









































Identification of the multiple bioactive derivatives and their endogenous molecular targets that may mediate the laxative effect of rhubarb in rats


CTMJ | traditionalmedicinejournals.com 

  

    ISSN : 2693 6356 

2024 | Vol 7 | Issue 1 

 

 

Chinese Traditional Medicine Journal | 2024 | Vol 7 | Issue 1 

 
 

Identification of the multiple bioactive derivatives and their 

endogenous molecular targets that may mediate the laxative effect 

of rhubarb in rats 

Xie, Ying, Ganqa Luo, Zhou, Jiayan Miao, Tang, Qin, Guan, Xiaoyan * 

School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, 102488, China 
 

 

Keywords: Rhubarb Laxative Anthraquinones Anthrones 

C-kit Aquaporin-3 

 

 

 

Introduction 

 

The Chinese herbal remedy rhubarb, scientifically 

known as Rheum tanguticum Maxim. ex Balf., is a 

popular choice for relieving constipation.1, 2 

Sennoside A, the main laxative component of 

rhubarb, is metabolised to rheinanthrone by gut  

 
 

Abstract— The goal of this study is to use a bioinformatics and in vivo method to figure out how rhubarb 

(Rheum tanguticum Maxim. ex Balf.) causes rats to go to the toilet more often.  

Methods: Using high-performance liquid chromatography in conjunction with linear ion-trap quadrupole 

Orbitrap high-resolution mass spectrometry, substances originating from rhubarb that were found in the 

colorectum were identified. Using databases and the literature, we identified targets with the potential to 

have laxative effects. Then, we employed compounds derived from rhubarb in molecular docking 

modelling. Then, using western blotting, we assessed the expression of potential endogenous target 

molecules that bind specific rhubarb components in rats that were either given or not given rhubarb for 

constipation. In the end, the components of compounds that showed promise as bioactive were identified 

We found 17 anthraquinones and 21 anthrones in the rat colorectum, which is a good indicator of the 

plant's medicinal value. Based on G-scoring, three proteins—c-kit, 5-hydroxytryptamine receptor 4 (5-

HT4), and aquaporin-3 (AQP3)—may mediate the laxative action. Furthermore, ten components derived 

from rhubarb—aloe-emodin, emodin, rhein, chrysophanol, physcion, sennoside A, sennoside C, 

physcionanthrone, aloe-emodinanthrone, and rheinanthrone—were chosen as compounds with high 

probability of activity due to their strong binding affinity for multiple potential targets. It is possible that 

the laxative action of rhubarb extract is mediated by the fact that it enhanced the expression of c-kit and 

5-HT4 while decreasing the expression of AQP3 in the colon of rats that were constipated. Additionally, 

we discovered that a single prototype component may undergo metabolism into many active metabolites, 

and that different molecules from the prototype can be combined to form a single active ingredient. The 

current research concludes that rhubarb's anthraquinones and anthrones may be metabolised into 

bioactive chemicals that enhance defecation via c-kit, 5-HT4, and/or AQP3. 

. 

 
Key words: Al7075 alloy composite, E-glass, MMCs, and short E-glass fibres 



But recent research has found that rhubarb only 

contains around 0.08% sennoside A,7,8 which is 

much too little to explain the plant's strong laxative 

effects. In addition, we found that rhubarb extract 

had a much stronger laxative effect than the same 

amount of sennoside A in our early trial. According 

to their research, the exact process by which 

rhubarb produces its laxative effects is still up for 

debate. Thus, we set out to better understand how 

rhubarb's laxative action works so that its 

therapeutic usage may be based on good theoretical 

grounding. 

For clinically-approved laxatives, the two main 

ways they work are by increasing the rate of 

intestinal motility and  

the stimulation of electrolyte and water secretion. 

One popular medicine, prucalopride, increases 

colonic motility by stimulating neuronal 5-

hydroxytryptamine receptor 4 (5-HT4), which in 

turn increases the contraction of the muscles lining 

the intestines.11th to 9th As an example, osmotic 

laxatives like magnesium sulphate control the 

expression of aquaporin-3 (AQP3) in colonic 

epithelial cells, which leads to water buildup in the 

intestinal lumen and promotes defecation.12, 13 

Since these laxatives act on specific molecular 

targets in the colon, it is vital to identify these 

targets and the specific rhubarb components 

involved in order to deduce how rhubarb exerts its 

laxative effects.  

