Human beings’ journey on this planet has never been comfortable, and they had learned instinctively that they have to fight for their survival, and one of the most significant wars that they fought has been against diseases. Human history with diseases goes back so long that the world’s oldest disease dates as far back as 600 BC, referred to as Leprosy disease, known as Hansen’s disease [
Cancer usually occurs when healthy living cells in a specific part of the body begin to grow without a restriction. Generally, cancer cells develop from normal cells due to damage to DNA. Inherited cancers can occur; this happens when the damaged DNA is inherited from the parents. However, sometimes the DNA damage may be transpired from environmental factors such as smoking, pesticides, and chemicals. Despite that, some people might think cancer is a new disease, but it is not. It dates back so far that some of the earliest evidence of human bone cancer was found in mummies in ancient Egypt around 1600 BC [
It is divided into different types based on where it began; all kinds of cancer disease continue to grow, divide, and redivide instead of dying and forming new abnormal cells. There are four main types of cancer, including carcinomas, which is the most common cancer type. They occur in the tissue that coats the surface of internal organs and glands, as in prostate and breast cancers; sarcomas begin in the membranes responsible for connecting and supporting the body. So, they usually happen in parts such as muscles, nerves, joints, and bone; leukemias are known as a cancer of the blood; and finally, lymphomas originate in the lymphatic system [
As mentioned before, cancer cells usually start at a particular part of the body; however, sometimes it continues to grow and can be migrated to other parts of the body to develop into new tumors. This process is called metastasis [
Cancer is the second leading cause of death in the world after cardiovascular diseases [
According to scientists, some cancers are contributed to humans’ lifestyle, including lung, skin, and colon cancers which have been widely increasing over the past few years. So, all the efforts are now directed at how to prevent them. The most important aspects that should be considered are the measures of primary prevention, which include teaching people about the damage of tobacco use, guiding them to healthy diets and food, and promoting health campaigns to raise knowledge about cancer [
Obesity is a disease determined by a measure called BMI (Body Mass Index) calculated based on weight (kg squared) and height (meter squared). A person is obese when his BMI score is between 30 and 40 and highly obese when it is above 40 [
Diabetes mellitus is a metabolic disorder of high blood sugar levels. Causes could be due to the pancreas’ inability to produce insulin (the case in type I), which is most common in children and young adults and makes up about 5% to 10% of all diabetes cases. In East Asia, the rate is almost 1 new case per 100,000 in one year. On the other hand, it could be due to insulin resistance or little production by the body (the case in type II), which makes up 90% of all cases [
Oxidative stress is an imbalance between free radicals and antioxidants in the body. The free radicals can start doing damage to fatty tissue, DNA, and proteins in the body. This damage could lead to various diseases over time, such as atherosclerosis, diabetes, inflammatory conditions, high blood pressure, heart diseases, cancer, neurodegenerative diseases, and aging. To avoid oxidative stress, one has to make sure he gets enough antioxidants in his diet, quit smoking, wear sunscreen, avoid overeating, get plenty of sleep, and do regular exercises [
There are many growing plants; some are considered poisonous, while others are safe to eat. Sometimes the benefit can originate from the whole plant or, in other cases, form one or more of its parts like the roots, rhizomes, leaves, fruits, stems, barks, and flowers. These particular parts are marked by including various chemical components such as volatile aromatic oils. Other components that may be used as a dietary supplement or have a pharmacological value can be used as a source for drug development; particularly today several plants derived drugs are used throughout the world [
Since the beginning of life, humans have depended on nature as a source of food and medicine; people knew the importance of plants and the massive benefits they contain. To this day, researchers are astonished by the amount of scientific data obtained from plants worldwide, such as the plant’s case that this study revolves on [
The leaves and roots are characterized by containing several chemical constituents such as tannins, caffeoyl sugar esters, terpenoids, and flavonoids [
To the best of our knowledge, no previous studies were established on (RS) plant roots. Therefore, the current study aims to estimate anticancer, antioxidant, and metabolic enzyme inhibitory of (RS) root four solvent fractions.
