INTRODUCTION:

Hemorrhoids are reported as the most common anorectal condition, with a prevalence of 4.4% up to 36.4%. It is more prevalent in those 45–65 years of age.^1,2 Alteration in the vascular tissue of the anal canal are the main characteristics of hemorrhoids.^3,4 The recto-anal cushions are crucial for the smooth passage of stool. However, when these cushions become inflamed, swollen, and displaced they develop into a pathological condition known as hemorrhoids.^5,6

Hemorrhoids are typically caused due to increased strain on the pelvic and rectal regions. This pressure causes the veins to dilate abnormally and become distorted, leading to blood leakage resulting in rectal bleeding.⁷ It typically manifests with signs and symptoms of bleeding, prolapse, edema, and pain, and severely affects the quality of life.⁸ Several risk factors have been associated with the pathophysiological alterations in hemorrhoids including diarrhea, constipation, pregnancy, obesity, alcohol, chronic straining, low-fiber diet, aging, sedentary lifestyle, and so forth.⁷

The research of various national organizations states that the incidence of hemorrhoids is 0.36% in India and 3.82 % in the United States. Furthermore, data show that hemorrhoids are a major issue in both developed and developing nations, affecting 36% to 40% of the population.^5,9 The treatments used for the management of hemorrhoidal disease include infrared photocoagulation, hemorrhoidectomy, cryotherapy, sclerotherapy, rubber band ligation, and laser treatment. However, these treatments are often associated with numerous side effects.^1,10

Hemorrhoids are the physiological and pathological problems largely caused by free radical production. The literature has provided ample evidence of the development of hemorrhoids due to free radicals.¹¹ Antioxidants play a significant role in neutralizing free radicals, thereby contributing to the management of hemorrhoids. Therefore, significant interest in utilizing antioxidants for their substantial health benefits while minimizing potential toxicities.¹²

L-carnitine L-tartrate (LCLT) is a nutritional compound that has potential health benefits¹³, particularly in the recovery and fat metabolism in mammals, plants, and specific bacteria. LCLT is a salt comprised of L-carnitine and L-tartaric acid. LCLT can be used as a nutrient source of L-carnitine and is also the most commonly used form of L-carnitine.¹⁴ Studies on LCLT's pharmacological properties have revealed that it has anti-diabetic, anti-wasting, anti-inflammatory, and anti-cancer properties.¹⁵ According to scientific reports, the LCLT exhibits antioxidant and anti-inflammatory properties. Thus, the current work used Croton oil-induced hemorrhoidal rats to assess the anti-inflammatory and antioxidant-mediated anti-hemorrhoidal activity.

MATERIALS AND METHODS:

Drug and Chemicals:

L-carnitine L-tartrate (LCLT) and Croton oil were purchased from Sigma Aldrich in the United States. Evans Blue was procured from Loba Chemicals, Mumbai, India. Formamide, pyridine, and diethyl ether were purchased from Merck Specialities Pvt. Ltd. in Mumbai, India. Pilex granules were procured from the retail pharmacy and all analytical-grade materials used in the studies came from reliable suppliers. A UV-visible spectrophotometer (UV-1800 Shimadzu) was used to take all analytical readings.

Experimental animals:

The National Institute of Biosciences Pvt. Ltd. provided Wistar albino rats weighing between 180-200gm, regardless of sex. Polypropylene cages with a 12hour light/dark cycle, regulated ambient temperature, and controlled relative humidity were used to house groups of six animals each. All of the animals were given free access to water and normal rat diet. Prior to the research, the animals were allowed a week to acclimate to the experimental conditions. The college's institutional animal ethics committee (IAEC) (Protocol no. DYPCOP/IAEC/2023/14/02) authorized all of the protocols used in the research, and they were all conducted following the accepted CPCSEA criteria.