Applying the notion of induced FIT and the lock-

and-key principle,14 One efficient method for 

identifying therapeutic targets and quickly 

screening possible active molecules is molecular 

docking modelling. The sole applications for this 

are the screening of bioactive compounds and 

potential targets. We wanted to conduct molecular 

docking analysis utilising active, biotransformed 

rhubarb derivatives and endogenous molecules that 

could mediate the laxative action to discover the 

active compounds in the col-orectum. Our goal was 

to increase the data's biological relevance. We used 

the in vivo metabolic understanding of the 

chemicals in rhubarb to discover the appropriate 

bioactive derivatives in the prototype by tracing the 

biotransformation products back to the original 

plant.15 Establishing suitable quality control 

procedures and providing a reference for the use of 

rhubarb derivatives in the clinic would be greatly 

aided by the identification of bioactive components 

in rhubarb.  

Our goal here was to find endogenous molecular 

targets that might modulate the actions of rhubarb 

derivatives that could have cumulative or 

synergistic effects to stimulate defecation. The first 

step was to use high-performance liquid 

chromatography in conjunction with linear ion-trap 

quadrupole Orbitrap high-resolution mass 

spectrometry to identify the rhubarb derivatives 

that were found in the colorectum. The next step 

was to find possible molecular targets by searching 

the literature, the Drug Bank Database (DBD), and 

the Therapeutic Target Database (TTD). As a 

further step, we ran molecular docking simulations 

with all of the rhubarb derivatives and all of the 

possible molecular targets to find binding partners. 

Next, we performed protein analysis of treated rats' 

colonic lysates to find out whether the molecular 

targets that were identified in silico were controlled 

in vivo. Lastly, the active elements in colorectum 

were identified by tracing them back to 

components of rhubarb, utilising knowledge of the 

metabolism of related chemicals. This study 

technique has greatly contributed to our 

understanding of the laxative mechanism of 

rhubarb by identifying essential components 

responsible for its therapeutic actions and their 

prospective molecular targets. Because herbal 

medicines include several bioactive components 

with various potential mammalian molecular 

targets, this technique might be valuable in future 

mechanistic investigations of these treatments.  

 
Materials and methods 

 
Ethical approval 

 
The study protocols were approved by the Ethics 

Committee of Beijing University of Chinese Medicine (BUCM-

4-2017102601- 1026; Beijing, China). 

 
Chemicals and reagents 

 
Rhubarb  was  provided  by  the  Modern  Research  
Center  of Traditional Chinese Medicine, Peking University 
(Beijing, China). 

Loperamide was purchased from Xian Janssen 

Pharmaceutical (Xi’an, China). Standard substances, 

including emodin (lot code: 110756-201512, 98.7%), rhein 

(lot code: 110757-201607, 99.3%), 

physcion (lot code: 110758-201616, 99.0%), aloe-emodin 

(lot code: 110795-201710, 98.3%), chrysophanol (lot code: 

110796-201721, 

99.2%), sennoside A (lot code: 110824-201702, 95.2%), and 

senno- side B (lot code: 110825-201603, 95.0%) were all 

obtained from the National Institutes for Food and Drug 

Control (Beijing, China). Standard substances, including 

emodin-8-O-b-D-glucopyranoside (lot code: 23313-21-5, 

>95%), aloe-emodin-8-O-b-D-glucopyrano- side (lot code: 

33037-46-6, >95%), and sennoside C (lot code: 37271-16-2, 



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— 

>95%) were purchased from Chengdu Biopurify Phy- 

tochemicals (Sichuan, China). Mass spectrometry grade 

acetoni- trile, methanol, and formic acid were obtained from 

Thermo Fisher Scientific (Fair Lawn, NJ). Ultrapure water 

was obtained using a Synergy UV water purification system 

(Millipore, Billerica, MA). 

 
Preparation of references and rhubarb extracts 

 
We weighed and dissolved compounds in suitable 

volumes of 0.5% sodium carboxymethylcellulose to prepare 

reference samples (Supplemental Table 1). 

The rhubarb herbal medicine (31.0 g) powder was 
weighed, 

300 mL ultra-pure water was added, then the suspension 

was boiled for 20 min at 100 ◦C. The extracted solution was 
evaporated to 100 mL. 

Rhubarb should be used at a dose of 0.05e0.25 g/kg/day 

in humans, according to the Pharmacopoeia of the People’s 

Republic of China (2015).16 According to the 

doseeconversion relationship be- tween human and rat, the 

dose for rats was 0.31e1.54 g/kg/day. In our previous study, 

we showed that the administration of 1.54 g/kg/ day of a 

rhubarb extract for 3 days exerted an effective laxative in 

rats. 

