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Effects of Ethanolic Crude Extract of Mangosteen (Garcinia mangostana) Linn. Pericarp on Irradiated Webster White Mice (Mus musculus) Linn.

ABSTRACT

Radiation exposure can cause damage to organ tissues if not observe. The researcher conducted the study on mangosteen (Garcinia mangostana L.) pericarp ethanolic extract which contains high antioxidant content, the xanthone. The study is about the effect of xanthone extract from the mangosteen fruit that may protect any pathological conditions caused by X-radiation. The xanthone extract was administered for two weeks on Webster white mice (Mus musculus) Linn. prior to irradiation and evaluation. Twenty-four mice were divided into four groups (control group, treatment with radiation exposure, no treatment with exposure, treatment with no exposure). A histopathological study was conducted to assess the protective activity of xanthones from the mangosteen extracts. Histopathological analysis found that the mice treated with xanthone extract and exposed to X-radiaton show no significant lesions in the mammary gland tissues of the white mice compared to the mice without treatment of mangosteen pericarp extract and exposed to x-radiation. The use of mangosteen pericarp extract showed indications of minimized presence of any pathological condition caused by radiation exposure, assessing the administration of supplement and placement and ecological aspect that may affect the health and behavioral status of the mice.

Keywords: Radiation, antioxidant, mangosteen, mammary gland, Webster white mice

INTRODUCTION

The advancement in technology and rapid industrialization increasingly placed everyone at risk of being exposed to ionizing radiation and its detrimental effects. This type of radiation is used both in the diagnosis and treatment of disease conditions through radiographic procedures or therapy in acute or chronic exposures. The popular utilization of this medium as a primary method of diagnosing disease (Callaway and Gurley, 2006) is beneficial in the medical field.

However, there are biological effects resulting either from direct or indirect exposures (Bushong, 2010). This interaction may lead to the transfer of energy to the atoms of the material. The greater the energy being transferred or absorbed, the higher the chances of biological effects in the patient's body (Statkiewicks, 2006). Since the human body is composed of 60 to 80 percent water of the body weight and is considered as the most abundant chemical substance in the human body (Marieb, 2006), this ionizing radiation interacts with the water molecules as the principal radiation interaction in the body resulting to dissociation thereby forming free radicals through radioloysis (Bushong, 2009). This indirect hit is vital in the induction of the oxidative stress which may further lead to cell damage and dysfunction of the organs (Berroud et al., 1996, Bushong 2008, and Bushong 2010).

For more than 50 years, the effects of antioxidants to potentially reduce cellular damage induced by ionizing radiation has been studied using animal models (Weiss and Lauder, 2003) and the results played an important role in providing protection to the cells against ROS species injury during radiation exposure (Zhang et al., 2001). There are dietary antioxidants that exhibit radioprotective effects that limit hematopetic cell death in animals receiving moderate exposure of whole body proton irradiation (Sanzari et al., 2011).

However, the application of antioxidant radioprotectors to human exposures including radiation exposures during diagnosis and treatment has not been extensively studied and established. Though the use of endogenous antioxidants is generally acceptable, such as cellular non-protein thiols and antioxidant enzymes are said to provide a certain degree of protection (Weiss and Lauder 2006).

This research study looked into the protective effects of the mangosteen fruit (Garcinia mangostana) L. which contains a compound called xanthone, which is thought to have antioxidant properties. Because of its many benefits, mangosteen has been known to provide a number of anecdotal effects for antioxidant protection against free radicals, maintains immune system health, promotes joint flexibility, provides positive mental support, reduces allergies, reduces joint inflammation, prevents cancer and others (Wadsworth et al., 2007).

The mammary glands are secretory tissues that have zinc (Zn) and is an essential micronutrient required for over 300 different cellular processes, including DNA and protein synthesis, enzyme activity, and intracellular signaling. Impairments in Zn secretion from the mammary gland are associated with disorders such as diabetes, infertility, and cancer, respectively (Kelleher, McCormick, Velasquez & Lopez, 2015).

This study is called for an experiment that it may successfully provide useful information and further means of radiation protection. It is our common knowledge that radiation is present in our natural environment for which every living thing is exposed in varied degrees especially radiologic technologists, patients and the rest of workers within the radiologic facility.

