The Effect of Salvia miltiorrhiza on the Memory of Dugesia dorotocephala

Abstract

In this experiment, the use of sage Salvia miltiorrhiza was assessed on its ability to improve memory retention and overall effect on the organism population. Salvia miltiorrhiza has been found to improve memory retention through aromatherapy and its active chemical properties to improve cognitive and neurological performance. Within this experiment, planarians were used as a neurological template to effectively showcase the effects of the properties of Salvia miltiorrhiza. The planarians were separated into five groups: Group A, B, C, D, and E, each with 10 individuals. Group A was the control group, B was 0.001g/mL, C was 0.002g/mL, group D was 0.003g/mL and group D was 0.004g/mL. They were treated in their respective sage concentrated solutions of 30 mL for 15 minutes on Mondays. It was predicted that the higher the tested sage concentration was, the better the memory retention and performance would be produced by the planarians. This experiment would provide the after treatment results of the memory retention examination and be compared to the before treatment values of the memory retention examination through the use of Excel and ANOVA with Tukey HSD and t-test. Results from trial 1 demonstrated that sage solution improved the planarians' ability to retain information. When compared to the values of the control group A and their pretreatment values, each group B and C displayed substantial variations. The differences between treatment groups B and C, however, were not significant, and the results for groups D and E remained inconclusive. [results/conclusion/issues/future work]

Introduction

Rationale

Sage is known widely for its application in culinary, fragrance, cosmetics, and medicinal purposes. It is recognized to have properties that strengthen immunity and reduce inflammation for many disorders. Testing sage on the memory of Dugesia dorotocephala can provide aid to humans to attain and remember specific information more efficiently. Though research about it has been conducted, it is not commonly known across the media. This study will be able to solidify the affirmations and benefits of sage on animal models and human brains. Memory may be improved by the use of sage aromatherapy and establish a more accessible alternative to memory repairment.

Sage Salvia miltiorrhiza

Several Salvia species were shown to have a wide range of active chemicals that may improve cognitive function and offer protection from neurodegenerative diseases, according to in vitro and animal studies. Salvia plants have historically been used to treat a variety of illnesses, and according to traditional wisdom, they may also be beneficial for neurological and cognitive disorders (Lopresti, 2017). Research has shown that a variety of Salvia species and each of their active components affect a number of biological functions that may have an impact on neurological and cognitive performance. Studies conducted in vitro, on animals, and in humans in the early stages have all supported the claim that salvia plants improve cognitive function and protect against neurodegenerative diseases. Nonetheless, additional study is needed in a number of areas.

Salvia miltiorrhiza has been used to treat a number of conditions, such as cancer, bone loss, hepatocirrhosis, coronary heart disease, cerebrovascular disease, Alzheimer's disease, Parkinson's disease, and renal insufficiency. The prevention and treatment of the said diseases derive from the active chemical components in Salvia miltiorrhiza. From SM, 49 diterpenoid quinones, 36 hydrophilic phenolic acids, and 23 components of essential oils were extracted and characterized, collectively representing a broad range of secondary metabolite groups. The main bioactive components of SM, according to chemical and pharmaceutical studies, are hydrophilic phenolic acids and diterpenoid quinones (Chun-Yan, et al, 2015). Two series of phenanthrofuran-10, 11-dione and phenanthrofuran-7, 11-dione diterpenoid quinones were identified. As condensation derivatives of caffeic acid with various linkage types and numbers, hydrophilic phenolic acids are thought of. While the majority of the essential oils are derived from flowers, these two types of active chemicals are typically isolated from roots. Therefore, Salvia miltiorrhiza can have astounding effects on multiple cognitive and neurological conditions (Chun-Yan, et al, 2015).

Planaria

Dugesia dorotocephala, commonly known as brown planaria, is the subject being investigated in this study. It is a planaria species that is a commonly used model for the research of developmental biology that displays behavior for data collection for pharmaceutical benefits. Planaria are acoelomates meaning they can live on land and in water while lacking a body cavity. Planarians feed on a variety of foods, such as fresh beef liver, hard-boiled egg yolk, pieces of earthworm, crushed aquarium snails, or Lumbriculus, a type of worm (Carolina, 2023). The planarians thrive with the aid of Poland spring water because tap water can contain metal ions that may be detrimental to planarians.