The roots of the (RS) plant were collected from the mountains of Jerusalem of Palestine in November 2018. The taxonomical characterizations and the voucher specimen code (Pharm-PCT-2065) were carried out at the Pharmacognosy Laboratory of An-Najah National University. The cleaned roots were first dried at ordinary conditions, and then an air-drying oven was used for five days with a regulated temperature (25 ± 2°C) and fixed humidity (55 ± 5 RH). Later on, the dried roots were powdered coarsely and kept in unique glassware at room temperature for future use.
Microplate reader (Unilab, 6000, Mandaluyong, USA), CO2 incubator (Esco, 2012-74317, Changi, Singapore), inverted microscope (MRC, IX73, Hong Kong, China), UV-Visible spectrophotometer (Jenway 7315, Staffordshire, UK), vortex (Heidolph Company, 090626691, Schwabach, Germany), ultrasonic cleaner (MRC Laboratory Equipment, 1108142200049, Haifa), autoclave (MRC Laboratory Equipment, A13182, Haifa), water bath (Lab Tech, 2011051806, S. Korea), stir-mixer (Tuttnaver, 300303159, China), cooled incubator (Gallenkamp, SG92/01/244, Loc, United Kingdom), micropipette (MRC Laboratory Equipment, MPC-1000, Haifa), multichannel micropipette (MRC Laboratory Equipment, MPC-8-50, Haifa), filter papers (Whatman no. 1, USA), shaker device (Memmert shaking incubator, Germany), rotatory evaporator (Heidolph vv2000 Heidolph OB2000, Germany), grinder (Molineux model, Uno, China), balance (Red wag, AS 220/c/2, Poland), flow cytometer (Becton-Dickinson LSR II, Immunofluorimetry systems, Mountain View, CA), and freeze dryer (Millrock Technology BT85, China) were used.
The following chemicals and reagents were used: methanol,
The powdered roots (75 g) were soaked in 500 ml of four solvents with various degrees of polarities. Briefly, (RS) root powder was fractionated sequentially by adding solvents of increasing polarity: first it was soaked in 500 ml of hexane (nonpolar solvent) and was placed in a shaker device at 25°C for two days, and then a suction filtration was used which worked by vacuum pressure to filtrate the mixture. The remaining (RS) plant residue was soaked sequentially in acetone (polar aprotic solvent), methanol (polar protic solvent), and water (polar protic solvent). Each of the obtained hexane, acetone, and methanol fractions were separately left in the rotavap to decrease the fractions’ volume, and then each fraction was dried in the incubator. In comparison, the aqueous fraction was dried in freeze-drier apparatus. The four dried fractions were stored in the refrigerator at 4–6°C for further use [
Methanolic stock solution (1 mg/ml) was set for (RS) roots fraction and for vitamin E analog (Trolox), which was utilized as a positive control with potent antioxidant activity. Then, various concentrations from the previous stock solution were prepared. Plant working solution (1 ml) was mixed with 1 ml freshly prepared DPPH (0.002 g/ml) methanolic solution, and 1 ml methanol was then added to the previous mixture. The blank solution contained DPPH and methanol only in a ratio of 1 : 1. The solutions were incubated at room temperature (25°C) in a dark place for 30 min. Then, their optical densities were measured by the UV/Vis spectrophotometer at 517 nm [
Antioxidant activity was calculated per the following equation:
The
Acarbose was used as a positive control following the same procedure. The
A plant extract solution (1 mg/ml) was made by dissolving 100 mg of each plant fraction in 100 ml of 10% DMSO, and then the produced solution was diluted to obtain different concentrations (5, 25, 50, 100, 200, 300, and 400
The dilution of (RS) roots extracts was done with the aqueous, methanol, acetone, and hexane solvents. Preserved extracts of 1 mg from each solvent were taken to obtain a concentration of 0.1 mg/ml. Three preparations at a final concentration of 10
