Short Term-Subacute and Chronic Toxicity Studies on The Mucilage of Plantago ovata, A Novel Natural Compound Administered by Oral in Wistar Rats

Chandra Sekhar Naik. D*, Shaik Abdul Rehman, Shaik Jafar Ali, K. Uday Kiran, Md. Zulekha

Nimra College of Pharmacy, Jupudi, Ibrahimpatnam, NTR -AP-521456.

*Corresponding Author E-mail: chandu.desavath@gmail.com

ABSTRACT:

The mucilage from Plantago ovata is being explored as a novel, highly effective disintegrating component in the creation of rapidly dissolving tablets for drugs that are not easily soluble, due to its enhanced compatibility with living systems and its attraction to water. This research aims to evaluate the immediate and delayed toxicity levels of Plantago ovata mucilage in Wistar rats. In the initial phase, the mucilage was given to Wistar rats in a single dose over a period of seven days. For the subsequent phase, both sexual Wistar rats have being subjected to a twenty-eight days study to evaluate the effects of oral dosages ranging from 50 to 600 milligrams per kilogram of body weight daily on various parameters including body mass, aliment consumption, death rates, biochemical tests, and histological examinations. The initial study showed that the highest dose of Plantago ovata mucilage that did not cause death in rats was above 2000 milligrams per kilogram of body mass. The follow-up study on subacute toxicity did not reveal any significant differences in contrast to the group under supervision, and there were no alterations in blood or biochemical levels. The pancreatic, kidneys, and liver weights were also consistent. From these results, it was determined that Plantago ovata mucilage at a dosage of 2000 mg/kg body mass daily was not chimeric or weakening, and did not affect reproduction or development, suggesting it is generally safe for oral consumption.

KEYWORDS: Plantago ovata, Biochemical tests, Wistar albino rats, OECD, Acute toxicity.

INTRODUCTION:

Superdisintegrants are substances that were added to tablets that dissolve quickly in order to speed up the disintegration process. For a while now, a wide range of excipients have been utilized as tablet disintegrants; nevertheless, formulators in the pharmaceutical sector currently only have access to a limited number of approved disintegrants. Among the most widely used disintegrants are simple starches¹. But sometimes a solid dosage form needs a disintegration time that is acceptable, sufficient, and quick.

Simple starch cannot provide this need. Thus, "superdisintegrants" have been created to improve the disintegration of immediate-release tablets. The extracellular matrix, the surface of cells, and the area beneath cells and tissues can all be impacted by toxicants. Therefore, it is now essential to ascertain a molecule's toxicity before using it as a medicinal excipient². The present study assessed the mucilage's acute and subacute toxicity profile. Unwanted effects that happen quickly following either one dosage of a drug taken orally or many doses given within a 24-hour period are referred to as acute toxicity³. The term "sub-acute toxicity" refers to the development of detrimental consequences that result from repeated or continuous therapy within a 24 to 28 day period. The incidence of negative side effects brought on by ongoing or repeated treatments—typically lasting weeks or months—is referred to as chronic toxicity. Plantago ovata seeds were esterified to create a novel superdisintegrant from their natural mucilage. Whether a superdisintegrant is made in a lab or is obtained from plants, it is crucial to review its toxicity information. Currently, the focus of research is on creating superdisintegrants containing mucilage to stop gel formation and to become soluble when they come into contact with water⁴.

MATERIALS AND METHODS:

Materials:

Plantago ovata seeds obtained from medicinal garden tirupathi, Acetone (Merk chemical, Mumbai, CAS 67-64-1), Potassium di hydrogen phosphate (Loba chemicals, Hyderabad, CAS 7778-77).

Methods:

Extraction of mucilage from Plantago ovate:

To isolate the mucilage, Plantago ovata seeds were utilized. After soaking 10grams of seeds in 500 milliliters of distilled water for 48hours, the seeds' mucilage was completely released into the water by boiling them for an hour at 80 to 120 degrees Celsius. To eliminate marc, the material was filtered by pressing it through a muslin cloth. The mucilage was then precipitated by adding 450 milliliters of acetone in an equal volume to the filtrate. The mucilage was separated, dried for 30 minutes at a temperature below 60°C in the oven, ground into a powder (#60) mesh, weighed, and kept in a desiccator until needed.