All vertebrates and many invertebrates share a body plan, and planarians are the simplest animals to display it. They have bilateral rather than radial symmetry, dorsal and ventral surfaces, and a rostrocaudal axis with a head and a tail, as well as specialized sense organs and a collection of nerve cells in the head. Planarian neurons exhibit characteristics of vertebrate neurons, such as a multipolar shape, dendritic spines with synaptic boutons, a single axon, expression of vertebrate-like neural proteins, and relatively low spontaneously generated electrical activity. These characteristics set planarian neurons apart from advanced invertebrate neurons (Buttarelli et al, 2008).

Short Term Memory vs. Long Term Memory

Short term memory is defined as systems that provide retention of limited resources for a limited time period (Vallar, 2017). On the other hand, long term memory are systems that provide retention of unlimited information for an indefinite period of time. In biological systems, memory is a crucial component of learning and decision-making. While long-term memory requires the expression of extra genes during learning, short-term memory includes the covalent alteration of pre-existing proteins. After a while of recollections of repeated stimulation, significance is transferred from short-term memory to long-term memory (Chang et al., 2011). This finding enables a molecular analysis of the induction and preservation of long-term memory (Goelet et al., 1986). The assumption that short and long term memory are functionally and neurobiologically distinct would be contraindicated if short term memory were to be compared to long term memory, which is a typical comparison. Short-term memory must accommodate the storage of many tokens of the same kind, the binding of variables, and the memory for previously unknown information (Norris et al., 2017).

Brain Regeneration of Planarian

While there is no direct evidence that sage can assist in the regeneration of the hippocampus in planaria, it is possible that the herb's memory-enhancing properties could indirectly assist in the retention of memories by the planarian. As it has been discovered that planarians can retain memories even after their heads were sliced off, it is plausible that incorporating sage into their environment could potentially aid in this process. However, further research is needed to determine the exact mechanisms by which sage could assist in the retention of memories in planaria.

Therefore, planarians were used for this experiment to assess the memory of the planarians after head regeneration. Planaria is known for its ability to regenerate their entire body, therefore including the regeneration of the hippocampus and memory. The planarian system may provide an opportunity to examine brain regeneration and memory in the same animal. Planarians have environmental memory that lasts for at least 14 days, which is sufficient time for the brain to rebuild. This is a demonstration of long-term memory (Shomrat et al., 2013).

After growing a new head, beheaded planaria show signs of memory retrieval in a savings paradigm. Shomrat et al 2013 suggested planaria as a crucial new model species for mechanistic studies of the biological tissues' recording of particular memories. Also, this technique is probably going to have a big impact on adult human stem cell treatments for degenerative brain illnesses. Moreover, their memory endures long enough for complete regeneration following amputation. The use of planaria as a model organism for current learning and memory research has a lot of potential because of this straightforward and promising method. Significantly, planarians are the only organism with a molecularly traceable mechanism that allows for the simultaneous study of memory and brain regeneration. This is a key benefit since it enables the investigation of novel hypotheses about the function of self-organization and epigenetic pathways in memory encoding and brain development and regeneration.

T-Maze Development

Figure 1, T-Maze construction by (Rice Jr et al., 1957)

Rice Jr et al., 1957, was able to use T-Maze to analyze the behavior of reactive inhibition. Lepley and Rice have extended this concept of reactive inhibition to account for variability of maze behavior in the paramecium. They stated that when any reaction occurs, the probability of its later occurrence is reduced. This current experiment presented in this article adapted this maze for memory testing as demonstrated effectively by Rice Jr. and colleagues. There is currently just one section of the multibranched maze, since including the whole maze may seem complicated in the situation of this experiment. The T-Maze also allows the planarians to rely more on memory retention rather than sight, therefore ensuring the accuracy of the experiment.

