Hepatoprotective and Wound Healing Activities of the Halymenia ceylanica Harvey ex Kutzing in Wistar albino mice

Fredrick Raja E.¹*, John Peter Paul J.², Noorjahan A³

¹Assistant Professor, Department of Botany, Sri Kaliswari College (Autonomous),

Sivakasi - 626130, Tamil Nadu, India.

²Assistant Professor of Botany and Director, Centre for Advanced Research in Plant Sciences (CARPS), Department of Botany, St. Xavier’s College (Autonomous), Palayamkottai – 627002, Tamil Nadu, India.

³Biofocus Research Center, Thanjavur – 613001, Tamil Nadu, India.

*Corresponding Author E-mail: fredysmash19@gmail.com

ABSTRACT:

The natural products used for pharmacotherapeutic effects have impressed among the sapiens globally. Marine macro algal components have their proven potential with positive affects in therapeutics substituted for the synthetic derived drugs in the modern allopathic medication system. The liver is one of the vital organs in the body, serving as a hub for nutrition metabolism and excretion of waste products. Hepatotoxicity is a toxic illness that harms the liver caused by an overdose of drugs such as acetaminophen. Red algae have numerous biologically active phytochemical compounds with specific biological functions including various therapeutic actions like anticancer, antiulcer, antioxidant, antimicrobial, hepatoprotective and wound healing activities. The present study was designed to screen the hepatoprotective and wound healing activity of Halymenia ceylanica Harvey ex Kutzing. The dosage of 200 mg/kg and 400 mg/kg was prepared by the ethanolic extract of Halymenia ceylanica for hepatoprotective activity. Acetaminophen (2g/kg body weight for 7 days) helps to induce the Hepatotoxicity for the wistar albino mice. The effects of hepatoprotective were shown for both the dosage groups and it is highly active against the dosage of 400mg/kg ethanolic extract. Histopathological studies have confirmed there is no toxicity in liver tissue at both doses of 200 mg/kg and 400 mg/kg of ethanolic extract. The two different concentrations of ointment ethanolic extract (5% and 10%) Halymenia ceylanica were used to examine the wound-healing activity. It was observed that a 10% ointment of ethanolic extract had remarkable wound healing activity as evidenced by the significant increase in the rate of wound contraction, a decrease in the epithelium period and an increase in tissue tensile strength. The results imply that ethanolic extract of Halymenia ceylanica has the potential hepatoprotective and wound healing activity with the bioactive compounds of pharmacological relevance.

KEYWORDS: Halymenia ceylanica, Bioactive compounds, Albino mice, Hepatoprotective, Wound healing.

INTRODUCTION:

Marine algae are one of the richest biodiversity in the marine environment¹. There are three types of plants found in the marine environment are phytoplankton, marine algae and sea grasses. Algae are important marine living renewable resources in the marine ecosystem².

It comes under the division thallophyta and primitive plants absence of any true root, stem and leaves. Marine algae are diversed into four groups based on the morphological, anatomical, pigments and reproductive systems namely Chlorophyceae, Phaeophyceae, Rhodophyceae and Cyanophyceae³. In ancient times, the people who live along the side of the ocean used the algae as food, medicine, minerals, fodder, fertilizer, etc⁴. The diverse algal species can produce wide varieties of chemical compounds that have unique structures with novel applications⁵. Marine algae are the richest source of carbohydrates, protein and lipids are the potentially healthy food supplement for the human diet with high nutrient value^6,7. Marine macro algae are the richest source of primary and secondary metabolites⁸. Marine algae have the properties of degradation of harmful content like nitrate to nitrite⁹, biofertilizers¹⁰. Marine algae produce a diverse of secondary metabolites with specific applications including cosmetic products, antioxidants, anticancer, anti-inflammatory, antimicrobial, anticoagulants, antiviral activities, etc^11,12,13.

Largest gland is noted as liver and most important organs that serve as a hub for the metabolism of nutrients and the excretion of waste products¹⁴. The main function is to regulate the flow and safety of chemical substances received from the digestive system to the circulatory system if this function is lag at any particular time it may lead to death¹⁵. Acetaminophen is a drug that can take overdose leads to hepatotoxicity which means damage to the liver¹⁶. The review of the algal research may lead to the development of useful medications for the treatment of liver problems in modern medicine¹⁷ but there are currently few reports on red algae about their hepatoprotective efficacy.