 
Rat model of constipation and rhubarb administration 

 
Thirty rats were acclimatized for 7 days, then allocated 

to three groups: control, model, and treatment. The control 

group was administered saline solution, and the model and 

treatment groups were administered loperamide at a dose 

of 1.5 mg/kg/day twice daily for 7 days by gavage to induce 

constipation. During the first 6 days, the wet and dry masses 

of the fecal pellets produced by the rats were measured 

each day.17 

After the model of constipation had been successfully 

estab- lished, the treatment group was fed with rhubarb 

extract at a dose of 1.54 g/kg/day for 3 days, during which 

the other groups were administered substances as above. 

The evacuation index (EI) was used to evaluate the laxative 

effect of rhubarb.18 In addition, the feces of rats in the 

treatment group were collected for analysis. All the rats 

were sacrificed on the 10th day, their mid-colons were 

collected, snap-frozen in liquid nitrogen, and stored at 80 ◦C 

until western blot analysis. 

 
Animals 

 
Specific pathogen-free male  Sprague-Dawley  rats  

weighing 200 ± 20 g were obtained from Beijing Vital River 
Laboratory An- imal Technology (Beijing, China) and 

maintained at a constant temperature (23 ± 2 ◦C) and 

humidity (60 ± 5%). 

Identification of rhubarb derivatives in feces 

Preparation of samples 

Fecal samples were collected over a 24-h period from 
another 20Sprague-Dawley rats that had been administered 
solutions of 10 reference substances. These feces were dried 
in   a   ventilator, crushed, and then placed in a centrifuge 
tube. Fecal   samples weighing 20 mg were extracted using 200 
mL 80% methanol and vortex-mixed for 30 min. Then, the 
samples were centrifuged at 13 522×g at 4 ◦C for 15 min and 

the supernatant was evaporated to dryness under nitrogen. 
The residue was reconstituted with 200 mL of 50% methanol, 
vortex-mixed for 3 min, then centrifuged at 13 522×g at 4 ◦C 
for 10 min. 

Chromatography and MS conditions 

We used previously optimized chromatographic and MS 

conditions.19 

 
Data processing 

 
The raw mass data generated by the HPLC-LTQ-Orbitrap MS 

analysis were processed using Qual Browser of Xcalibur 2.1 

(Thermo Fisher Scientific, San Jose, CA). Online chemical 

databases (http://www.chemspider.com/and 

https://www.ncbi.nlm.nih.gov/) were used to identify the 

compounds obtained. The identification of potentially novel 

compounds was accomplished using the Sci- Finder database 

(https://sso.cas.org/). 

 
Creation of a laxative target database 

 
First, we used the TTD (https://db.idrblab.org/ttd/) to identify 

molecular targets for the treatment of constipation and  the  Drug 

Bank Database (https://www.drugbank.ca/) to summarize the 

tar- gets of existing laxatives.  Then,  we  searched  the  literature  

to further screen for molecular targets with a possible laxative 

effect. 

 
Homology modeling 

 
The target sequences of 5-HT4 and AQP3 were acquired 

from Uniprot (http://www.uniprot.org/; Uniprot IDs Q13639 [5-

HT4] and Q92482 [AQP3]). Template crystal structures for 

these target se- quences were identified using BLAST and 

downloaded from the Research Collaboratory for Structural 

Bioinformatics Protein Data Bank (RCSB PDB, 

http://www.rcsb.org/), using the PDB IDs of 6H7J for 5-HT4 and 

6F7H for AQP3.20 MOE v2014.0901 software (Mo- lecular 

Operating Environment (MOE); Montreal, Canada) was used for 

homology modeling. The QuickPrep module in MOE was used to 

determine the likely sites of protonation of the proteins and the 

orientation of hydrogen atoms in pHs up to 7 and temperatures 

up to 300 K. First, the target sequence was aligned with the 

template sequence, and 10 independent intermediate models 

were estab- lished. These models, with differing homologies, 

were the result of permutational selection of different loop 

candidates and side-chain rotamers. Then, we selected the 

intermediate model with the highest generalized born/volume 

integral score as the final model, which was subjected to further 

energy minimization using AMBER10: EHT force field (MOE). 