Anti-Hemorrhoidal Activity:

Induction of Hemorrhoid:

Evans blue (EB) dye (30mg/kg i.v.) was first injected into the tail vein of rats who had fasted all night in order to cause hemorrhoid. After 30minutes of administration of EB dye croton oil preparation (COP), was applied in anorectal region of rats (except normal control). 100µL of COP was soaked in the area where sterile cotton swabs (4mm in diameter) had been used to puncture a rat's anus, which was around 20mm deep. The 10 seconds, were spent with the swabs in place. Following 7-8hours of COP induction, edema showed a linear progression. A hemorrhoid was defined as edema that persisted for up to 12hours. Relevant treatment was administered to each group for seven days following a 24hour induction period.^7,16

Grouping of animals:

After 24hours of induction, hemorrhoids were induced in all animals. Afterward, the animals were split into five groups: Distilled water was administered to Group 1, which was the normal control (NC) group. Group 2 was given distilled water as the hemorrhoid-induced negative control group. Group 3 received Pilex granules (400mg/kg) as the standard treatment group, while Groups 4 and 5 received LCLT (300 and 500mg/kg) as treatment groups. The rats received LCLT once every 24 hours for seven successive days.

Body weight of rats:

Hemorrhoids directly affect the food consumption of rats. Hence there is a need to record changes in the body weight of rats. For that, purpose record the body weight of the rat before the initiation of the study, and at the end of the study.

Hemorrhoidal Parameters:

Severity score:

On the 7^th day, the degree of inflammation in the anorectal region was assessed as per the method described by Azeemuddin (2014). Take a transverse section of anorectal tissue 1 hour after the appropriate treatment. After mounting the tissues on white paper, the macroscopic severity assessment was completed.⁷

Assessment of Evans Blue Exudation:

To measure the amount of plasma exudation caused by Croton oil, the anorectal tissue of the rat was examined for the presence of Evans Blue (EB) dye. The technique for determining EB exudation in anorectal tissue has been described by Azeemuddin (2014) and Dey (2016).^7,16 One hour after the appropriate treatment, on the 7^th day, 20mm of the animal’s anorectal tissue was removed and weighed. The formamide was used to remove the EB dye from the tissue. To determine how much EB dye was in the sample, a standard calibration curve was used and a UV spectrophotometer was used to record the peak light intensity (λ max) at 620nm. The concentration of EB dye was expressed in anorectal tissue in µg/mg.

Estimation of rectoanal coefficient (RAC):

Rats' anorectal tissues were weighed, and the results were compared to the rats' body weights to calculate the RAC. The following formula was then used to determine the RAC.⁷

RAC = Weight of tissue (recto-anal in mg)/Body weight (g).

In-vivo Antioxidant Activity:

In vivo, antioxidants such as lipid peroxidation (LPO) and catalase (CAT) are also evaluated using conventional techniques as mentioned by Dubey (2023).⁵ An hour after the proper treatment on the 7^th day, a sample of anorectal tissue from the animals that were sacrificed was taken, and it was properly cleaned with an phosphate buffer. The material was centrifuged at 16,000g for an hour at 0°C after the tissue had been homogenized in 1.15percent potassium chloride (KCl) to produce a 10% w/v suspension. This solution was used to assess antioxidant activity in vivo.

Assessment of Lipid Peroxidation Activity:

To find the lipid peroxidation activity of LCLT, the procedure described by Dubey (2023) was used.⁵ 2mL of TBA-TCA-HCl reagent and 0.1mL of tissue homogenate were mixed in equal amounts. After 15 minutes of incubation in a water bath, the sample was cooled. After centrifuging the mixture for ten minutes at 3500g, the clear supernatant was gathered. The result was given as mg per 100g of tissue after the absorbance at 535nm was measured.

Assessment of Catalase Activity:

The procedure described by Dubey (2023)⁵was used to measure the catalase activity of LCLT. To accomplish this, make a reaction mixture of 0.1mL of tissue homogenate, 1.0mL of 0.01 M phosphate buffer (pH 7.0), and 0.4 mL of H₂O₂. Additionally, get a 5% ready dichromate-acetic acid reagent by mixing potassium dichromate and glacial acetic acid in a 1:3 ratio. Then, add 2.0mL of this reagent to the reaction mixture to halt the reaction (Figure No. 2). The catalase activity of the LCLT is expressed in milligrams of H₂O₂ absorbed per minute per milligram of protein.