Experimental animals:⁵

Acute and sub-acute toxicity studies were conducted using mature, healthy male and female Wistar albino rats (body weight: 150-200g for males, 120-150g for females) at 8-12 weeks of age. Three rats were placed in cages made of polypropylene. The male and female rats were separated, and the chosen female rats were not pregnant and had not given birth. Before the experiments began, the animals were kept in the laboratory animal room for a minimum of a week in clean polypropylene cages equipped with stainless steel top grills. Standard cage conditions were a 12hour light-dark cycle, a temperature of 25±2°C, and a relative humidity of 65%. The bedding consisted of stainless-steel sipper tubes attached to polypropylene water bottles that were constantly on hand. Every day, the water bottles and the cage bedding were cleaned. All experimental techniques have been approved by the IAEC of KVSR SCOPS, located in Vijayawada, NTR, India (CPCSEA Number 01/KVSRSCOPS/IAEC/2021). When conducting tests with brand-new paddy husk animals, the guidelines for the care and use of laboratory animals were adhered to. When conducting studies on animals, the Protocols for the handling and application of laboratory animals were followed.

Acute toxicity study^5-6

With a few minor adjustments, the acute toxicity tests were carried out on Wistar rats in accordance with OECD guideline 423. The experiment employed rats of both sexes. Then, five rats of each sex were selected from the male and female rats to form the usual control and treatment groups. The test sample was given orally to the test group rats at a dosage of 2000 milligrams per kilogram b.mass. Water was the only vehicle provided to the rats in the control group. The night before the experiment started, all of the rats were weighed, registered, and allowed unrestricted having access to water. Following the dose, the rats were fasting for an additional four hours. Throughout the first four hours and the next twenty-four hours, findings were continuously made for every group's rat to search for any abnormalities or deaths. Following that, the subjects were observed twice a day for seven days in order to search whichever negative results, including convert in the skin, hair, or eye colors; adjustments to the amount of food or liquids consumed; quivering; paroxysms; slobbering; loose motions; inactivity; respiring; abnormal behavior; muscular action; resting; or hibernation. To get information on the test materials degree of short-term toxicity, which helps ascertain dosages for the oral toxicity trial that is repeated, acute toxicity was conducted.

Sub-chronic toxicity studies:^5-6

A subacute toxicity study of Plantago ovata mucilage was carried out using Wistar albino rats, adhering to the guidelines provided in OECD recommendation four hundred seven.

Variation in body weight:^5-6

For the duration of the trial, the body weight of each rat was measured once, on Day 0, the day before treatment began, and then once per week after that. The night before blood was drawn, the animals were fasted. On Day 28, the body weight was measured.

Variation in weight of the organ:⁷

After being removed, the liver, thymus, kidneys, and lungs were cleaned with filter paper and weighed with an analytical scale. Producer: Mettler Toledo All organ weights were recorded as absolute values.

Body and organ weight evaluation:⁸

The body weights of the control and treated animals were compared in order to screen for possible toxicity. A macroscopic examination was performed on three doses of the target organs (200, 400, and 600mg/kg) to search for any abnormalities in size, texture, or shape that would suggest harmful consequences. The rat liver, kidneys, lungs, and thymus are the main organs that are attacked.

Hematological estimation:^8-9

Blood was extracted into capillary tubes from Wistar rats while they were sedated. Hematological indices such as Hb count, RBC, WBC and platelet count.

Estimation of Biochemical tests:^9-10

To perform a biochemical assay, blood was centrifuged for 10 minutes at 4°C at 3000RPM. After centrifugation, the serum was extracted from the blood and kept cold until further examination. Traditional methods were used. to assess the levels of albumin, serum creatinine, urea, total protein, aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), and urea.

Histology study:^10-11

Changes in the liver, stomach, and kidney were assessed histopathologic ally. Following homicide, the kidney, liver, and stomach were surgically extracted and kept in normal condition using 20 percent formalin. salty. Five-millimeter pieces were cut on a rotary microtome, and then they were stained with HE 40. After that, the slices were looked at under a microscope to check for pathological abnormalities.