 
Molecular docking between rhubarb derivatives in the 

colorectum and molecules mediating a laxative effect 

 
The 2D structures of the rhubarb derivatives were drawn in 

ChemBioDraw 2014 and chemically standardized, which 

consisted of the addition of hydrogens, ionization at pHs from 

5.1 to 9.1, and the formation of stereoisomers and effective 

single 3D conforma- tions using the LigPrep module in Maestro 

(version 9.4, Schro€dinger). 

The 3D structures of the proteins were downloaded from 

RCSB PDB. In addition, homology modeling was conducted 

for proteinstructures that were not found in the database. The 

http://www.chemspider.com/
https://www.ncbi.nlm.nih.gov/
https://sso.cas.org/
https://db.idrblab.org/ttd/
https://www.drugbank.ca/
http://www.uniprot.org/
http://www.rcsb.org/


× × 

3D structure of c-kit was downloaded from the RCSB PDB (PDB 

ID: 6GQK),21 and those of 5-HT4 and AQP3 were obtained by 

homology modeling. 

The  Glide  module  in  Schro€dinger  v2015.09  was  used  for  
mo- 

lecular docking modeling and to predict the binding affinity 

of li- gands for target proteins. The structures of the 

proteins were manipulated using the “Protein Preparation 

Wizard” workflow in Maestro. The main operations were 

exclusion of water molecules, protonation, and optimization 

via OPLS_2005 force field. The Glide module was used to 

generate a grid file for each protein receptor, to determine 

the location of the active site of the protein, in order to 

model the binding of ligands and proteins. The grid enclosed 

a box centered on the binding site with dimensions of 10 10 

10 Å. A scaling factor of 0.8 was used for the van der Waals 

radii of receptor atoms with a partial atomic charge of 

<0.15. 

An extra-precise Glide docking procedure (Glide-XP) 

was employed to model the docking of the compounds to 

the binding sites of the proteins and the optimal orientation 

of the compound, determined using the Glide scoring 

function (G-score), was recor- ded. The more negative the 

G-score is, the stronger is the expected binding affinity of the 

compound and receptor. Molecular graphics were 

generated using PyMOL (https://www.pymol.org/). 

 
Western blotting 

 
Protein lysates of colonic tissue were prepared using 

RIPA Lysis Buffer (Solarbio, Beijing, China) containing 

protease inhibitors. The protein content of each was 

quantified using the bicinchoninic acid method (Beyotime, 

Shanghai, China), then the samples were separated by 

sodium dodecyl sulfate-polyacrylamide gel electro- 

phoresis on 10% polyacrylamide gels, and transferred to 

poly- vinylidene fluoride membranes (Millipore, Billerica, 

MA). The membranes were then blocked with 5% skimmed 

milk powder (BioRuler, Danbury, CT) in tris-buffered saline 

for 2 h, and incubated overnight with primary antibodies 

targeting c-kit (sc-365504, 1:100; Santa Cruz, San Francisco, 

CA), 5-HT4 (TA323344, 1:200; Origene, Rockville, MD), AQ3 

(ab125219, 1:1000; Abcam, Cam- bridge, UK), or 

glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:20 

000; Proteintech, Chicago, IL) at 4 ◦C. The mem- branes were 

then incubated with a secondary antibody, after three 

washes. Specific protein bands were detected using 

Enhanced chemiluminescence solution (Proteintech) and 

imaged using a ChemiDoc MP Imaging System (Bio-Rad, 

Hercules, CA). 

 
Statistical analysis 

 
Data are shown as means (SEMs). Student’s t-test was 

used to compare data between the two groups. P < .05 was 

accepted as indicating statistical significance. Data analysis 

was performed using GraphPad Prism 8.0 software 

(www.graphpad.com). 

 
Results 

 
Laxative effect of rhubarb extract in constipated rats 

 
To confirm the laxative effect of rhubarb, we 

administered rhubarb extract to Sprague-Dawley rats in 

which constipation had been induced by loperamide gavage 

for 7 days. Loperamide is commonly used to increase 

mucosal contact time, which has the effect of inducing 

additional absorption of electrolytes and water, thereby 

reducing fecal mass and the number of bowel movements. 

Twenty-four-hour fecal water content was monitored for 

the first 6 days of loperamide administration, and a 

reduction was identified in the model group from the second 

day (Fig. 1A), which confirmed that constipation had been 

successfully induced. Then, rhubarb 

 

 
 

Fig. 1. Laxative effect of rhubarb extract in constipated rats (A) The 24-h fecal water content of the control and model groups (B) The evacuation indices of the three groups (C) 

https://www.pymol.org/
http://www.graphpad.com/


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The contents of the colorectum and the shape of the feces. 