OBJECTIVES OF THE STUDY

The general objective of this study is to determine the effects of ethanolic crude extract of mangosteen (Garcinia mangostana) L. pericarp on the irradiated Webster white mice (Mus musculus) Linn. Specifically it aimed to: 1) to determine the effects of ethanolic crude extract of mangosteen (Garcinia mangostana) L. pericarp on the irradiated mammary glands of Webster white mice (Mus musculus) Linn.; and 2) to determine the histopathological findings of the effects of radiation to the mammary glands of the Webster white mice (Mus musculus) Linn.

MATERIALS AND METHODS

Research Protocol

The researcher sought approval from the Vice President for Academic Affairs. The Director of the RPO thoroughly assessed and reviewed the manuscript for quality assurance and quality control then, an approval was given. The researcher accomplished a standard form from the University Ethical Board in compliance with the ethical standards and consideration in securing and use of animals for research purposes then, approval will be made.

Training on the proper caging and disposal of experimental mice based on RA. 8485, Section 6, humane procedures on the use of the most scientific method available as may be determined and approved by the Committee. The care and feeding of the experimental animals were done in the University. The exposure of the animals was conducted at the Liceo de Cagayan University X-ray Clinic/Laboratory. The researcher requested the services of the pathologist from the University to interpret the histopathological data.

Preparation of the Cage

Cage was made of aluminum screen nailed on plywood borders. The height of the cage is 0.25 meter, the width is 0.5 meter and the length is 1 meter and made of 10 divisions. Another cage was bought from the local sellers with 10 compartments; this is to prepare for the pregnant female mice to be separated from their mates.

Acquisition of mice

The researchers purchased the white mice in a local mice seller at Bolonsore, Camaman-an, Cagayan de Oro City for the purpose of the study. The researchers bought 10 pairs of white mice and were housed at Opol, Misamis Oriental. The mice were placed in a cage with 10 compartments. Each compartment contains a pair of white mice, 1 male and 1 female for breeding.

Breeding and reproduction of white mice

The researcher fed the 10 white mice with food and plenty of fresh water twice daily (morning and evening) to ensure that the mice will be healthy. This is to increase the likelihood of breeding. The breeding of the white mice lasts within 3 to 6 days. After days of breeding, the female mice were pregnant after almost a week of mating. When the female mice were pregnant the researchers immediately separated the male and female mice. The researchers waited for 3 weeks for the mothers to give birth to its offsprings. After giving birth, the researchers waited for 3-4 weeks for the pups to be weaned then be separated them from their mothers. After then, the researchers waited another three weeks for the pups to reach its maturity for the experimentation study.

Separation of the mice for experimentation

After 3 weeks of waiting, the pups were now matured and ready for experimentation. The researchers divided the matured white mice into four groups. Each group was composed of 3 female and 3 male white mice and placed in a different compartment according to their gender to avoid mating. The first group of 6 white mice were placed as the control group (T0Rn). The second group was placed in a different compartment, 6 white mice was administered mangosteen pericarp extract and was also exposed to radiation (Treatment with Exposure - T1Rn). The third group with 6 white mice, were administered with mangosteen pericarp extract but was not exposed to radiation (Treatment without Exposure--T2Rn). The fourth group of 6 white mice were not administered with mangosteen pericarp extract but was exposed to radiation (No Treatment with Exposure--T3Rn).

Preparation of the extract

The researcher bought 1 kilogram of mangosteen in the local fruit stand. The most abundant source of antioxidant xanthone in mangosteen is its pericarp (Tohi, 2013). The researchers then separated the pericarp from the seeds. Dried mangosteen (G. mangostana L.) pericarps were obtained from sun drying. The dried sample was crushed by a hammer until it was grinded into at least 3 mm particle size. The dried sample powder was packed in plastic bags and stored in darkness for one night (Yoswathana, 2012).

Maceration

The researcher underwent a seminar on the proper extraction of the important component of the mangosteen fruit at AVR 3, Liceo de Cagayan University. The maceration was performed in a 250 ml beaker filled with 5 grams of dried powder soaked in a 100 ml of 95% ethanol. The extraction was carried out at room temperature for 30 minutes to 2 hours without shaking. The amount of mangosteen pericarp extract during the 2 hours maceration was about 0.06 mg/g dried sample. It expires for 3-5 days (Yoswathana, 2012).