Figure No. 1 Lipid Peroxidation Activity

Figure No. 2 Catalase Activity

Assessment of serum cytokines:

Blood samples were taken from the retro-orbital sinus 1 hour after the appropriate treatment on the 7^th day. The purpose of this procedure was to estimate the concentration of inflammatory cytokines in serum, including PGE-2, TNF-α, 6 IL-6, and MMP-3. Enzyme linked immune sorbent assay kits were utilized for this analysis, and the manufacturer's instructions were adhered to.^3,17

Histopathological studies:

On the 7^th day, a transverse section of the anorectal tissue was obtained, 1 hour after the appropriate treatment, to assess the degree of inflammation in the rectoanal region. Inflammation, necrosis, congestion, bleeding, and vasodilatation were then examined.^7,16

Statistical Analysis:

Graph Pad Prism version 5 software was used, and P< 0.05 was considered statistically significant for all statistical comparisons. The data are presented as mean ±SEM and were analyzed using the Dunnett test and one-way ANOVA. Each group consisted of six rats.

RESULTS:

Anti-hemorrhoidal Activity:

After 24 hours of application of COP, hemorrhoids were significantly developed in rats, which were visible as blue color spots on the anorectal region (Figure No. 3). The normal control group showed a normal anorectal region (Figure No. 3 A). Hemorrhoid growth was visible in the rats in the induction control group (Figure No. 3 B). However, in the treatment groups, rats given LCLT (300 mg/kg) showed low recovery on treatment (Figure No. 3 D). Rats administered 500 mg/kg of LCLT and 400 mg/kg of Pilex granules showed a significant recovery in comparison to the induction control group (Figures No. 3 E and C).

7.3.5 Biochemical Parameters:

The effects of LCLT treatment on several biochemical parameters are shown in Table No. 3. The level of biochemical parameters was expressed more frequently in the induction control group but following LCLT treatment, there was a significant drop in the level of cytokines, PG-E2, MMP-3 and a notable effect seen in the case of LCLT (500 mg/kg) as compared to (300 mg/kg), respectively.

1. Serum cytokines (TNF-α, IL-6):

Figure No. 7 shows how L-carnitine L-tartrate (LCLT) affects TNF-α and IL-6 in the Croton oil-induced hemorrhoid, respectively. When compared to the normal control, TNF-α was considerably (###p<0.001) higher in the induction control group. In contrast, there is a significant difference (p<0.001) between the reference standard group and the induction control, and treatment-2 (500mg/kg) was found to reduce TNF-α equipotently (Figure No. 7 A).

When comparing the induction control group to the normal control, IL-6 was considerably (###p<0.001) higher. On the other hand, a reduction in TNF-α was observed with treatment-2 (500mg/kg) having significance (p<0.01), and the reference standard group has significance (p<0.001) when compared to the induction control (Figure No. 7 B).

Figure No. 7 Effect of LCLT on (A) TNF-α and (B) IL-6 cytokine levels in the Croton oil-induced hemorrhoid model. Graphs show that the highest effective dose level of LCLT for managing hemorrhoids is 500 mg/kg.

(NOTE: Dunnett's multiple comparison test was used to examine the results following a one-way ANOVA test, where ###: p < 0.001 compared to the NC group and *: p < 0.05, **: p < 0.01 and ***: p < 0.001 compared to the IC group. Results expressed as mean ± SEM (n = 6))

2. PG-E2 and MMP-3:

Figure No. 8 shows how L-carnitine L-tartrate (LCLT) affects PG-E2 and MMP-3 in the Croton oil-induced hemorrhoid, respectively. Comparing the induction control group to the normal control, PG-E2 was considerably (###p<0.001) higher. In contrast, a decrease in PG-E2 was seen with treatment-2 (500 mg/kg) having significance (p<0.01) and the reference standard group has significance (p<0.001) when compared to the induction control (Figure No. 8 A).

Comparing the induction control group to the normal control, MMP-3 was considerably (###p<0.001) higher. In contrast, a significant decrease in MMP-3 was seen with treatment-2 (500mg/kg) having significance (p<0.001), and the reference standard group has significance (p<0.01) when compared to the induction control (Figure No. 8 B).

Figure No. 8 Effect of LCLT on (A) PG-E2 Level and (B) MMP-3 Level in the Croton oil-induced hemorrhoid model. According to graphs, 500 mg/kg of LCLT is the maximum dose level that is helpful for treating hemorrhoids.