Note: Data are presented as the mean (SEM). n ¼ 10. **P < .01 vs. the control group. 
extract was administered to the treatment group for 
3 days. A significant increase in EI occurred in this 
group (Fig. 1B), which confirmed that rhubarb 
extract has a laxative effect in constipated rats. We 
then compared the water content of the colorectal 
con- tents of the control, model, and treatment 
groups (Fig. 1C) and found that the rhubarb extract 
increased this. These results imply that rhubarb may 
have its laxative effect by increasing the retention of 
water in the colorectum. 

 

Identification of rhubarb derivatives in rat feces 

 
We used HPLC-LTQ-Orbitrap MS to identify the 

derivatives of rhubarb in the colorectum that might 

contribute to its laxative ef- fect. Using the established 

strategy of characteristic fragment filtration for the detection 

of rhubarb derivatives,19,22 a series of anthraquinones and 

anthrones were identified. The identification process can be 

illustrated using the example of the metabolite emodin-O-

glucuronide (Fig. 2). The full ion chromatogram of a fecal 

sample is shown in Fig. 2A and the extract ion chromatogram 

for emodin-O-glucuronide is shown in Fig. 2B. In negative 

mode, the diagnostic ion fragments of m/z 269.0451 and 

225.0552 were detected, which suggested that the skeleton 

of the target com- pound was the same as that of emodin (Fig. 

2C). In addition, the detection of a quasi-molecular ion of m/z 

445.0765 [M H], which differs from the expected m/z 

269.0451 for emodin by 176.0814 Da, implied that the 

compound was emodin-O-glucuronide (Fig. 2D). Overall, 38 

derivatives of known rhubarb components, including 17 

anthraquinones and 21 anthrones, were identified in 

colorectal samples, including prototypes and various 

metabolites (Fig. 3), and the compound identification data 

are presented in Supplemental 

Tables 2‒4. 

Construction of a target protein database 

 

To determine the mechanism of the laxative effect of 

rhubarb, the molecular targets of clinically-approved 

laxatives were identi- fied using the TTD, Drug Bank 

Database, and published literature. As shown in 

Supplemental Table 5, there are 17 types of molecular target 

for laxatives. All of these, which include channel proteins and 

receptors, have been identified to mediate effective 

treatments for constipation. For instance, magnesium 

sulfate, which is classified as an osmotic laxative, increases 

AQP3 expression in the epithelial cells, which has the effect 

of retaining water in the intestinal lumen and promoting 

defecation.23 In addition, linaclotide is a secreta- gogue that 

activates the guanylate cyclase-C receptor on intestinal 

epithelial cells to increase fluid secretion into the intestinal 

lumen.24,25 Some previous studies have shown that the 

number of interstitial cells of Cajal (ICCs) is lower in the 

colons of patients with slow-transport constipation,26,27 and 

c-kit is a specific marker of ICCs, which have been shown to 

be intestinal pacemaker cells.28 Finally,  prucalopride,  a 

highly selective  5-HT4  receptor agonist, 
directly   activates   afferent   neurons   and   improves   
intestinal 

motility.29,30 The creation of a molecular target database for 

laxa- tives provided a tool for us to identify potential targets 

and bioac- tive derivatives of rhubarb compounds for the 

treatment of constipation. 

 

Candidate colorectal molecular targets and potentially bioactive 

compounds 

 
To screen the candidate targets and bioactive rhubarb 

de- rivatives, molecular docking modeling was performed 

using 38 rhubarb derivatives from the colorectum and 17 

endogenous 

 

 
 

Fig. 2. The identification process for emodin-O-glucuronide (A) The total ion chromatogram of a fecal sample (B) The extract ion chromatogram (C) Mass spectrometry 

(MS)/MS spectrum (D) The identification of an ion with m/z 445.0765 in the fecal sample. 



 
 

 
Fig. 3. Structures of the 38 rhubarb derivatives identified in fecal 
amples. 

 

molecules that mediate laxative effects. First, homology modeling was conducted to predict the structures of proteins that were not 

found in the database, such as 5-HT4 and AQP3. The models generated for 5-HT 

4 and AQP3 are 

depicted in Fig. 4A and B, 

 



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Fig. 4. Structural modeling of molecules mediating laxative effects (A) Homology model of 5-HT4 (B) Homology model of AQP3 (CeD) Ramachandran plot for 5-HT4 and AQP3. 