Wounds are physical injuries that cause the skin to open or break¹⁸. Restoring the skin compromised functional state and broken anatomical continuity requires proper wound healing. Healing is a multi-step process that helps wounded tissues regains their integrity and function. The collaboration of several different tissues and cell types is necessary for healing¹⁹. The ability to heal wounds more quickly is enhanced by a study on the mechanics of wound healing that describes various phytochemical substances from marine algae and their target influence on the cascade of wound repair²⁰.

With the information from the research papers, the red algae are rich in phytochemical compounds that have the potential to serve from pharmacological perspectives. The commercial drug is causing side effects so nowadays scientists preferred natural medicine. The objective of the research is to evaluate the hepatoprotective and wound healing activities of the ethanol extract of Halymenia ceylanica Harvey ex Kutzing.

MATERIALS AND METHODS:

Algal Sample:

Halymenia ceylanica Harvey ex Kutzing, a fresh red algal sample, was taken from the Mandapam in the Ramanad district of Tamil Nadu, India, at 9° 16' 48.00" N and 79° 07' 12.00" E. The sample was authenticated and placed at the Xavier's College Herbarium, Centre for Biodiversity and Biotechnology, St. Xavier's College (Autonomous), Palayamkottai-627002. The voucher number for Halymenia ceylanica Harvey ex Kutzing was XCH20504. The collected samples were extensively rinsed with marine water to get rid of the epiphytes and silt particles. The samples were well cleaned in tap water to get rid of the salt off the surface of the thalli and rinsed with distilled water. Then the sample moves to the lab in polythene bags²¹.

Preparation of extract:

Halymenia ceylanica Harvey ex Kutzing was extracted with ethanol for 8hours from 30g of fine powder. The sample was shaken at regular intervals for 72hours in the dark. After incubation, the solution was filtered using filter paper, and the filtrate was recovered as crude extracts ²².

Experimental animal:

Wistar albino mice (160-200g) of either gender were obtained from Venkateswara Enterprises, Bangalore, Karnataka, India. The animals were acclimated for seven days under standard husbandry circumstances, which included a 351°C ambient temperature, 45-55 percent relative humidity, and a 12/12h light/dark cycle. The mice were fed a standard mouse pellet diet and had unlimited access to water during the experiment. Dietary components include 10% protein, 4% Arachis oil, 1% fibre, 1% calcium, 1000 IU/g vitamin A and 500 IU/g vitamins D. All of the animals were acclimatized to the laboratory conditions before the testing. All of the studies were carried out between the hours of 10 a.m. and 17 a.m., in compliance with the ethical rules of the International Association for the Study of Pain's ethical guidelines²³. All of the experiments adhered to the Committee for Control and Supervision of Animal Experiments' standards for the care and use of experimental animals (CPCSEA).

Acute toxicity test:

The research was carried out using the OECD-423 criteria for acute oral toxicity²⁴. A random sampling method was used to choose Wistar albino mice (n=6) of either sex for the acute toxicity research. The animals were fed just water for one night while fasting, and then the extract (a 50% ethanolic extract) was administered orally at a dosage of 5mg/kg body weight during the course of a 14-day observation period. If two out of every three animals per dosage die, the dose is considered hazardous. If an animal dies, the same amount is administered twice more to confirm the toxic dose. If no one dies, the treatment will be repeated with greater doses of 500, 1000, 1500, and 2000mg/kg body weight.

Hepatoprotective activity:

a) Induction of acetaminophen hepatotoxicity and experimental design²⁵

Thirty male and female wistar albino mice were selected randomly and evenly splitted into five groups. All of the other mice, except from the control group, received acetaminophen (2g/kg) for seven days. It was determined that all of the animal's liver cells were destroyed after seven days. Group I treated as normal control. Animals in group II were given an oral dose of acetaminophen (2g/kg) powder dissolved in water. Group III treated with Silymarin 100mg/kg orally as the positive control for seven days. Group IV and V were feed oral ethanol extract of the selected algae at 200mg/kg and 400mg/kg respectively for seven days. All of the experimental animals were euthanized on the eighth day by cervical dislocation receiving a minimal amount of ether anesthesia.