Other Studies

Rosemary (Rosmarinus officinalis L.) and sage (Salvia officinalis L.) are similar in both antimicrobial and antioxidant properties (Bozin et al., 2007). For instance, both rosemary and sage derive from the family Lamiaceae. Both rosemary and sage also demonstrated stimulatory effects of and affected brain wave activity, autonomic nervous system activity, and mood states. A study by Filiptsova et al. 2018 tested the use of rosemary oil on the short term memory of humans. Before and after the treatment, subjects had taken their electroencephalograms, which are similar MRI scans of brain activity. The results showed that following aromatherapy, blood pressure, heart rate, and breathing rate all significantly increased. The volunteers reported feeling more rested. The stimulating effects of rosemary essential oil on the brain wave activity were also revealed by an analysis of EEG. However, sage is still different from rosemary, such that rosemary is more widely known and applied more than sage. Rosemary and sage also possess different structural components that contribute to cognitive growth. For example, rosemary possess diterpenes, which are derived from the terpenoids or mevalonate pathway and hence composed of repeating 5-carbon backbone skeleton, isoprene units (Habtemariam, 2016). While sage contained 1,8-cineole, camphor, α-thujone, β-thujone, borneol, and viridiflorol (Hamidpour et al., 2014). Although they possess different structures, they both demonstrate similar effects on cognitive growth, sharing a major constituent carnosic acid.

Another study by Tildesley et al. 2003 also shows the effect of the Spanish sage on the memory of participating healthy young adults. The current study's findings suggest that healthy young adults who consume single doses of S. lavandulaefolia (sage) may have a dose-dependent improvement in memory. The impact on instantaneous word recall was the most notable. This shows that Sage ingestion allowed the quickest memory retention within word recall. As acetylcholine is essential for cognitive processes like learning and memory, it's possible that the memory improvement seen in these results is a result of Salvia's anticholinesterase properties. However, given Salvia's rich pharmacology, which includes many other potential beneficial mechanisms, more mechanistic studies are required. These findings provide the first comprehensive proof that salvia can modify cognition acutely in healthy young adults. Therefore, sage can provide novel bioengineering in improving memory retention, and continuous research on other varying sage species should be explored to expand the horizon of sage medicine in cognitive growth.

In this experiment, the use of sage solution on the memory retention of neurologically structured organisms such as humans and planarians was assessed. Due to the limited amount of scientific studies on the direct impact of sage on memory retention, this experiment highlights novelty and seeks more originality than others. Additionally, this experiment suggests the inclusion of more species of sage in order to test its whole effect on the cognitive development of neurological organisms.

Materials and Methods

Maintaining Planaria

The planarias underwent habitual adaptation for about a week straight in order to recover from delivery difficulties and strains. Before and after operations, the countertops were bleached, hands should be washed, and gloves, goggles, and aprons should be worn. Poland Spring water was used to fill two Petri dishes with 30mL each, then one cubic centimeter of chopped beef was added into each Petri dish. The planarians were separated into each Petri dish evenly. The feeding time was around 30 minutes and then the planarians were retransferred to new separate Petri dishes with 30mL Poland Spring water. The old Petri dishes were disposed of to prevent infections and unwanted bacterial growth.

Preparation of Solution

The countertops were bleached with 10% vol bleach before and after the operation. To prepare the Sage solution, 0.3g of sage powder was added and then mixed to 300mL of Poland Spring water. After diffusing the powder solute to the water solvent, transfer the mixture to a Erlenmeyer flask and label it with a masking tape as group B. For the following groups, group C, D, and E, use 0.6g, 0.9g, and 1.2g of sage powder and 300mL of Spring water each, respectively. This is also shown in Table 1. After preparing all required solutions for all groups, the flasks were sealed with plastic wrap or parafilm.

Table 1. Experimental Setup

Group	Concentrations (g/mL)	Sage Extract (g)	Spring water (mL)	Planaria
A	0	0	300	10
B	0.001	0.3	300	10
C	0.002	0.6	300	10
D	0.003	0.9	300	10
E	0.004	1.2	300	10

Treating Planarian with Sage Extract

Before completing this section, the preparation of the solution was made first. This way, there was time for efficient and simple treatment administration. After a week of adapting to the synthetic habitat, the planarians were treated with the specific sage solution of each group for 30 minutes each on Monday according to Table 1. The countertops were bleached and gloves, goggles, and aprons were worn beforehand. Then, 30mL of each solution was transferred to separate Petri dishes, with each Petri dish labeled with its assigned group letter. Afterwards, carry the planarians using a pipette into each Petri dish carefully. Concluding treatment, the planarians were carried from each group into Poland Spring water individually, with the Petri dishes labeled. The water should not be changed for a day, and stored at room temperature.