Dark green dots represent the residues in favored regions, yellow dots represent the residues in permitted regions, and red crosses represent the residues in irrational regions. 

Abbreviations: 5-HT4: 5-hydroxytryptamine receptor 4; AQP3: aquaporin-3. 

 

respectively. Ramachandran plots for 5-HT4 and AQP3 showed that more than 99% of their amino acid residues were in permitted re- 

gions, which indicates that the predicted 3D structures were reasonable (Fig. 4C and D). Then, on the basis that the more negative the G-

score is, the stronger is the binding affinity between 

 

 
Table 1 

G-scores for the binding of the top-ranked rhubarb derivatives with their potential molecular targets. 
 

 

No. Ligand Receptor (kcal/mol) 
 

 

c-kit 5-HT4 AQP3 
 

 

1 Aloe-emodin —8.476 / —5.289 

2 Emodin —9.378 —5.541 —5.743 

3 Rhein —8.453 —5.963 —5.270 

4 Chrysophanol —8.530 —6.221 —5.514 

5 Physcion —8.275 —5.241 —5.987 

6 Sennoside  A —6.854 —8.891 / 

7 Sennoside  C —9.019 —8.810 / 

8 Physcionanthrone —9.069 —5.246 —6.809 

9 Aloe-emodinanthrone —9.278 —5.163 —6.165 

    10 Rheinanthrone —9.747 —5.914 —5.763  

Abbreviations: 5-HT4: 5-hydroxytryptamine receptor 4; AQP3: aquaporin-3. 

the compound and receptor, we selected 
candidate targets and rhubarb derivatives. 
The G-scores are listed in Table 1, and show 
that three molecules, c-kit, 5-HT4, and AQP3, 
represented candi- date targets. 
Furthermore, 10 rhubarb derivatives (aloe-
emodin, emodin, rhein, chrysophanol, 
physcion, sennoside A, sennoside C, 
physcionanthrone, aloe-emodinanthrone, 
and rheinanthrone), were found to have 
strong binding affinities for the three targets, 
and were therefore selected as potentially 
bioactive rhubarb derivatives. 

To gain insight into the binding affinity of rhubarb 

derivatives for the binding sites of the three candidate 

molecular targets, the ligandereceptor interactions were 

analyzed in silico, and we found that all 10 of the derivatives 

are likely to bind strongly to c-kit. Multiple oxygen atoms in 

hydroxyl and carbonyl groups in the basic skeleton of the 

anthraquinones and anthrones were shown to be located in 

the binding site and to form hydrogen bonds with the 

Cys673, Lys623, Glu671, and Leu595 residues of c-kit. The 

com- pound with the strongest predicted binding affinity for 

c-kit was rheinanthrone ( 9.747 kcal/mol). The predicted 

interaction is shown in Fig. 5A, which involves three 

hydrogen bonds and one H- p conjugation: the oxygen 

atoms at the 9- and 3-sites of the carbonyl group, and the 

oxygen atom at the 1-site of the hydroxyl 



— 

— 

Fig. 5. Interaction models for the three candidate molecular mediators of the laxative effect of rhubarb. The interaction mod el for rheinanthrone and c-kit (A), sennoside A 

and 5- hydroxytryptamine receptor 4 (B), and physcionanthrone and aquaporin-3 (C). The ligands are yellow and the surrounding residues in the binding pockets are cyan. The 

backbone of the receptor is depicted as a light blue ribbon. 

group in rheinanthrone formed hydrogen 

bonds with the Cys673, Lys623, and Glu671 

residues of c-kit, respectively. In addition, 

the carbon atom at the 4-site in 

rheinanthrone formed an H-p conju- gation 

with the Phe811 residue of c-kit. 
Sennoside A and sennoside C were shown to have the 

strongest binding affinities with 5-HT4 of the bioactive 

derivatives. They were predicted to bind via hydrogen bonds to 

the Asp84, Val188, Lys174, Glu80, and Asn279 residues of 5-

HT4. The strongest pre- dicted  binding   affinity   for   5-HT4   

was   that   of   sennoside   A ( 8.891 kcal/mol). The five hydrogen 

bonds are shown in Fig. 5B: the oxygen atom at the 8-site of the 

hydroxyl group in the glucose group of sennoside A formed a 

hydrogen bond with the Glu80 and Asp84 residues of 5-HT4, 

separately. In addition, two oxygen atoms of  at  the  80  site  of  the  

hydroxyl  group  in  the  glucose  group  con- nected with the 

backbone and side-chain of Val188, and the oxygen atom at the 

90  site of the carbonyl group of sennoside A interacted with the 

Lys174 residue of 5-HT4. 

