Edward Y. Zhang, a Samantha S. Cope, a Yi Zheng, b Bethany A. Reinecke, b Yan Zhang b#
a. Todd Allen Phillips Center for Medical Sciences, Godwin High School, 2101 Pump Rd, Richmond, VA 23238
b. Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, 800 E. Leigh Street, Richmond, VA 23298
Peer Reviewer: Janice Rateshwar
Professional Reviewer: Yan Zhang
# Corresponding Author: email@example.com. (804)8280021
Parkinson’s disease (PD) is a neurodegenerative disorder that attacks midbrain dopaminergic neurons and reduces motor skills. In Asia, Zingiber officinale (ginger) is used as an herbal medicine against PD. The purpose of this study was to evaluate different methods of extracting potentially active constituents from ginger, 6-shogaol and 6-gingerol, and their putative neuroprotective effects, particularly in a cellular PD model. These effects were assessed by measuring the absorbance of nitric oxide (NO), a biomarker of PD. Ginger was first extracted by 95% ethanol or water, the two independent variable levels. After component analysis, the extracts were added to the neuronal cells that were then treated with 1-methyl-4-phenylpyridinium (MPP+), a neurotoxin used to induce PD symptoms, and tested for their absorbance of NO. Cells given only MPP+ were the positive control, and cells given no ginger extract were the negative control. It was hypothesized that the 95% ethanol ginger extract would show better neuroprotective effects. The results of this study supported this hypothesis, with ethanol showing higher levels of neuroprotective effect than water. After a t-test analysis, the data showed that the ethanol levels were significantly different from the MPP+ level control indicating meaningful neuroprotective effects. However, the same analysis of the water level showed no significant effects against the MPP+ level. The results of the experiment may be supported by the reported mechanism of 6-shogaol on inhibiting the inflammatory signaling pathways activated by MPP+. Future research may be conducted by using pure 6-shogaol and 6-gingerol to treat or prevent PD.
The plant Zingiber officinale is widely used throughout Asian cuisine and is renowned for the spicy flavor that it adds to dishes. Zingiber officinale, commonly known as ginger, has also been used as an herbal medicine in Eastern cultures to treat a variety of illnesses such as stomach and gastrointestinal disorders (Ok and Jeong, 2012). When it comes to the use of ginger in Eastern medicine, one study has reported “a moderate to high level use in clinical settings” (Cho et al., 2018). Recently, there have been reports that suggest Zingiber officinale or more specifically, 6-shogaol, a pungent compound found in ginger that gives its spice, may have anti-inflammatory and neuroprotective capabilities (Park et al., 2013). Parkinson’s disease (PD), in particular, attacks the brain’s motor abilities and neurons in the midbrain. It is hypothesized that if 6-shogaol has been shown to have neuroprotective abilities, then it is possible that it could help to prevent the onset of PD.
PD is the second most common neurodegenerative disease, affecting around 10 million people across the world. Despite its prevalence, there is no cure and the cause of PD is still unclear. Since the cause itself is unknown, ways to prevent PD are also quite ambiguous (Mayo Clinic, 2018). Therefore, any research or drug development involving the prevention of PD would be immensely beneficial, especially if it involves products or material that can be found as commonplace as in a local grocery store. Currently, some published studies have successfully tested the abilities of 6-shogaol in in vivo and in vitro PD models (e.g. Park et al., 2013). However, these reports have used the purified natural product, 6-shogaol, not a direct extract from ginger. Herein the current report has tried to focus on the ability to replicate similar beneficial results in a kitchen, hopefully as much as in a laboratory setting, essentially simulating the ability to prevent PD while cooking with ginger at home.