(NOTE: Dunnett's multiple comparison test was used to examine the results following a one-way ANOVA test, where ###: p < 0.001 compared to the NC group and *: p < 0.05, **: p < 0.01 and ***: p < 0.001 compared to the IC group. Results expressed as mean ± SEM (n = 6))

Histopathological studies:

In a histopathological study of anorectal tissues, normal control group showed no abnormality, and normal tissue architecture (Figure No. 9 A). Induction control group showed degradation of the mucosal layer, significant edema, and inflammatory zones in their anorectal tissues (Figure No. 9 B). However, in the treatment groups, rats given LCLT (300mg/kg) showed weak to moderate protection in their anorectal tissues (Figure No. 9 D). Rats administered 500mg/kg of LCLT and 400mg/kg of Pilex granules showed a significant reduction in inflammatory zones, yet normal tissues and normal blood vessels remained (Figures No. 9 E and C) as compared to the induction control group.

Figure No. 9 Histopathological analysis of anorectal tissue from rats administered with LCLT. The anorectal tissue in the normal control group is shown as being in good condition (figure A), the induction control group is shown as having severey mononuclear cells (MNC) infiltration (Arrow) and edema (Arrowhead) in rectoanal tissue, (figure B), the reference standard is shown as having minimal congestion (arrow) in rectoanal tissue, after receiving standard Pilex treatment (400 mg/kg) (figure C), the treatment group (300 mg/kg) is shown as having mild congestion (Arrow) and MNC infiltration (Arrowhead) in rectoanal tissue after receiving LCLT treatment (figure D). The treatment group (500 mg/kg) is shown as having minimal congestion (arrow) in rectoanal tissue, (figure E).

DISCUSSION:

Hemorrhoids, referred to as piles, are a frequent anorectal condition that is mainly defined by changes in the anal canal's vascular tissue. This leads to increased vascular permeability and inflammatory cytokine extravasation into interstitial spaces¹⁸. Croton oil-induced hemorrhoids is a widely used preclinical model due to the presence of active phorbol esters responsible for releasing inflammatory mediators such as PG-E2, IL-6 hence produce inflammatory reactions similar to hemorrhoids.^1,7

The present research study revealed a significant anti-hemorrhoidal activity of L-carnitine L-tartrate in experimental models of hemorrhoids induced by croton oil, respectively. Previously published acute oral toxicity studies of LCLT reported LD₅₀ more than 5000mg/kg¹⁴, as well as other pharmacological activities, found more significant results at 300 and 500mg/kg p.o.¹⁵ Hence, these doses were selected as low-dose and high-dose for the present study.

The results of in-vitro activities conducted on LC¹⁹, showed its usefulness in the treatment of disorders linked to oxidative stress and inflammation. Initially, there were no noticeable differences in body weight. Rats' body weight significantly decreased when hemorrhoids were induced. However, administration of L-carnitine L-tartrate (LCLT) subsequently increased body weight.

The findings of our research demonstrated that LCLT (500mg/kg) has a promising effect on lowering severity scores, EB exudation, and RAC; compared to the induction control group, and the outcomes were comparable to the conventional treatment with Pilex granules.

The result of in-vivo antioxidant activity conducted on hemorrhoidal rats showed its usefulness in the treatment of hemorrhoids. The findings of our research demonstrated that dose-dependent decreases in oxidative stress. LPO is a major contributor to the development of hemorrhoidal illness because it causes lipid peroxidation, which damages lipids oxidatively. However, these altered enzyme levels (LPO, CAT) significantly restored after treatment with 500mg/kg of LCLT, demonstrating LCLT's antioxidant capacity, which is crucial for anti-hemorrhoidal activity.

Induction of hemorrhoids was confirmed by the application of COP, which resulted in severe inflammation and a significant rise in RAC and macroscopic severity levels.^7,16 It causes a significant increase in inflammatory mediators like PG-E2, TNF-α, IL-6, and MMP-3 which increases vascular permeability and causes oxidative stress in the anorectal tissue, which eventually leads to vascular damage.²⁰

In comparison to the normal control group, the administration of COP in the induction control group resulted in a substantial rise in the inflammatory mediators PG-E2, TNF-α, IL-6, and MMP-3.^7,21 The anti-inflammatory potential of hemorrhoids was validated by the dose-dependent significant reduction in PG-E2, TNF-α, IL-6, and MMP-3 levels observed with LCLT treatment at 300mg/kg and 500mg/kg in comparison to the induction control group.