RESULTS AND DISCUSSIONS:

The acute toxicity investigations did not result in any deaths. both 24hours after the initial 4hours of continuous observation. Furthermore, no fatal effects were observed throughout the seven days when the mucilage was provided. Normal morphological traits are displayed. There was no evidence of any aberrant behaviors, such as drooling, diarrhea, or lethargic behavior. weight, amount of food and water consumed, and typical lung activity were all noted. Ten to fifteen minutes after the test dose was administered, significant sedation was observed.

This proves that the mucilage dosage of 600mg/kg, b.wt. was safe. The LD50 figure was believed to be higher than the maximum as there was no data on mortality for all doses. a 600mg/kg body weight test dosage.

Throughout the experiment, every animal remained alive and presented itself normally on Day 28, the conclusion day of the study. Each animal's body weight grew. The body weight findings are displayed in Table no 1.

There were no variations in the males' organ weights at the end of the treatment period. The ladies' relative liver/body mass was considerably greater than the control group's at the 600 mg/kg/day treatment dose it indicates Table no 2.

Table No 2: Effects of 28 Day Oral Administration of Plantago ovata mucilage on Organ Weights in Rats

Section	Mean ± S. E. organ weight (g/100 g body weight), n = 6.
Section	Liver	Kidney	Heart
1	3.22 ± 1.2	.64 ± 1.1	.27 ± 1.1
2	3.24 ± 0.2^ns	.65 ± 1.2^ns	.26 ± 1.1^ns
3	3.42 ± 0.1^ns	.64 ± 1.2^ns	.28 ± 1.2^ns
4	3.41 ± 0.2^ns	.67 ± 1.1^ns	.27 ± 1.1^ns

ns = non-significance

After 28 days of treatment, the hematological data were analyzed shown table 3 figure 1, and no changes of toxicological significance were discovered. When compared to the control group, a few metrics revealed statistically significant differences in some treatment groups. However, as the dose-response was absent. connection and no equivalent alterations in females, it was concluded that these differences had little biological importance. The parameters of the blood biochemistry examinations did not show any statistically significant differences. There were no visible lesions seen during the necropsy that could be linked to the therapeutic regimen. There were no variations in the males' organ weights at the end of the treatment period. At the 600 mg/kg/day dosage level, females had a considerably greater relative liver/body weight than the control group. The absence of any histological correlations in the liver, the somewhat lower liver weights in control females, the lack of a comparable effect in males receiving large dosages, and the lack of any effects on liver function markers, such as blood parameters and serum biochemistry results, made this finding not biologically significant indicate Picture 2 and table no 4.

Table No 3: Effects of 28 Day Administration of Plantago ovata mucilage on Hematological Parameters in Rats

Section	ESR (mm/h)	Hb (%)	RBC (X 10⁶ mm³)	WBC (X 10³ mm³)	Differential Count Percentage
Section	ESR (mm/h)	Hb (%)	RBC (X 10⁶ mm³)	WBC (X 10³ mm³)	N	L	E	M	B
1	7.22 ± 1.11	11.24±1.2	7.11±0.2	8.21±0.2	68.99±0.2	29.2±0.1	11±1.1	2.55±1.2	-
2	7.23 ± 1.19^ns	11.22±1.1^ns	7.21±0.1^ns	8.32±0.1^ns	68.32±0.1^ns	29.3±0.2^ns	1.2±1.2^ns	2.32±0.1^ns	-
3	7.27 ± 1.11^ns	11.23±1.2^ns	7.24±0.1^ns	8.27±0.2^ns	68.99±0.1^ns	29.7±0.3^ns	1.3±1.3^ns	2.37±0.2^ns	-
4	7.26 ± 1.13^ns	11.22±1.3^ns	7.37±0.2^ns	8.33±0.1^ns	68.51±0.2^ns	29.6±0.4^ns	1.4±1.1^ns	2.34±0.1^ns	-

Data are the mean of 6 animals ± S.E, , ns = non-significance

Figure 1: Rats' Hematological Parameters After 28 Days of Mucilage Administration (*n=6, Mean ± S.E.M.)