The independent variable of the experiment is the level of 6-shogaol taken from ginger using different liquids. When cooking in a kitchen, many different ingredients can be used, e.g. water, alcohol, and oil, all of which can also be used to extract 6-shogaol from ginger. When it comes to cooking with ginger, it has been prepared in a variety of ways. Traditionally, ginger is cooked with many Chinese dishes, such as stir-fries, which use high heat with oil. However, as ginger incorporates more into mainstream Western culture, it can find itself next to water in hot soups or with alcohol in meat dishes. Extracting 6-shogaol from ginger may take heat and an extraction substance, therefore, part of the experiment was to assess which substance is capable of extracting the most 6-shogaol (Ok and Jeong, 2012). However, it is important to note that 6-shogaol is not the only compound in ginger. Among the possible components in ginger, closely related to 6-shogaol is 6-gingerol, which when dehydrated produces 6-shogaol (seen in figure 1). 6-Gingerol has been used more frequently than 6-shogaol across medicine, but the two share similar qualities (ScienceDirect, 2018). Therefore, the independent variable for this experiment is the different solvents to extract 6-shogaol and 6-gingerol from ginger.
Figure 1. The chemical transformation between 6-gingerol and 6-shogaol.
The dependent variable is the level of protection that the extract affords against PD in the cells. PD affects the substantia nigra of the midbrain, a section of the brain that controls dopamine releases and motor functions. MPP+, or 1-methyl-4-phenylpyridinium, is a neurotoxin that can cause Parkinsonism and eventually cell death. In this experiment, MPP+, as previously mentioned, has been used to simulate PD within cells and allow studying PD in the experiment. To quantify the presence of PD, a biomarker, or an indicator of a specific disease, can be measured. In particular, this experiment has been focused on measuring the absorbance of nitric oxide (NO), the dependent variable (DV) and a biomarker of PD. In essence, the lower the absorbance of NO, the more the cells would be protected from neurotoxic effects. NO is measured at 540 nm, and its absorbance is how the DV is measured (Park et al., 2013).
In this experiment, different levels of the independent variables were given the neurotoxin MPP+, except for the negative control. The levels were 95% ethanol-based extraction, water extraction, only MPP+, and the no treatment level. Ethanol is commonly used for herbal extraction because of its compatible solubility to a lot of chemical entities. Water is a polar solvent that is used in many extraction related experiments. The other two levels were the positive (only MPP+) and negative (no treatment) controls, used to show the possible outcome of the two testing levels. The MPP+-only level simulated a cell with parkinsonism-like symptoms. On the other hand, the level receiving no treatment represented a healthy cell that should contain no symptoms of PD.
To summarize, the purpose of this experiment was to evaluate different methods of 6-shogaol and 6-gingerol extraction and the extracts’ effects on the protection of dopaminergic neuronal cells simulating Parkinsonism in cells. The independent variable levels, as stated previously above, were 95% ethanol with MPP+, water with MPP+, cells with MPP+ only, and no treatment at all. The levels have been chosen because they have been used in common cooking methods with ginger. The positive control was the cell group treated only with MPP+, while the negative control was the group with no treatment. The dependent variable, the absorbance of NO, was measured in absorbance units (AU) using a spectrophotometer. It was hypothesized that the 95% ethanol group would result in the most neuroprotection for the PD cell model. This was based on the fact that 95% ethanol has been most successfully used for extraction purposes regarding 6-shogaol from ginger (Ok and Jeong, 2012).
Study Methods and Materials
Reagents and Instruments
All reagents (including 6-gingerol, 6-shogaol, and MPP+) were purchased from Sigma-Aldrich or as otherwise stated. Rat embryonic mesencephalic dopaminergic neuron was purchased from Cleprogen, CA. Griess reagent kit (G7921, ThermoFisher, Grand Island, NY, USA) was applied for nitrite quantitation of extracellular NO levels. MPP+ was applied as the positive control compound. HRMS (ESI) analysis was performed on the Perkin Elmer AxION 2 TOF mass spectrometer. HPLC analysis of the samples was achieved on Varian ProStar 210 system on Microsorb-MV 100-5 C18 column (250 mm × 4.6 mm) at 254 nm eluting with acetonitrile (0.01% TFA):water, 40:60 – 0:100 (1 & 3) or 45:55 – 0:100 (2), at 0.65 mL/min over 30 min. Nitric oxide reading was recorded in the microplate reader (FlexStation3, Molecular Devices) and peak values were obtained using SoftMaxPro software (Molecular Devices).