b) Biochemical assay:

The intracardiac puncture was needed to collect blood samples in glass tubes, which were then used to analyze liver enzymes. The activity of Serum Glutamic Pyruvic Transaminase (SGPT) and Serum Glutamic Oxaloacetic Transaminase (SGOT) was determined using the Reitman and Frankel method²⁶, the activity of Alkaline Phosphatase (ALP) was determined using the Kind and King²⁷ method, and bilirubin were determined using the Malloy and Evelyn²⁸ method. The livers of the animals were immediately removed and rinsed in ice-cold saline following scarification. A part of the liver was promptly cut into thin cross-sections and examined under the microscope.

Wound healing activity:

a) Formulation preparation:

The extract was used to make two types of ointment formulations, 5% and 10% (w/w), by mixing 10 and 20 g of the extract with 100g of simple ointment base British Pharmacopoeia (BP), respectively²⁹. Povidone-iodine ointment (5 percent w/w) was employed as a control treatment to compare the extract's wound-healing abilities. Animals were split into five groups, each with six animals, the group I, which served as a control and was left untreated; group II, treated as a negative control (ointment base treated); group III, treated as a standard and was treated with 5 percent (w/w) povidone-iodine ointment; and groups IV and V, which were treated with 5 and 10 percentage of ethanolic extract ointment. All of the therapies were administered once a day.

b) Excision wound model:

Totally from the five groups of animals, each including six rats, were shaved on the dorsum section using depilatory cream and sedated with ketamine hydrochloride (50mg/kg, i.p., body weight). An imprint was made on the dorsal area that had been shaved, and the location of the wound was noted. Using toothed forceps, a surgical blade, and sharp scissors, a full-thickness excision incision with a circular area of 314mm² was produced along the marking³⁰. Rats were exposed as undressed to the open environment. From the first day till the experimental completion, the simple ointment base, designed extract ointment, and standard medicine were used once daily. The wound contraction and epithelialization period were assessed in this model. Wound contraction was measured as a percent contraction every fourth day after the initial formation of the wound All of the rats were sedated at the end of the trial, and samples were taken from the wounds, leaving a 4mm margin of normal skin surrunding the borders of the healed wounds. Histopathological and biochemical tests were conducted on specimen tissues preserved in a 10% formalin solution.

Measurement of Wound Contraction and Epithelialization Period:

The wound area of the excision model was determined by tracing the wound with the transparent sheet using millimeter-based graph paper. The percentage of wound contraction was calculated by the following formula

% Wound contraction =

(Initial wound area – Specific Day wound area)

------------------------------------------------------------ × 100

Initial wound area

The number of days necessary for the dead tissue remains of the wound to come off without any remaining raw wound was determined as the epithelialization period

RESULTS AND DISCUSSION:

Acute toxicity study:

At a dosage level of 2000 mg/kg, there were no indicators of toxicity or fatality in the acute toxicity trials. Therefore, the therapeutic dosage was 200 mg/kg b.w. and 400mg/kg b.w.

Hepatoprotective Activity:

The hepatoprotective efficacy against acetaminophen induced Wistar albino mice is analyzed in the ethanolic extract of Halymenia ceylanica Harvey ex Kutzing. The ethanolic extract of Halymenia ceylanica Harvey ex Kutzing was given in two different doses of 200 mg/kg and 400 mg/kg body weight for liver damage. Both the doses of ethanolic extract of Halymenia ceylanica Harvey ex Kutzing clearly showed the hepatoprotective activity against the experimental animal.

In the hepatoprotective activity assay of the ethanolic extract of Halymenia ceylanica Harvey ex Kutzing the levels of Alkaline Phosphatase (ALP), Serum Glutamic Oxaloacetic Transaminase (SGOT), Serum Glutamic Pyruvic Transaminase (SGPT) and bilirubin levels were significantly higher in the treated wistar albino rats compared to the control group. The levels of SGPT, SGOT, ALP, and bilirubin in the control group were 64.03 IU/L, 71.13 IU/L, 14.21 IU/L, and 1.28mg/dL, respectively, whereas the acetaminophen (2g/kg) induced group of animals had 131.13 IU/L, 142.01 IU/L, 55.01 IU/L, and 2.72mg/dL, respectively The standard drug Silymarin (100mg/kg) reduced the levels of SGPT (69.02 IU/L), SGOT (77.4 IU/L), ALP (16.24 IU/L), and bilirubin (1.36mg/dL) levels.