Application of croton oil results in severe vasodilation, inflammatory cell filtration, and bleeding.⁷ The histopathological examination of the induction control group showed degradation of the mucosal layer, significant edema, and inflammatory zones in the anorectal tissue. However, administration of LCLT (300 and 500mg/kg) resulted in dose-dependent recovery from inflammation, and tissue examination revealed nearly normal architecture with minimal damage.

We have effectively shown that LCLT has positive impacts on hemorrhoid management. The LCLT's anti-inflammatory and antioxidant properties were linked to its apparent anti-hemorrhoidal potential, with L-carnitine possibly having a significant impact on tartaric acid conjugation. However, to determine the anti-hemorrhoidal potential of LCLT with a particular mode of action, a thorough investigation of the formulation is necessary.

CONCLUSION:

The significant anti-inflammatory properties of LCLT have, in general, validated its potential use in the treatment of croton oil-induced hemorrhoids. Furthermore, antioxidant activity is helpful in the management of hemorrhoids. In addition, a noteworthy impact of LCLT was observed on the levels of inflammatory markers. However, further research, including clinical and formulation studies, is needed to determine LCLT's therapeutic potential for use in humans.

ACKNOWLEDGMENTS:

We would like to sincerely thank everyone who helped us finish this research article. Additionally, we are grateful for the opportunity and support that Dr. D. Y. Patil College of Pharmacy in Akurdi, Pune, gave us so that we could take part in and contribute to this study project.

DECLARATION OF CONFLICTING INTERESTS:

Conflicts of interest are not present concerning the research work conducted by the authors.

ETHICAL APPROVAL:

The College's Institutional Animal Ethics Committee (IAEC) authorized all of the protocols (Protocol no. DYPCOP/IAEC/2023/14/02) used in the research, and they were all conducted following the accepted CPCSEA criteria.

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Received on 23.08.2024 Revised on 19.03.2025

Accepted on 04.07.2025 Published on 01.12.2025

Available online from December 06, 2025

Research J. Pharmacy and Technology. 2025;18(12):5891-5898.

DOI: 10.52711/0974-360X.2025.00851

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Sr. No.	Hemorrhoidal parameters	Normal Control (NC)	Induction Control (IC)	Reference Pilex granules (400 mg/kg)	LCLT (300 mg/kg)	LCLT (500 mg/kg)
1.	Severity score (Grade)	0.141±0.022	1.963±0.078^###	1.520±0.059***	1.665±0.066**	1.556±0.067***
2.	Evans blue dye Exudation	0.301±0.011	4.866±0.238^###	3.742±0.160***	4.012±0.182**	3.802±0.147***
3.	Recto-anal coefficient	1.414±0.080	3.576±0.062^###	2.834±0.110***	3.183±0.055**	3.030±0.074***

Sr. No.	Parameters	Normal Control (NC)	Induction Control (IC)	Reference Pilex granules (400 mg/kg)	LCLT (300 mg/kg)	LCLT (500 mg/kg)
1.	Lipid Peroxidation (LPO)	7.172±0.366	10.89 ±0.43^###	8.658±0.225***	9.345±0.264**	8.822±0.255***
2.	Catalase (CAT)	11.812±0.247	6.917±0.264^###	11.133±0.308***	8.375±0.176**	10.118±0.191***

Sr. No.	Biochemical parameters	Normal Control (NC)	Induction Control (IC)	Reference Pilex granules (400 mg/kg)	LCLT (300 mg/kg)	LCLT (500 mg/kg)
1.	TNF-α	74.075±2.865	110.67±3.275^###	90.958±1.835***	95.923±2.891**	92.450±2.524***
2.	IL-6	16.720±1.763	57.115±2.194^###	41.437±2.272***	47.327±2.063*	44.512±2.638**
3.	PG-E2	34.558±2.316	64.927±3.776^###	46.678±1.918***	54.588±2.694*	50.707±1.927**
4.	MMP-3	0.138±0.019	0.368±0.015^###	0.282±0.017***	0.300±0.019**	0.256±0.013***