Several large pieces of ginger were bought from a local grocery store and washed with water. Total 250 grams of ginger was sliced into circular pieces about 3 centimeters in diameter using a knife. The pieces were then placed onto a baking paper over a metal tray and baked in an oven at 80° Celsius. Every half hour, the tray was taken out and the weight of the ginger was measured to find out how much water had evaporated. Once the weight did not reduce anymore, the tray was removed from the oven. The ginger pieces were ground using a blender into 30 grams of powder. Five (5) grams of the powder was mixed with 50 mL of 95% ethanol, which was heated at 80 degrees Celsius and a pH of 4 for 24 hours, to produce the first extract and level of the independent variable (IV). The pH was adjusted using 5 mL of acetic acid and handled with gloves. The ginger powder extract was then filtered through a layer of diatomaceous earth. Afterward, the filtrate was concentrated to dryness using a rotary evaporator and 980 mg total of oily residue was obtained. After the first powder level was completed, the extraction and filtration process was repeated with water, the second level, in a similar fashion, and 201 mg total of oily residue was obtained. After the extractions were completed, they were then tested for 6-shogaol and 6-gingerol contents using high-performance liquid chromatography (HPLC) and mass spectrometry (MS) before the cell-based studies.
Rat midbrain dopaminergic neurons were purchased from Celprogen and cultured for 3 days in a Biosafety Level 1 laboratory at Biotech One at Virginia Commonwealth University School of Pharmacy (Celprogen, 2018). Briefly, after thawing the cells following the protocol coming along with the shipment of cryovial of cells, the 500 µL of cells was added to flask pre-coated with Rat Embryonic Mesencephalic Dopaminergic Neuron Extra-cellular Matrix with 15 mL of Rat Embryonic Mesencephalic Dopaminergic Neuron Complete Growth Medium. The cells were then incubated in the flask in a 370C in 5% CO2 humidified incubator. 100% Media change was performed every 48 hours. Sub-culturing ratio was 1:2. Once the cells were grown to confluence (75-80 % of a petri dish was covered), they were separated into four groups (one for each level). Each group was then further split into six wells, one for each trial. Two of the cell groups were treated with either the ethanol or water extract at defined concentrations (see Results section) for 1 hour. After one hour, the two groups were incubated with MPP+ at a concentration of 0.01 µM for 23 hours. The third cell group was only incubated with MPP+ for 23 hours without any extraction, as the positive control. The last group was given no treatment for the 24-hour period, as the negative control.
Once the 24-hour incubation period was complete, each cell group was tested for absorbance of nitric oxide (NO) by collecting the supernatants separately from testing wells. The accumulated level of NO in culture supernatants was measured using the colorimetric reaction with the Griess reagent for 10 min at room temperature in the dark. Absorbance was measured at a wavelength of 548 nm in the microplate reader (FlexStation3, Molecular Devices, Sunnyvale, CA, USA) and concentrations of NO were determined by NaNO2 standard curve (see Supporting Information).
Once the experiment was complete, the cells were bleached, diluted, and put into lab specific containers for the research institution to be disposed of. The MPP+ was put in the proper biohazard container to be later disposed of by the institution. Finally, statistical analysis (mean, variance, t-tests) was carried out on the collected data using Excel.
All handling of the cells and MPP+ was done in a biosafety cabinet. MPP+ is an irritant to skin at the experimental dose, and can cause Parkinsonism at higher doses. Gloves, goggles, and a lab coat were constantly worn throughout the experiment. A permission form was signed by a parent of the student that indicated that they had read and understood the risks and possible dangers involved in the research and that they consented to their child participating in this research.
Experimental Results and Analyses
Mass Spectrometry and High Performance Liquid Chromatography Analyses
It was critical to evaluate how successful each extraction was, which would directly influence the observed neuro-protective effects. As shown in the mass spectra (Figures 1 and 2), the 95% ethanol level contained a significant level of 6-shogaol, represented by the peak at 299.1621 (M+Na)+ on the graphs, and 6-gingerol, represented by the peak at 317.1731 (M+Na) + on the graphs. On the other hand, the water level only showed a high level of 6-gingerol without significant level of 6-shogaol.
