhibit anticancer properties by inhibiting cell proliferation through regulation of oxidative stress in colon cancer cells [42
]. However, remaining compounds are yet to be explored for their biological potential.
The bezoars are formed from the reaction of undigested porcupine food and enzymes from porcupine itself. The bezoar itself is a medical illness (gastrolith) for porcupines and needs to be surgically removed for the porcupine to survive. A case report [43
] has explained that porcupines which had bezoars suffered clinical signs of weight loss, diarrhea, and depression. Ignoring the condition leads to death as the bezoar causes gastric perforation. Additionally, it is crucial to understand that each PB is unique in each porcupine. The chemical profile of each PB depends on the food habits of individual porcupine; the age of the bezoar in the porcupine and the location of the bezoar collected could also play major roles in the different chemical profiles of the bezoar.
The findings in this study have reported PB aqueous extract’s chemical profile, antioxidant activity, and anticancer activity on A375 cells for the first time. PB aqueous extract revealed 5,10-diethoxy-2,3,7,8-tetrahydro-1H,6H-dipyrrolo [1, 2-a: 1′, 2′-d] pyrazine, ursodeoxycholic acid, cholest-5-en-3-ol (3.beta.)-, carbonochloridate, and pentadecyl acrylate as the major constituents. The compounds that may be responsible for the anticancer activity of PB aqueous extract are ursodeoxycholic acid and stearic acid. However, other unidentified compounds which were not present in the library of the GC-MS system may also play some roles towards its antioxidant and anticancer effects. The PB showed low TPC and TFC values, which play major roles in the antioxidant activity (ABTS > DPPH > β-carotene). PB extract was shown to inhibit cell growth through induction of apoptosis via the mitochondria pathway with minimal toxicity to NHDF cells. However, further study is still necessary to isolate active compounds of PB and evaluate their antioxidant and anticancer properties along with safety profile.