However, in contrast to the situation with c-kit and 5-HT4, 

in which the bioactive compounds interacted with amino acid 

resi- dues via numerous hydrogen bonds, AQP3 was predicted 

to have few, weak interactions with the rhubarb derivatives. 

The strongest binding affinity with AQP3 was shown by 

physcionanthrone ( 6.809 kcal/mol): the oxygen atom at the 1-

site of the hydroxyl group in physcionanthrone formed a 

hydrogen bond with the Ala80 residue of AQP3 (Fig. 5C). Taken 

together, the results of the mo- lecular docking modeling 

showed that various oxygen atoms of basic skeletons and 

glucose groups of the 10 bioactive compounds derived from 

rhubarb can form hydrogen bonds with the three molecules 

that mediate laxative effects. 

 
Effects of rhubarb extract on the expression of c-kit, AQP3, 

and 5- HT4 in the colons of rats 

 
In order to increase the biological relevance of the 

molecular docking data, the effects of rhubarb extract on c-

kit, 5-HT4, and AQP3 expression were evaluated by western 

blot analysis. Colonic lysates prepared from rats in the 

control, model, and treatment groups were prepared and 

immunoblotted for each of the proteins. As shown in Fig. 6, 

the expression of c-kit and 5-HT4 was much lower, and that 

of AQP3 was higher in model rats than in the control group. 

When constipated rats were treated with rhubarb extract, 

these changes in expression were all reversed. The increase 

in c-kit expression in the colon would tend to maintain the 

phenotype of ICCs, thereby promoting intestinal 

motility.31,32 In addition, the 

 

 
 

Fig. 6. The effects of rhubarb extract on the expression of c-kit, 5-HT4, and AQP3 in the colons of rats (A) c-kit, 5-HT4, and AQP3 protein expression in the colon, measured 

using western blotting (BeD): c-kit, 5-HT4, and AQP3 protein expression, normalized to that of GAPDH, respectively. 

Note: 5-HT4: 5-hydroxytryptamine receptor 4; AQP3: aquaporin-3; GAPDH: glyceraldehyde 3-phosphate dehydrogenase. Data are expressed as mean (SEM). n ¼ 3. *P < 
.05, 

**P < .01 vs. the control group. 

 



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Fig. 7. Potential metabolic pathways for the anthraquinones and anthrones (AeC) Biotransformation of emodin, sennoside A, and rhein as a product of the metabolism of 

various prototype compounds. The prototype components are shown in red and the active metabolites are shown in blue. 

 

increase in 5-HT4 expression in the colon would be expected to increase intestinal transport.11,33,34 Finally, and as expected, lower 

expression of AQP3 was shown in the colon of treated rats, which would be expected to inhibit water absorption, which would also 

have a laxative effect.35‒37 Thus, rhubarb extract regulates the expression of the identified potential mediators of a laxative effect. 

 

 
 

Fig. 8. Network describing the prototype components of rhubarb, their colonic derivatives, and the endogenous molecular targets potentially mediating a laxative effect. 

The prototype components are shown in pink and the rhubarb-derived components present in the colorectum are shown in blue. 

 

 



Tracing of the candidate bioactive derivatives back to the 

components of rhubarb 

 
To trace the biotransformation products to its prototype, 

we explored the in vivo metabolic transformation of the 

compounds in rhubarb. We studied the metabolism of 10 

representative com- pounds, including both anthraquinones 

(emodin, rhein, physcion, aloe-emodin, chrysophanol, 

emodin-8-O-b-D-glucopyranoside, aloe-emodin-8-O-b-D-

glucopyranoside) and anthrones (sennoside A, sennoside B, 

and sennoside C). As shown in Fig. 7A, anthraquinone-type 

components  are  metabolized  to  glycosides in vivo, and the 

principal metabolic pathways involved are hy- droxylation, 

methylation, glucuronidation, and sulfonation. For anthrone-

type compounds (Fig. 7B), the glucose group is typically lost as 

a result of hydrolysis. In addition, it was predicted that  

anthrone-type compounds would principally undergo 

C10eC10’ bond cleavage in vivo and be further metabolized to 

anthraquinone-type metabolites. Overall, similar metabolic 

pat- terns were predicted for the 10 representative 

compounds. We found that a prototype component, for 

instance sennoside A (Fig. 7B), may be metabolized to form 

several active metabolites (rheinanthrone, emodin, 

chrysophanol, aloe-emodin, rhein, and physcion), and a single 

bioactive molecule, such as rhein (Fig. 7C), can also be 

generated from various prototype compounds (senno- side  A,  

sennoside  B,  sennoside  C,  physcion  [10  / 100 ]  rhein  type 

aglycone,   chrysophanol   [10   /  100]   rhein   type   aglycone,   

aloe- emodin, emodin, physcion, and chrysophanol). 