T-maze Test

The countertops were bleached beforehand, and gloves, goggles, and aprons were worn. To create the T-Maze, obtain one piece of 3 x 0.5 in², two pieces of 1.75 x 0.5 in², and two pieces of 1.25 x 0.5in² of non-clear High-Density Polyethylene. Then using the water-resistant glue, glue the 3 x 0.5in² for the top of the T on the Petri dish, then glue one of the 1.25 x 0.5in² pieces below it on either side of the Petri dish, leaving a space in the middle. Lastly, glue a piece of the 1.75 x 0.5in² piece perpendicular to the 1.25 x 0.5in², and repeat this for the other side. This process was repeated three times to make enough T-Mazes for use (Figure 2). The structure of the T-Maze was based on the study by Rice Jr. and colleagues, where a multibranched maze was utilized. However only one branch was selected to use as reference in creating the maze applicable to this experiment.

Figure 2, T-Maze scale print

This process of the procedure was performed a week after treating the planarians with the sage solution. For conducting the before values of memory retention, treatment was not administered for the planarians yet. The countertops were bleached before and after the procedure and gloves, goggles, and apron should be worn. The T-Maze was prepared by rinsing it thoroughly with water. Then, one vertical centimeter of Poland Spring Water was filled into the maze. Then one cubic centimeter of raw minced beef was placed on one end of the T-Maze. This was used as the role of reward to motivate the planarians to finish the maze and receive a redeemable souvenir.

The countertops were bleached before and after the procedure and gloves, goggles, and apron were worn. If needed, synthesize additional sage solutions for the treatment medium. After exposure to the treatment, a pipette was used to transfer the planarians into the stem of the T-Maze. Data was collected on the turn the planarian takes, and the time used for the planarians to complete the maze from the second it was placed into the maze. After collecting the data, the planarians were replaced back into their Petri dishes with Poland Spring water. This was conducted separately for each group.

Experimental Trials

The planarians were assessed on their ability to improve memory with the guidance of sage aromatherapy and solution. The T-Maze assessment was performed 24 hours after treatment for Day 2, 48 hours from treatment for Day 3, 72 hours from treatment for Day 4, and then 96 hours from treatment for Day 5. For the original experimental trial and Days 2, 3, and 4, food reward was placed at one end of the maze for all days. Then on Day 5, the planarians were required to complete the maze without any outside stimuli; this consolidated the memory benefit of the sage solution on neurological functions.

Population count

The planarians have become accounted for population changes after application of the treatment sage solution. Throughout the trials, the overall condition of the population was recorded. Analysis of the overall change in population allows for the overall assessment of the additional treatment of the sage solution to the planarians. Ergo, if the population shows a positive trend, then the administration of sage was able to have beneficial effects on all aspects of the neurological model. If there is a negative trend, then it can indicate that the treatment size may be out of range and further research should be conducted in order to understand the effect of the treatment size on the overall health.

Data Analysis

For one, the planarians were recorded and their time to complete the maze was collected. They were recorded the second they are placed in the maze, and the video was analyzed afterwards for explicit timing for graphical and data analysis. The time for each individual was collected then averaged to form the distinct group time. All values were stored on MS Excel spreadsheets. The mean values were calculated by the standard function of Excel. This was demonstrated in a bar graph, with the x-axis representing the groups and the y-axis representing the time. ANOVA tests were used to compare the results between the groups and their time. ANOVA followed up by Tukey HSD was conducted to determine the significant difference using www.astatsa.com. This was done before the treatment and with the food reward to understand the memory of the planarians before treatment with an outside stimulus. Then, after treatment, this process was repeated and analyzed for resulting values. By the use of the t-test, the comparison between before and after values of the groups was assessed to understand the improvement in the memory of the planarians. The t-test was organized by placing before and after treatment values of each group with each other to more directly analyze the improvement in memory based on each concentration of sage.

The before-and-after treatments were also plotted on a bar graph, although no ANOVA was conducted for before values, since there would be no factor that would change the significance between the groups. The paired t-test was conducted to determine the significant difference between the memory values but at different times: before and after treatment. The mean values for each group were calculated by the standard function of Excel. All values for before and after values were stored on MS Excel spreadsheets. ANOVA was used for after treatment values because significance between the groups were demonstrated in the difference in sage concentrations they were administered. ANOVA followed up by Tukey HSD was conducted to determine the significant difference using www.astatsa.com. This would support the favorable range of sage concentration in benefitting the memory the most.