Following cultures, the harvested Hep3B cells’ adjustment to 106/ml in staining buffer (in saline consisting of 1% bovine albumin was achieved. For viability measurements and apoptosis, the staining of fragmented DNA by propidium iodide (PI) and phosphatidylserine staining using annexin-V conjugated to FITC was done according to the instructions of the manufacturer. Early apoptosis was defined as annexin-V (+) but propidium iodide (-), late apoptosis was defined as annexin-V (+) but propidium iodide (+) while necrosis was defined as annexin-V (-) but propidium iodide (+). On the other hand, viable cells were defined as annexin-V (-) but propidium iodide (-). Unstained controls were used in each of the experiments, such as IgG isotype controls and FMO controls. The analysis of the cell cycle by quantization of DNA content was achieved by employing propidium iodide. The Hep3B cells’ fixation was performed in cold 70% ethanol at 4°C for at least 30 min. After that, the cells were washed 2x in PBS. It was calibrated to spin at 2000 rpm to dispose of the supernatant. To ensure that only DNA was stained, the treatment of cells with ribonuclease (50
Statistical differences were analyzed with either the 2-tailed unpaired Student’s
Throughout history, people have been struck by several diseases, that some even shaped human history like the Black Death: Bubonic Plague, which spread across Europe during the fourteenth century killing 75–200 million people, and Malaria, which claimed the lives of 200 million people in the nineteenth century alone [
Antioxidants are the substrates that prevent molecules from oxidation inside the living cells and the presence of excess oxygen species that develop lethal diseases such as cancer, Alzheimer’s, atherosclerosis, and diabetes mellitus [
Figure
DPPH free radical scavenging property by
the IC50 values of the evaluated fractions of
IC50 ( | |||||
---|---|---|---|---|---|
Enzymes | Hexane | Acetone | Methanol | Aqueous | +ve controls |
DPPH | 4.71 ± 0.28 | 0.67 ± 0.25 | 0.078 ± 0.22 | 6.55 ± 0.23 | 2.04 ± 0.52a |
50.90 ± 1.23 | 29.11 ± 0.39 | 20.12 ± 0.34 | 26.90 ± 0.19 | 6.56 ± 0.3b | |
Lipase | 8.33 ± 0.30 | 6.03 ± 1.23 | 6.51 ± 1.01 | 10.31 ± 0.67 | 0.39 ± 0.45c |
aTrolox; bacarbose; corlistat.
A study conducted by Serteser et al. found that (RS) methanolic fruit extract has free radical scavenging activity with IC50 values of 1320
Moreover, an investigation conducted by Zengin et al. found that (RS) leaf ethyl acetate, methanol, and aqueous extracts have a free radical scavenging property with Trolox equivalent values of 24.12 ± 0.81, 347.61 ± 13.21, and 386.39 ± 10.97 mg TE/g extract, respectively [
Additionally, an investigation conducted on
One of the methods to lower postprandial hyperglycemia, one of the leading clinical goals to control blood glucose levels in diabetic patients, is to reduce glucose absorption in the gastrointestinal tract by repressing the enzymes responsible for the hydrolysis of carbohydrates, such as pancreatic
Table
A study conducted by Zengin et al. found that the leaf (RS) ethyl acetate, methanol, and aqueous extracts have
Lipase is one of the essential metabolic enzymes that endorse the hydrolysis of lipids to free fatty acids and glycerol. For that, the inactivation of lipase is one of the main goals in the pharmaceutical practice to treat overweight, obesity, hypertriglyceridemia, and hypercholesterolemia. About 60% of total dietary fat hydrolysis was established by the pancreatic lipase enzyme [
In fact, (RS) root methanol, aqueous, hexane, and acetone fractions inhibited potentially the porcine pancreatic lipase with IC50 values range 6.03–10.31
Porcine pancreatic lipase inhibitory activity by
A study established by Jamous et al. found that the ethanol extract of (RS) leaves inhibited the porcine pancreatic enzyme with an IC50 dose of 88.1 ± 4.6 mg/ml [
One of the current study aims was to detect whether (RS) affects cell cycle disturbances in liver cancer cells. As a result, propidium iodide-stained nuclei cells were assessed through flow cytometry analyses. Parameters of cell cycles were investigated for (RS) roots fractionated with (10
The proportion of cells in the G1, S, and G2-M phases after processing the different fractions of the