Figure 2. 95% Ethanol Mass Spectrometry Reading (6-shogaol at 299.1621)
Figure 3. Water Mass Spectrometry Reading (6-gingerol at 317.1737)
To further quantify the amount of 6-shogaol and 6-gingerol, an HPLC analysis was conducted by first establishing the standard curve of each compound, then evaluating the contents of each sample (see Supporting Information).
As shown in Table 1, the ethanol level managed to extract 49.70 mg of 6-shogaol and 94.50 mg of 6-gingerol out of 5 grams of the ginger sample. On the other hand, it did not show the presence of 6-shogaol for the water level, but rather 9.38 milligrams (mg) of 6-gingerol out of 5 grams of the ginger sample.
Table 1. Total amount of 6-shogaol and 6-gingerol in the 5 grams of ginger used for each trial.
Data Collection and Analysis
The effect of different methods of extraction of 6-shogaol and 6-gingerol from ginger on the protection of dopaminergic neurons in a Parkinson’s disease (PD) model was studied and the results of the statistical analysis were shown in Table 2 and Figure 4 below. Due to the fact that different solvent systems gave significantly different extraction capacity, the components of each tested extraction sample were normalized based on the concentration of 6-gingerol. In detail, from the 95% ethanol extract (980 mg of oil), 20.0 mg of oil was dissolved in the 1 mL of testing buffer to give us a stock solution with the concentration of 6-gingerol at 6.6 µM. Similarly, from the water extract (201 mg of oil), 41.3 mg of oil was dissolved in the 1 mL of testing buffer to give us a stock solution with the same concentration of 6-gingerol. For the ethanol extract, the final concentration of 6-gingerol in the cell incubation solution was 0.10 µM and the final concentration of 6-shogaol was 0.056 µM, respectively, based on the dilution factor. The final concentration of 6-gingerol in the cell incubation solution was 0.10 µM for the water extract as well.
Table 2. Statistical analysis of the effect of different methods of extraction of 6-shogaol and 6-gingerol on the protection of neurons in a PD model.
Figure 4. A comparison of the absorbance of NO from each group produced at the IV levels (data normalized based on the blank group).
Table 2 summarized the NO absorbance data collected from the experiment and the statistical analysis results. The means, ranges, variances, standard deviations, as well as the number of trials were all shown in Table 2. There was no unit for any of these points because the dependent variable measured was UV absorbance, which carries no unit. The data shown in the Figure 4 was normalized by taking all the means and converting them into percentages of the cells receiving no treatment (blanks or negative controls). This allowed the IV levels to be compared directly to one baseline.
The research hypothesis was that the 95% ethanol extract would provide better neuroprotection of PD cells. Protection was shown by a lowering in absorbance of nitric oxide (NO), a biomarker for PD. Comparing the means of 95% ethanol’s absorbance (0.173) and water (0.257), the independent variable (IV) levels, to the MPP+’s absorbance (0.323), the positive control, showed that the IV levels both had lower absorbance of NO, seen in Table 2. Shown in Figure 4, 95% ethanol had better protection against NO in terms of UV absorbance. Therefore, the results demonstrated that the research hypothesis was supported by the data.
The standard deviation (SD) was relatively high throughout the levels. However, no data point was outside of the 2 SD range, so there were no outliers in the data. The null hypothesis in the experiment was that the different methods of extraction would have no statistically significant differences in protecting the cells from MPP+. The t-test performed was at a level of significance of 0.05 with 10 degrees of freedom. The calculated t-values for the ethanol comparison (3.387) were higher than the t-value from the table of 2.228. This implied that the null hypothesis should be rejected for this level. Meanwhile, the water comparison’s t-value (1.444) was lower than the table value of 2.228, which implied that the null hypothesis should not be rejected. The data was statistically significant for ethanol, but not for the water levels. These comparisons assessed whether a statistically meaningful neuroprotective effect was observed, since MPP+ is the control. The other t-values were shown in Table 2 but were not as crucial to the experiment.
