Effects of Trichosanthes Root Extract on Regeneration Rate, Behavior, and movement Activity in Dugesia tigrina

Abstract

Planarians of the species Dugesia tigrina are established model organisms for studying stem cell-driven tissue regeneration due to their pluripotent neoblasts and low husbandry cost. Trichosanthes kirilowii root, a herb used extensively in traditional Chinese medicine, contains bioactive compounds—including trichosanthin, flavonoids, and polysaccharides—with documented antioxidant and anti-inflammatory properties. Because oxidative stress and inflammation are known barriers to tissue repair, we hypothesized that exposure to Trichosanthes root extract would accelerate regeneration in D. tigrina. Planarians (n = 10 per group) were assigned to one of four treatment concentrations (0%, 0.01%, 0.03%, or 0.05% v/v extract) for seven days, then transversely amputated. Head and tail fragments were monitored for 11 days across two independent trials, with body length, eyespot regeneration, locomotor activity, c-shape behavior, and touch responsiveness recorded at regular intervals. Statistical analysis used one-way ANOVA followed by Tukey's HSD post hoc test. Although statistically significant between-group differences in tail regeneration length emerged at several time points (Day 1: p < 0.01; Day 3: p < 0.0001; Day 5: p < 0.02; Day 10: p < 0.0001; Day 11: p < 0.01), no dose-dependent trend was observed and no differences were detected for eyespot regeneration, locomotor activity, c-shape curling, or touch responsiveness (all p > 0.05). These results indicate that Trichosanthes root extract at the tested concentrations does not meaningfully enhance regeneration in D. tigrina, providing useful negative evidence for future screens of traditional herbal compounds in regenerative biology.

Introduction

Rationale and Significance

Identifying natural compounds capable of enhancing tissue regeneration represents a central challenge in translational medicine. Planarians of the species Dugesia tigrina offer an experimentally tractable model for this endeavor: when bisected, each fragment activates a population of pluripotent adult stem cells called neoblasts that proliferate and differentiate to restore the entire organism within days (Rink, 2013). The neoblast-driven regenerative program shares molecular features with vertebrate stem cell systems, making planarians informative for understanding the cellular prerequisites of tissue repair (Rink, 2013).

Traditional Chinese medicine (TCM) has documented anti-inflammatory and antioxidant interventions for centuries, yet most of these compounds remain uncharacterized in living regeneration assays. Because inflammation and oxidative stress suppress stem cell activity and delay wound healing (Zhang et al., 2022), compounds that mitigate these stressors are rational candidates for promoting regeneration. Trichosanthes kirilowii root represents one such candidate: its bioactive constituents—including the ribosome-inactivating protein trichosanthin, flavonoid antioxidants, triterpenoids, and immunomodulatory polysaccharides—are well-documented in cell-culture and clinical contexts (Lo et al., 2017; Luo et al., 2013), yet no study has examined their effect on in vivo tissue regeneration in a whole-organism model.

The present study was therefore designed to test whether continuous exposure to graded concentrations of Trichosanthes root extract (0–0.05% v/v) would accelerate regeneration in D. tigrina, as measured by body-length recovery, eyespot reformation, and indices of nervous system function. A positive result would justify further mechanistic investigation; a null result would provide valuable negative data guiding future compound selection in this growing research area.

Trichosanthes kirilowii Root

Trichosanthes kirilowii (family Cucurbitaceae) has been prescribed in TCM for respiratory inflammation, fevers, and metabolic conditions for over a millennium (Lo et al., 2017). The root is pharmacologically complex. Trichosanthin, its most studied protein, inactivates ribosomes and has demonstrated antitumor and antiviral effects in vitro (Luo et al., 2013). Flavonoids scavenge reactive oxygen species, protecting cells from oxidative damage that accumulates in injured tissue. Triterpenoids and polysaccharides contribute anti-inflammatory and immunoregulatory activity, respectively, collectively creating conditions proposed to be conducive to cellular repair (Zhang et al., 2022). Despite these properties, the regenerative potential of Trichosanthes root in a living, whole-organism system has not previously been evaluated.