Finally, using this information regarding the likely metabolic 

pathways involved, the active compounds identified in 

colorectal contents were traced back to the prototype 

components of rhubarb. As shown in Fig. 8, anthraquinone 

derivatives, anthrone derivatives, and their glucosides in 

rhubarb can all be metabolized to generate the 10 potentially 

bioactive compounds identified, which have the potential to 

provide three complementary molecular mechanisms for the 

laxative effect. 

 
Discussion 

 
In the present study, we have used an MS-based 

approach and molecular modeling to determine the 

mechanisms involved in the laxative effect of rhubarb. We 

have shown that anthraquinone and anthrone derivatives in 

rhubarb can be metabolized in the rat in- testine to generate 

various bioactive compounds, which are all capable of 

regulating molecular targets in the colon that mediate the 

laxative effects of known drug substances (Fig. 9). Thus, de- 

rivatives of rhubarb may have additive or synergistic effects 

to promote defecation. 

This strategy represents a valid approach to ensure 

quality control and to provide a reference for the rational 

medicinal use of rhubarb in the clinic. Because of the 

complexity of potential me- dicinal compoundetarget 

interactions, the identification of inter- action domains is 

still in its infancy. However, high-affinity ligandereceptor 

interactions can be validated using surface plas- mon 

resonance studies,38,39 and his method may be used to

 
 

Fig. 9.  Summary of the study findings. 

Abbreviations: 5-HT4: 5-hydroxytryptamine receptor 4; AQP3: aquaporin-3. 

 

 



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2024 | Vol 7 | Issue 1 

 

 

further characterize the molecular mechanisms involved in 

the laxative effect of rhubarb and to identify lead compounds 

for further investigation. 

Most herbal medicines contain numerous  components  

that may be bioactive or metabolized to form bioactive  

derivatives, such as flavonoids, alkaloids, and organic acids. 

Rhubarb is a common Chinese medicine that also contains 

several components of this type, including anthraquinone 

derivatives, anthrone de- rivatives, glucosides, tannins, and 

organic acids. In the present study, rhubarb was used as an 

example to illustrate an approach to the determination of the 

mechanisms of a medicinal effect of a plant product. In it, we 

identified bioactive compounds, deter- mined their origin in 

the plant, and identified potential binding partners that are 

known mediators of the medicinal effect. This strategy can 

provide insights into the mechanisms whereby multiple 

components of herbal medicines may have additive or 

synergistic effects, exerted via multiple molecular targets, to 

achieve a therapeutic endpoint. 

Conclusion 

 

By binding to c-kit, 5-HT4, and/or AQP3, the 

current research showed that bioactive chemicals 

with a laxative action might be generated by 

metabolism of anthraquinones and anthrones in 

rhubarb. It was indicated using molecular docking 

modelling that five anthraquinones (aloe-emodin, 

emodin, rhein, chrysophanol, and physcion) and 

five anthrones (sennoside A, sennoside C, 

physcionanthrone, aloe-emodinanthrone, and 

rheinanthrone) interact with c-kit, 5-HT4, and 

AQP3. Importantly, we have shown that rhubarb 

extract, which is believed to mediate the laxative 

action of the plant, raised the expression of c-kit 

and 5-HT4, while reducing that of AQP3 in the rat 

colon. Additionally, we discovered that a single 

proto-type component in rhubarb, like sennoside A, 

can be metabolised into multiple active metabolites 

(rheinanthrone, emodin, chrysophanol, aloe-

emodin, rhein, and physcion), as well as one active 

ingredient,  

 

 

compounds including physcion, emodin, 

chrysophanol, aloe-emodin, sennoside C, and 

sennoside A are potentially potential building 

blocks for compounds like rhein. Our results 

suggest that rhubarb's laxative action could be due 

to the interaction of several derivative chemicals 

with various molecular targets in the colon, which 

might lead to cumulative or synergistic effects.  
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