Figure 2: Effects of 28 Days Administration of mucilage on biochemical Parameters in Rats

Table No 4: Effects of 28 Day Administration of Plantago ovata mucilage on Hepatic Function in Rats

Section	Liver glycogen (mg%)	SGPT (IU/L)	SGOT (IU/L)	ALP (IU/L)	Bilirubin (mg/dL)
1	132.2 ± 0.2	43.42 ± 0.2	116.44 ± 0.1	207.22 ± 0.1	.72±0.11
2	131.4 ± 0.1^ns	41.44 ± 0.1^ns	116.74 ± 0.2^ns	208.41 ± 0.2^ns	.73±0.13^ns
3	132.5 ± 0.3^ns	41.32 ± 0.2^ns	116.20 ± 0.5^ns	208.52 ± 0.3^ns	.74±0.14^ns
4	132.2 ± 0.4^ns	43.48 ± 0.1^ns	116.66 ± 0.4^ns	207.44 ± 0.4^ns	.75±0.15^ns

Data are the mean of 6 animals ± S.E,

Repeated dose administration was used to evaluate the 28-day oral mucilage treatment in Wistar rats. There were no statistically significant variations found in the parameters of the blood biochemistry and urinalysis investigations. Figure 2 and displays the biochemical parameter levels for each group that were evaluated.

The histopathological study did not reveal any evident adverse effects associated with the treatment. There appeared to be a tendency for the incidence of stomach inflammation to increase in females receiving large doses. Figure 3 table shows a slice of the stomach with erosions and surface necrosis of the mucosal epithelium. In a small number of animals, the stomach's serosal surface thickened and deposited fibrinous exudates. The serosal surface exudates showed a significant neutrophil infiltration.

Figure 3: Effects of 28 Days Administration of Plantago ovata mucilage on Kidney Function in Rats (*n=6, Mean±S.E.M)

Figure 4 illustrates a portion of the kidney with some mild tubular parenchymatous degenerations. but there were no discernible changes in the glomeruli. When the liver was compared to control animals, no histopathological changes were discovered. Some animals, though, experienced changes to their fatty livers.

Table No 5: Effects of 28 Day Administration of Plantago ovata mucilage on Kidney Function in Rats

Section	Blood urea (mg%)	Serum creatinine (mg/dL)
1	41.24 ± 1.2	.73 ± 0.1
2	41.55 ± 1.1^ns	.72 ± 0.1^ns
3	41.64 ± 1.2^ns	.74 ± 0.3^ns
4	41.77 ± 1.4^ns	.74 ± 0.2^ns

Figure 4: Effect of Plantago ovata mucilage stomach, Control rats and rats treated with the Plantago ovata mucilage (600 mg/kg)

Figure 5: Effect of Plantago ovata mucilage on liver, rats given 600 mg/kg of the mucilage, and rats used as controls

CONCLUSION:

Mucilage's oral LD50 was more than 600mg/Kg. Furthermore, studies on Mucilage's sub-acute toxicity have revealed that, at test levels, it had no detrimental effects on body mass. biochemical parameters, or hematological assays. Through toxicity testing, Mucilage was found to be well tolerated. The findings demonstrate the safety of Mucilage, and it is recommended to take 600mg of this novel super disintegrant every day.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

AUTHORS’ DECLARATION:

The writers hereby state that the material shown in this piece is unique and that they will take responsibility for any claims concerning the content of this piece.

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Received on 20.07.2024 Revised on 11.12.2024

Accepted on 21.02.2025 Published on 01.07.2025

Available online from July 05, 2025

Research J. Pharmacy and Technology. 2025;18(7):3268-3272.

DOI: 10.52711/0974-360X.2025.00471

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

Group	Body mass in grams, n= 6 Mean ± S.E
Group	Initial	1 days	14 days	21 days	28 days
I	143 ± 1.2	145 ± 1.1	148 ± 1.2	150 ± 1.1	154 ± 1.3
II.	142 ± 1.1^ns	145 ± 1.3^ns	147 ± 1.1^ns	149 ± 1.1^ns	152 ± 1.3^ns
III	142 ± 1.2^ns	147 ± 1.3^ns	149 ± 1.3^ns	150 ± 1.2^ns	155 ± 1.3^ns
IV	145 ± 1.2^ns	147 ± 1.3^ns	149 ± 1.3^ns	151 ± 1.3^ns	154 ± 1.2^ns