Also, a slight decrease of cells in the S phase, which is in charge of DNA replication, was seen after the processing of the different fractions of the (RS) roots with averages of 12.1 ± 0.8%, 11.8 ± 2.5%, 10.3 ± 1.5%, and 12.6 ± 2.08% for the aqueous, methanol, acetone, and hexane fractions respectively, compared to the average of the DOX which was 17.6 ± 0.57%; Figure
Cells proportion in the G2/M phase, in which mitosis commonly takes effect, was increased as the averages obtained after treatment of the aqueous, methanol, acetone, and hexane fractions were 10.1 ± 2.2%, 24.5 ± 2.2%, 6.9 ± 1.6, and 19.6 ± 3.0% respectively, compared to 7.4% ± 1.8 in the DOX (
These results show that (RS) roots fractions had effects on the cell cycle of the Hep3B cells. As it was noticed, methanol fraction seems to be the most potent inhibitor since it decreased the cell proliferation rate by prolonging the G2-M phase in a time-dependent process, thus slowing cancer progression. In conclusion, these data might imply that (RS) root extracts have anticancer properties.
The next question was whether the aqueous, methanol, acetone, and hexane fractions of the (RS) roots that disturb the DNA content could provoke programmed cell death known as apoptosis. To investigate that, it was acknowledged that cells going through apoptosis have their phatidylserine (PS) phospholipid moved from the plasma membrane’s inner face to the cell surface; hence identifying apoptotic cells can be managed by the presence of PS on the cell surface. As stated in materials and methods, the staining of PS with a fluorescent conjugate of annexin-V, a protein that is known to have a high affinity for PS, was done. The detection of the PS can be accomplished; after that, flow cytometry analysis was performed. Also, cells were stained with propidium-iodide (PI), capable of entering the cell only when the plasma membrane is impaired. Early apoptosis was appraised by positive for PS, however negative for PI. Nevertheless, it was specified from the late apoptotic and necrotic cells phase, positive for PS and PI.
Figure
(a) The apoptotic cell population and the late apoptotic/necrotic cells’ population alone and under treatment form the different fractions of the
Figure
AFP is a tumor marker and protein synthesized in the fetal liver and has been speculated to be the fetal analog of serum albumin. High AFP levels might hint at different types of cancers, such as liver cancer. Averages of the AFP’s secretion from the Hep3B cells alone or under treatment from the different fractions of the (RS) roots are demonstrated in Figure
Averages of the AFP’s secretion from the Hep3B cells alone or under treatment form the different fractions of the
A study conducted by Selmi et al. found that the cytological analysis showed a decline in the mitotic indices in the treated (RS) roots compared with their corresponding controls at all concentrations and durations tested. This notable decrease was time- and concentration-dependent. The toxic influence of
No previous studies were conducted on the roots of the (RS) plant to the best of our knowledge, and our obtained results were distinguished from previously conducted results on other (RS) parts used. These facts suggest that the (RS) roots four extracts could be used as a potential antioxidant, anti-
The obtained results showed that the (RS) plant roots have potential antioxidant, antilipase, and anti-
The data used to support the findings of this study are available from the corresponding author upon request.
The authors have declared no conflicts of interest.
N.J. and M.D. designed the current study while N.J., J.A., F.H., L.I., J.A.A., H.H., D.N.A., H.Z.M., K.O., M.S., and R.K. conceived this study. M.H. and M.Q. analyzed the data obtained. This paper was written by N.J., M.D., and M.H., and drafted by all authors. All authors read and approved the final manuscript.
The authors would like to thank the Faculty of Medicine and Health Sciences at An-Najah National University.