The percent of planarians that scored under three minutes were also analyzed. There was a timer set for three minutes and the amount of planarians that had completed the maze under that time was calculated over the total individuals in the group. The mean values for each group were calculated by the standard function of Excel. All values were stored on MS Excel spreadsheets. This was also demonstrated in a bar graph, with the x-axis representing the groups and the y-axis representing the percentage calculated. ANOVA test was also used to determine the significance of completing the maze under three minutes among the groups. ANOVA followed up by Tukey HSD was conducted to determine the significant difference using www.astatsa.com.

Population was also analyzed from each treatment group by the end of each week. All values were recorded and stored on MS Excel spreadsheets. This was represented in a line graph, with the y-axis representing the population in numbers and the x-axis representing the time in weeks. Each line of the graph had represented each group of the initial population of ten individuals. This graph thus showed the overall population assessment of the planarians under treatment conditions. If there is a population decay, it can be concluded that the concentration in sage was lethal while if there is a population growth, it can be concluded that the concentration in sage was beneficial in the preservation of and development of the overall population.

Results

Figure 3, Trial 1 After treatment values with groups A, B, and C. Values represent mean ± SD. Values with different superscripts are significantly different, p<0.01.

This graph showcases the values of the groups after treatment with the exception of group A, since it is the control group. Each bar represents the mean time plus and minus the standard deviation value of each group. ANOVA followed up by Tukey HSD was conducted to determine the significant difference using www.astatsa.com. Groups B and C were both significantly different from control group A. However, there was no significant difference between groups B and C. On average, group A had a mean time of 2.25 minutes while groups B and C had a mean time of 1.75 minutes.

Figure 4, Trial 1 Comparison between Before and After values.

The graph contains before and after values for groups A, B, and C only due to complications with groups D and E. Groups D and E may have been handled haphazardly, resulting in the elimination of the members and thus the groups. However, when looking at the bars of treatment groups B and C, there was a significant reduction in the time spent in the maze after the sage solution treatment. Each bar represents the mean time plus and minus the standard deviation value of each group. A t-test was conducted for both groups B and C to analyze the significance between the before and after values. The p-value for group B was 0.000327 which is significant since p<0.01. The p-value for group C was 0.001664 which is also significant since p<0.01.

Figure 5, Trial 1 of the total percent of planarians scoring the maze under 3 minutes.

This line graph shows the amount of planarians that completed the maze over the total amount of planaria in each group. Each line represents their respective groups. According to the graph, control group A had the lowest percentages scored compared to other groups B and C. Group B and C had similar results of percent scored under three minutes over the course of four days. When the mean of the percent scored under three minutes were taken, both groups B and C had a mean percent of 82.5% score the maze in the given time frame over the course of the days of the experiment.

Figure 6, Trial 1 population growth of the planarian groups.

Discussion and Conclusions

The application of sage solution on planarian memory recall resulted in a positive connection. In trial 1, groups B and C were able to spend the maze for around a minute less than the control group and their pre-trial values. In comparison to their pretreatment values, the outcomes of both groups are dramatically different. The majority of each group's memory capacity allowed them to remember enough information to finish the maze in less than three minutes. The ANOVA test between group B or group C does not yield significant findings, despite the fact that they differ significantly from the control group and from their baseline values. This demonstrates that there is no discernible difference between the treatment value of 0.001 g/mL and the treatment value of 0.002 g/mL.

It is necessary to conduct another experiment after concluding that Groups D and E in Trial 1 were dead after treatment. This conclusion may have been reached due to a lack of rigorous instruction. This conclusion may have resulted from several actions, including neglect, cross contamination from objects on the lab table, sedimentation in the sage solution treatment, and other such actions. Future experiments should carefully consider rigorously cleaning the utensils, preparing more sage solutions to prevent significant sedimentation, and closely monitoring the treatment progress in order to stop this from occurring.

Acknowledgements

We thank Ms. Amy Zhu, Dr. L. Wang, Dr. J. Cohen, Dr. S. X. Lin, Mr. Z. Liang, Ms. N.

Jaipershad, Dr. D. Marmor, and the FLHS Science Department for funding.

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