Discussion and Conclusions
The purpose of the experiment was to investigate the effects of different methods of extraction of 6-shogaol along with 6-gingerol from ginger on the protection of dopaminergic neurons in a Parkinson’s disease (PD) model. Two methods of extracting these two natural products from ginger (95% ethanol and water) were tested for their ability to protect neurons from MPP+. The research hypothesis was that if 95% ethanol was used to extract 6-shogaol from ginger, then its extract would provide better protection of the neurons. The results showed that 95% ethanol did show greater protection for the neurons from MPP+ and therefore, the research hypothesis was supported by the data.
The data from the results indicated that both extracts from 95% ethanol and water had positive neuroprotective effects. When a t-test was performed on the data to determine the data’s significance, the data was shown to be significant when comparing the 95% ethanol extraction and the positive control (MPP+ only), which was the most important tests since they directly relate to the neuroprotective effects of the extract against MPP+. The t-tests showed that the data for the IV levels and the control (MPP+) were significant in the case of 95% ethanol, implying that the results were likely due to the IV. Meanwhile, the t-test for water extract versus the MPP+ was not statistically significant, so it seemed that the results for that variable were due to chance or other causes than the IV.
Trying to prevent or treat PD with herbal medicines dates back thousands of years to the times of the ancient Chinese and is still practiced around the world (Kim et al., 2012). More specifically, 6-shogaol was found to exhibit neuroprotective abilities on dopaminergic neurons when the neurons were dosed with MPP+ (Park et al., 2013). One study in particular, found the optimal extraction method of 6-shogaol from ginger was with 95% ethanol (Ok and Jeong, 2012). These results can be explained by the proposed neuroprotective abilities of 6-shogaol. When MPP+ is induces reactive gliosis, or the activation of glial cells functioning as macrophages (microglia), it contributes to neuronal degeneration because the activated microglia will produce many inflammatory cytokines (small, cell signaling proteins) as well as elevated levels of nitric oxide (NO). The elevated levels of NO will often result in cell death. 6-Shogaol is able to inhibit those inflammatory pathways and therefore protect the cells (Park et al., 2013).
One possible reason that the water level did not succeed as well is that the water mostly extracted 6-gingerol, which has been reported carrying lower anti-inflammatory abilities than 6-shogaol (Ok and Jeong, 2012). Water extracted 6-gingerol but not 6-shogaol possibly because 6-gingerol is a more polar substance than 6-shogaol, and has better solubility in the water. In addition, under acidic conditions, 6-gingerol is more stable in the water media than it is in the ethanol one.
Compared to the literature (Park et al., 2013), the results of our experiment showed relatively lower activity of 6-shogaol in protecting neuronal cells. One explanation could be that the extraction yielded a mixture of a variety of components, which may interfere with the activity of 6-shogaol, whereas the literature report used the pure chemical entity, 6-shogaol.
Several improvements may be implemented in this experiment. First, when cutting up the ginger for drying, the slices varied in size, (though most were around 3 centimeters in diameter), which may have caused the drying process to slow down. Second, some extracts may have got lost between extractions due to mishandling of flasks. For these errors, better management of equipment and newfound knowledge would clear up most of these issues. Finally, regarding cell management, the neurons took a long time to culture to confluency, so the cells could have been obtained earlier to enhance this process.
Looking into further research, ginger has other active components that can be looked into, for example, 6-gingerol, which the water level extracted significant quantities of, or zingerone. Zingerone, like 6-shogaol, has seen some benefit in protecting against PD (Parkinson’s Foundation, 2018). Second, tumor necrosis factor alpha is another biomarker of PD that may be applied for further PD research (Leal et al., 2013), and to compare with the results from NO levels. Finally, the field of herbal medicine can and should be considered as a promising therapeutic and medicinal option for several other diseases, e.g. Alzheimer’s disease.