Treatment

Twenty-four (24) white mice were needed for experimentation. Twelve (12) of which were administered with mangosteen pericarp extract as the treatment groups. Treatment with exposure--T1Rn and 6 white mice (3 females and 3 males). Treatment without exposure--T2Rn. The researcher continued to administer 1mL of mangosteen pericarp extract using a plastic syringe for 2 weeks. Another 6 white mice (3 females and 3 males) were assigned in the control group. Controlled Group --T0Rn was neither administered with mangosteen pericarp extract nor irradiated. The other 6 white mice (3females and 3males) No treatment with Exposure--T3Rn group were not administered with mangosteen pericap extract but was exposed to radiation.

Experimental Stage

The researcher ensured that the mice were all in a healthy condition from the beginning of the treatment until 2 weeks of administration. The researchers then exposed the 11 white mice with the use of technical exposure factor 60 kvp, 100 mA 1 second at 25 inches of SID with the interval of 1 minute (Martinez et al., 2014). The other 6 white mice was fed with mangosteen pericarp extracts but was not exposed to radiation. The last group was the control group which was neither administered with mangosteen pericarp extract nor irradiated.

Observation

All white mice were then observed by the researcher if there were any physiological reactions during treatment. During the first day of administration, the female mice under mangosteen pericarp extract without exposure (T2) were very active and kept on going the same path in a circular motion while male mice under mangosteen pericarp extract with radiation exposure (T3) became frail. During the 11th day of administration, one female mouse was not able to survived under the group treatment without radiation exposure (T2) due to asphyxia.

Histopathology of the mice organs

The mice were brought to Central Mindanao University- College of Veterinary Medicine for preparation and reading of slides. The organs were preserved in 10% formalin solution and placed in the slides and were interpreted by the pathologist.

RESULTS AND DISCUSSION

Objective 1: To determine the effects of the anti-oxidant activity of mangosteen pericarp extract on specific organs mammary and prostate gland to the irradiated Webster white mice.

Based on the study of MacNulty (1996), figure 7, the normal appearance of the mammary gland is when the inner layer are the actual ductal epithelial cells whereas the outer layer of cells is, in fact, a layer of myoepithelial cells. It composed mostly of pale white connective tissues, interlobular septa with scattered lobules containing small dark cross cuts of many intralobular ducts. There were very few, if any, secretory alveoli in the inactive gland most of the interlobular tissue is adipose tissue. As demonstrated in the histopathological appearance of the Replication 1, Plate 6, it shows presence of many adipose tissue and myoepithelial cells, which implies that the mammary gland in the control group has no significant lesion. Replication 1, Figure 8 is a representation of replication 2 and 3 of the control group since it appears to be of the same result according to the pathologist.

Figure 8 and 9 are cross-sectional views of the female Webster white mice mammary gland showing histopathological appearance of the replication one ([R.sub.1]) and two ([R.sub.2]) mangosteen pericarp extract with radiation exposure ([T.sub.1]). Based on a study, mangosteen pericarp extract was found to significantly inhibit the proliferation of human breast cancer cell. An ethanolic extract from mangosteen pericarp was found to have anti-proliferative activity on cells (Moongkarndi et al., 2004). As demonstrated in the histopathological appearance of figure 8, [T.sub.1] [R.sub.1] shows presence of alveoli and adipose tissue in which there were no pathological signs shown as similar in Plate 5 the control group. It implies that plate 9 has no significant lesion. In figure 9, [T.sub.1][R.sub.2] shows presence of congestion in the vessel, which means it has an excessive accumulation of blood in the vessel due to improper dissection of the organ, thus it is an exemption if there were any pathological indication caused by radiation, according to the pathologist. This indicates that the mangosteen pericarp extract has a radioprotective effect against radiation exposure. Replication 1 and 2, figures 8 and 9 are representations of replication 3 of the treatment with radiation exposure group since it appears to have the same result according to the pathologist.

Objective 2: Identify the histopathological condition of the mammary gland of the white mice administered with mangosteen pericarp extract without radiation exposure.