Dugesia tigrina as a Model Organism

D. tigrina is a freshwater planarian favored in undergraduate and professional research alike for its transparent body, short regeneration timeline, and readily observable behavioral outputs. Critically, its neoblast system—the only dividing somatic cells under homeostatic conditions—underpins regeneration through mechanisms including Wnt signaling gradients and positional information cues that determine anterior-posterior identity (Rink, 2013). Previous work has used D. tigrina to assess the effects of diverse exogenous compounds, including caffeine and nicotine, on regeneration rate (Nekliudov et al., 2018), providing methodological precedents for the present study. Eyespot reformation in tail fragments and body-length recovery in both head and tail fragments serve as established proxy measures for neoblast function and nervous system reconstitution, respectively.

Materials and Methods

Organism Husbandry and Experimental Design

Forty Dugesia tigrina planarians (Carolina Biological Supply, catalog no. 132954) were randomly assigned to four groups (n = 10 per group) immediately upon receipt. Animals were maintained individually in loosely sealed glass jars containing Poland Spring water and stored at room temperature (~22°C) in a darkened cabinet to minimize phototactic stress. Planarians were fed ground beef liver three times per week (Monday, Wednesday, Friday); following each feeding, the jar was emptied, rinsed, and refilled with fresh water to prevent ammonia accumulation. A seven-day acclimation period preceded all experimental manipulations. All work surfaces were decontaminated with 70% isopropanol before each session. Experiments were replicated across two independent trials (Trial 1 and Trial 2) using separate cohorts to assess reproducibility.

Preparation of Treatment Solutions

Trichosanthes root extract (HerbalTerra, purchased via Amazon) was prepared as a stock and diluted to target concentrations in Poland Spring water. Working solutions (50 mL total volume) were prepared for each group according to Table 1 under sterile conditions; the appropriate volume of extract was measured with a calibrated micropipette and combined with water in a beaker, then gently swirled to homogenize. Solutions were prepared fresh on each treatment day.

Table 1. Composition of treatment solutions by group.

Group	Extract Conc. (% v/v)	Extract Vol. (µL)	Water Vol. (mL)	n (organisms)	A (Control)	0	0	50	10
B	0.01	5	50	10
C	0.03	15	50	10
D	0.05	25	50	10

Treatment Administration

Following the acclimation period, each group was immersed in its corresponding treatment solution for approximately 24 hours. Planarians remained in treatment solution throughout the pre-amputation period. Water quality was maintained by replacing the treatment solution at each feeding interval.

Locomotor Activity and Behavioral Assessment

Baseline locomotor activity was recorded before treatment. For each assay session, 30 mL of Poland Spring water was added to four sterile Petri dishes placed over graph paper grids. Individual planarians were transferred to their respective dishes by group, allowed a 60-second acclimation period, and then video-recorded for 30 seconds. Two behavioral measures were quantified from video: (1) the number of grid lines crossed, as a proxy for overall locomotion, and (2) the frequency of c-shape body curvature events, a stereotyped aversive response used as a stress indicator. Recordings were scored by a single observer blinded to group assignment where possible.

Transverse Amputation

Planarians were cold-anesthetized on ice for one minute to reduce movement before dissection. Each animal was transversely bisected anterior to the pharynx using a scalpel sterilized with 70% isopropanol; the scalpel was re-sterilized after every three cuts to prevent cross-contamination. Head and tail fragments from each group were placed into separate labeled containers (e.g., AH = Group A heads; AT = Group A tails) containing the appropriate treatment solution.

Regeneration Assays

Body length (mm) of both head and tail fragments was measured with a ruler every other business day for 11 days. Eyespot regeneration in tail fragments was assessed daily under a dissecting microscope using a standardized three-point scale: 0 = no visible eyespot; 0.5 = partial pigmentation or incomplete structure; 1 = fully formed, bilaterally symmetric eyespots. Touch responsiveness was evaluated in tail fragments only after pharynx regeneration was confirmed; a soft-bristle brush was lightly applied to the anterior margin of each fragment and the presence or absence of a directed avoidance response was recorded. Planarians were not fed until pharynx regeneration was complete, to avoid confounding feeding behavior with regeneration status. At the conclusion of both trials, all organisms were ethically euthanized by autoclaving at 121°C for 20 minutes under faculty supervision.

Statistical Analysis

All data were entered into Microsoft Excel. Group means and standard deviations were calculated for each measure at each time point. Differences among groups were assessed with one-way ANOVA at each time point, followed by Tukey's HSD post hoc test for pairwise comparisons when the omnibus test was significant (significance threshold: α = 0.05). Analyses were conducted using the online platform at https://www.socscistatistics.com/tests/anova/default2.aspx.