Table 1 shows the impacts of the ethanolic extract of Halymenia ceylanica Harvey ex Kutzing on serum enzyme parameters such as SGPT, SGOT, ALP and bilirubin. The wistar albino mice treated with the acetaminophen (2g/kg) had significantly greater levels of the serum enzymes SGPT, SGOT, ALP, and bilirubin levels were considerably higher than the control group. Acetaminophen-induced increases in serum SGPT, SGOT, ALP, and bilirubin levels have been linked to hepatic structural damage since these enzymes are typically located in the cytoplasm and released into the bloodstream following cellular injury extract of ethanol. Mice treated with 200mg/kg ethanol extract of Halymenia ceylanica Harvey ex Kutzing had significantly lower levels of SGPT (74.01 IU/L), SGOT (83.01 IU/L), ALP (19.02), and bilirubin (1.52mg/dL) than treated with acetaminophen. Mice treated with 400mg/kg ethanol extract of Halymenia ceylanica Harvey ex Kutzing had significantly lower levels of SGPT (67.17 IU/L), SGOT (76.03 IU/L), ALP (15.01) and bilirubin (1.34mg/dL)than treated with acetaminophen. The restoration of SGPT, SGOT, ALP, and bilirubin levels to normal indicated that the plasma membrane had stabilized and that the hepatic tissue damage induced by paracetamol had been repaired. The ethanolic extract of Halymenia ceylanica Harvey ex Kutzing was shown to exhibit hepatoprotective effects at both doses. The 400mg/kg ethanolic extract of Halymenia ceylanica Harvey ex Kutzing had a greater hepatoprotective effect than the 200mg/kg ethanol extract.

Histopathological studies in liver sections (100X) from the experimental groups were shown in Figure No.1. The histopathological profile of the liver sections of control group revealed a normal cellular structure with usual hepatic cells, sinusoidal spaces and central vein, liver sections treated with acetaminophen produced disruption or misarranging of hepatic cells with centrilobular necrosis, vacuolization of cytoplasm and fatty degeneration. The experimental animal of the liver sections treated with ethanolic extract of Halymenia ceylanica Harvey ex Kutzing showed a sign of protection as it was evident by the considerable changes and back to the normal hepatic cells and the arrangement in the pathological changes when compared with acetaminophen induced animals.

DISCUSSION:

Marine algae are rich in phytochemical compounds that have the potential to be employed as nutritive food or as a therapeutic effect for chronic illness. Marine algae are the major source of synthetic substances that could contribute to improving consumer well-being by being incorporated into new functional foods or medications. Natural biologically active phytochemical compounds receive a lot of attention from consumers as a functional food and medicinal perspectives³¹. Hepatotoxicity is induced in Wistar albino rats and leads to an increase in the level of serum enzyme which is attributed to the liver cell membrane destruction and the release of transaminase from the cytoplasm into the bloodstream. The brown algae Padina boergesenii is perform the antioxidant activity against CCl₄ due to the presence of carotenoids-induced free radical damage. It proved its activity through the stabilization of the cellular membrane³². Spirulina platensis, Chlorella vulgaris, Laminaria japonica and Sargassum species also revealed hepatoprotective activity against the CCl₄-induced Wistar albino rats and also had antioxidant activity. The histopathological analysis also proves the decline of liver toxication by the stabilization of cell membrane³³. The hepatoprotective, antiulcer and wound healing activities are shown in the Turbinaria ornate, Gracilaria crassa, and Laurencia papillosa. Among these Gracilaria crassa was significant as it showed prominent and noteworthy wound healing and hepatoprotective activities³⁴. The freshwater algal species also have the properties of wound healing activity. Oedogonium intermedium and Oedogonium globosum are freshwater algal species are good candidates for medical use including wound healing activity. Oedogonium intermedium shows the maximum percentage of wound healing³⁵. Cystoseira trinodis, brown algae plus polyherbal extract have the properties of wound healing activity. The experimental medicinal extract mixture promoted the activity of wound healing and tissue granule formation in the experimental animal³⁶.