Figure 10 is a cross-sectional view of the female Webster white mice mammary gland showing a histopathological appearance of replication one ([R.sub.1]) of mangosteen pericarp extract with no radiation exposure ([T.sub.2]). As demonstrated in the histopathological appearance of figure 10, it shows the presence of mostly pale white connective tissue interlobular septa with scattered lobules containing small dark cross cuts of many intralobular duct with intact alveoli. Most of the interlobular tissue is adipose tissue. This implies that there is no significant lesion as a result of administrating mangosteen pericarp extract. Various studies have shown that phytoceuticals in mangosteen (in some cases known to be its xanthones) have properties such as: anti-tumor (shrinks tumors), anti-leukemia, antifungal (critical for all cancer patients), antibacterial (to protect DNA), antioxidants (at least two dozen different kinds of xanthones are in the mangosteen fruit), anti-proliferation, kills cancer cells and causes apoptosis (programmed cell death) for some types cancer (Kehr, 2012). Replication 1, figure 10 is a representation of replication 2 and 3 of the treatment with no radiation exposure since it appears to have the same results according to the pathologist.

Figure 11 is a cross-sectional view of the female Webster white mice mammary gland showing the histopathological appearance of replication one ([R.sub.1]) of no mangosteen pericarp extract and with radiation exposure ([T.sub.3]).

The effects of ionizing radiation resulted to lesions that occur in multiple organ/tissues and affected the parenchyma or epithelia and the blood vessels. The vascular lesions are quite consistent. Most often they affect the micro vessels (capillaries, sinusoids) producing lethal and sublethal damage to the endothelial cells, with capillary rupture. Damage in large vessels is less common; it occurs more in arteries than in veins (Fajardo, 2005).

As demonstrated in the histopathological appearance of the [T.sub.3][R.sub.1] presence of hemorrhage (Hem.) and presence of artifact (A) is shown. Figure 11 indicates that radiation exposure has a severe effect to the tissue of the mammary gland of the Webster white mice. This may also indicate that cutting of the organ has not been properly done due to the artifact present. Replication 1, figure 11 is a representation of replication 2 and 3 of the no treatment with radiation exposure group since it appears to have the same result according to the pathologist.

CONCLUSIONS

The presence of antioxidant mangosteen (Garcinia mangostana) L. significantly minimizes pathological condition and decreases the appearance of free radicals caused by radiation exposure. According to the pathologist, the histopathologic findings also show that the presence of congested vessels found in the mammary gland do not affect the progress of enhancement in the treatment. Therefore, the amount and concentration of antioxidant extracted from mangosteen fruit (Garcinia mangostana) Linn. is sufficient enough to manifest the enhancement of the selected organs caused by radiation exposure. The technique used and the amount of concentration that was extracted from mangosteen pericarp in this study played a vital role on the effects on the white mice. The result was indicative that mangosteen pericarp extract protect the selected organs such as the mammary gland against radiation. Exposure to radiation without treatment could cause damage or death to the tissues or organs in the body. Based on the results, the researchers concluded that mangosteen pericarp extract may have the potential to minimize pathological conditions caused by radiation exposure. The coloration of the organ tissue during the histopathologic findings is due to failure of removal of ethanol on the mangosteen pericarp extract solution via evaporation.

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EDZEN A. ESPINA

ORCID No. 0000-0001-5686-1860

eaespina@liceo.edu.ph

Liceo de Cagayan University Cagayan de Oro City, Philippines
Table 1. Control Group (TO) Mammary Gland

Subject                                Result

Female mice- Replication 1([R.sub.1])  Normal
Female mice- Replication 2([R.sub.2])  Normal
Female mice- Replication 3([R.sub.3])  Normal

Table 2. Treatment with Radiation Exposure (T1) Mammary Gland

Subject                                          Result

Female mice- Replication 1([R.sub.1])            Normal
Female mice- Replication 2([R.sub.2])  Congestion of blood vessel
Female mice- Replication 3([R.sub.3])            Normal

Table 3. Treatment with No Radiation Exposure ([T.sub.2]) Mammary Gland

Subject                                 Result

Female mice- Replication. 1([R.sub.1])  Normal
Female mice- Replication. 2([R.sub.2])  Normal
Female mice- Replication. 3([R.sub.3])  Normal

Table 4. No Treatment with Radiation Exposure ([T.sub.3]) Mammary Gland

Subject                              Result

Male mice- Replication 4([R.sub.4])  Hemorrhage
Male mice- Replication 5([R.sub.5])  Hemorrhage
Male mice- Replication 6([R.sub.6])  Hemorrhage
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Author:Espina, Edzen A.
Publication:Asian Journal of Health
Article Type:Author abstract
Geographic Code:9PHIL
Date:Jan 1, 2015
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