With the respect to above research, the present study examined the hepatoprotective and wound healing activity of Halymenia ceylanica. A widely used and effective treatment model for assessing the efficacy of hepatoprotective natural substances is acetaminophen-induced hepatotoxicity in mice. The liver injury in this model generally develops after 3-5 hours and peaks after 12 hours with Acetaminophen doses up to 2g/kg. The ethanolic extract of Halymenia ceylanica showed a significant hepatoprotective effect. It has been observed that an ethanol extract of the Halymenia ceylanica might stop the biochemical alterations of liver damage caused by acetaminophen. The intracardiac puncture was used to assess liver enzyme serum for the test of SGPT, SGOT, ALP, and Bilirubin, which gives a fair idea of the functional status of the liver. The dosages of Halymenia ceylanica ethanolic extract employed in the studies were 200mg/kg and 400mg/kg. The extract's power to restore anti-oxidative enzyme function may account for its ability to protect the liver from damage brought on by acetaminophen. The wound healing activity shows the maximum results in the extract of Halymenia ceylanica compare to the standard povidone. The 10% ointment of ethanol extract of Halymenia ceylanica shows a considerable rise in the rate of wound contraction which concluding the significant wound healing activity. It is possible to draw the conclusion that the ethanol extract of Halymenia ceylanica possesses hepatoprotective and wound-healing capabilities, which might result in the development of a novel drug.

CONCLUSION:

Halymenia ceylanica Harvey ex Kutzing showed the acceptable hepatoprotective action and wound healing activity in wistar albino mice in this study. For the research of hepatoprotective activity, it was concluded among the two doses of ethanolic tested; 400mg/kg was shown to be more effective than 200mg/kg. Histopathological analysis of liver sections from the mice treated with the toxicant showed significant misarranging of the hepatic cells. The lack of the necrosis and vacuoles in the mice treated with the doses of ethanolic extract. The tested sample was revealed to exhibit significant dose-dependent hepatoprotective effectiveness as a consequence. For the research of wound healing activity, showed that 10% ointment of aqueous extract of ethanol extract possesses significant wound healing activity as evidenced by the significant increase in rate of wound contraction. The presence of phytochemical constituents such alginates, saponins, alkaloids, tannins, and terpenoids in the plant extract may be responsible for the wound healing activities. To justify its usage in herbal formulations prescribed for the treatment of liver problems, further extensive research is needed to discover and isolate the specific compounds for this activity of hepatoprotective and wound healing in the ethanolic extract of Halymenia ceylanica Harvey ex Kutzing. Furthermore, public and medical education is sought to raise public awareness of the possible toxic effects of the commercial drug and the importance of natural medicines.

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Received on 11.07.2022 Revised on 15.12.2023

Accepted on 26.10.2024 Published on 20.01.2025

Available online from January 27, 2025

Research J. Pharmacy and Technology. 2025;18(1):273-279.

DOI: 10.52711/0974-360X.2025.00042

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

S. No	Animal Group	SGPT (IU/L)	SGOT (IU/L)	Alkaline Phosphatase (IU/L)	Bilirubin (mg/dL)
1	Control	64.03± 1.21	71.13±1.02	14.21±0.03	1.28±0.21
3	Acetaminophen (400mg/kg)	131.13±1.22	142.01±3.12	55.01±0.98	2.72±.43
2	Silymarin (100mg/kg)	69.02±1.21	77.14±1.11	16.24±0.12	1.36±0.37
4	200mg/kg dosage	74.02±0.97	83.01±0.51	19.02±0.11	1.52±0.27
5	400mg/kg dosage	67.17±0.81	76.03±0.23	15.01±0.13	1.34±0.11

Group	Experimental Groups	Remaining original excision wound area (mm²)
Group	Experimental Groups	0^th day	10^th day	20^th day
I	Control (Simple ointment)	521.16±2.23	281.31±3.12	40.21±1.27
II	Standard (Povidone iodine ointment)	518.14±4.10	230.13±1.98	11.02±0.27
III	5% ointment (Test sample)	521.03±4.15	274.53±2.10	17.22±0.97
IV	10% ointment (Test sample)	520.19±3.97	226.53±3.43	9.57±0.16