The Effect Of Durian Skin Extract ( Durio zibethinus Murr.) On Histological Features Of The Lungs Rats ( Rattus norvegicus ) Induce 7,12-Dimetylbenz (Α) Anthracene (Dmba)

Durian peel extract is one of the raw materials for herbal medicine that contains large amounts of flavonoids, such as flavones and flavanols, which function as antiproliferative and antioxidant. The research was conducted from September 2022 to March 2023 which aimed to analyzed the effect of durian peel extract on the morphological and histological of rat lung induced by 7.12-Dimethylbenz (α) Anthracene (DMBA). The samples used were 24 male rats (Rattus norvegicus) with a weight ranging from 150 to 200 grams. This method of the study used a Completely Randomized Design consisting of six treatments and four replications. Each treatment group was divided into a negative control (normal); positive control (DMBA 10 mg/kgBW); P1 (DMBA 10 mg/kgBW + methotrexate 9 mg/kgBW), P2 (DMBA 10 mg/kgBW + durian peel extract 150 mg/kgBW); P3 (DMBA 10 mg/kgBW + durian peel extract 300 mg/kgBW), and P4 (DMBA 10 mg/kgBW + durian peel extract 450 mg/kgBW). The results of this research showed that durian peel extract had significant (p<0.05) to improved the morphological damage (color, texture, weight, and volume) as well as its histology from the degeneration, inflammation, congestion


Introduction
The incidence of cancer in the world at the end of 2020 reached 19.2 million with 9.9 million deaths, so it is estimated that the number of cancer patients in 2030 will be 26 million and 17 million of them can die.The data shows that cancer ranks second as a cause of death worldwide with a prevalence of 13% after cardiovascular disease.The most common types of cancer found generally in men are lung, prostate, colorectal, stomach and liver cancer, while breast, cervical and thyroid cancer often occur in women [1].Based on statistical data obtained, the number of cancer prevalence in Indonesia is 1.79% [2].The high prevalence of cancer can be caused by genetic factors, sex, age, chemicals, drugs and immunological nutrition [3].
Cancer is a disease caused by abnormal proliferation of body tissue cells with damaged mechanisms.Cancer cells grow rapidly, uncontrollably and infiltrate (invasive) in the tissues of an organ, then spread (metastasize) to other organ tissues so as to inhibit the growth of biologically normal cells.Under normal conditions, cancer cells come from the division of cells that have died and damaged, vice versa Cancer cells cause unnecessary buildup of new cells due to continuous division under abnormal conditions.Excessive buildup of abnormal cells can damage normal tissue in certain organs rapidly in the body [4].
7,12-Dimethylbenz (α) Anthracene (DMBA) belongs to chemicals from the Polycyclic Aromatic Hydrocarbon (PAH) group [5].DMBA has mutagenic, teratogenic, carcinogenic, cytotoxic and immunosuppressive properties.DMBA is produced through a natural and incomplete combustion process in tar combustion from cigarette smoke, wood burning smoke and gas combustion smoke [6].The resulting DMBA can enter if injected through the skin (subcutaneous) and further accumulates in vital organs in the body, namely the kidneys, liver, heart and lungs [7].The entry of DMBA will form a receptor bond with the Aryl Hydrocarbon Receptor (AHR).AHR is an important receptor in the process of binding DMBA with the nitrogenous base malonialdehyde so that it can damage DNA (Kleiner et al., 2002).DMBA binding with AHR activates enzymes in the liver with the help of cytochrome P450 to form DMBA 5,6-dihydrodiol and 7,12epoxid which play an important role in generating DNA adducts that cause early cancer [8].Induction of carcinogenesis using DMBA is highly specific for the creation of models of breast, lung, skin and mouse liver cancer.Each male wistar rat (Rattus norvegicus) experimentally induced using a single dose of DMBA as much as 4.2 mg / day for 30 days can trigger cancer in mouse lung cells [9].According to [10], cancer induction using DMBA involves free radical reactions or Reactive Oxygen Species (ROS) and lipid peroxidation so as to damage the structure of the alveoli and damaged cells causing inflammation (inflammation) in mouse lung cells.
The lungs are respiratory organs that function as a place of oxygen and carbon dioxide metabolism, so it is easier to experience interference due to particles, toxic substances and pathogenic microorganisms transported by the blood [11].Blood is generally highest distributed in lung tissue, followed by liver, kidneys, heart, brain, fat and bone.The large blood supply in lung tissue causes a drug and carcinogen-induced chemicals to be distributed quickly [12].
Cancer in general can be treated through surgery, agent chemotherapy and radiotherapy.In long-term chemotherapy exposure, the drug causes various physiological complications and some cancer cells can be resistant to certain chemotherapy drugs [13].In addition to inhibiting the proliferation of cancer cells, the treatment carried out has side effects that are not good for normal cells.Methotrexate drugs have toxic side effects, namely hair loss, bone marrow suppression, drug resistance, gastrointestinal lesions, neurological dysfunction and cardiac toxicity.
Durian bark is one of the herbal medicinal raw materials that has been studied to have anticancer, anticardiovascular, antidiabetic pharmacological effects [14], antioxidant [15], antibacterial [16], antiobesity [14] and antihypertensive [17].In the bark of Durio zibethinus Murr.there are secondary metabolite compounds namely flavonoids, phenolics, alkaloids, steroids, saponins and a little terpenoid [18].The largest number of flavonoid compounds in durian skin are flavones and flavonols that function as antiproliferatives and antioxidants.Flavonoids as antiproliferatives are able to prevent apoptosis, deactivate the cancer cell cycle through the mechanism of inhibition of topoisomerase and cytochrome P450 enzymes and stimulate the activation of the enzyme gluthatione S-transferase to eliminate and destroy toxic substances in the body [19].Other flavonoids in durian skin are catechins and quercetin [20].Quercetin inhibits the proliferation of prostate, lung, liver, breast and cervical cancer cells [21].Based on the description above, a study has been conducted to describe and analyze the effect of durian bark extract (Durio zibethinus Murr.) on histological features of rat lungs (Rattus norvegicus) induced by 7,12-Dimethylbenz (α) Anthracene (DMBA).

Making Durian Fruit Skin Extract
Durian peel extract is made using the extraction method.Durian skin is dried using an oven and then cut into small pieces.The cut skin is smoothed using a blender.The results obtained are in the form of powder and then macerated with 70% ethanol solvent and placed on a shaker for 2 hours.The resulting solution extract can be filtered and concentrated with a rotary evaporator vacuum at 40 °C so that a thick extract is obtained.Phytochemical screening tests on viscous extracts are carried out to determine the chemical compounds contained in the skin of durian fruit [22].

Cancer Induction in Experimental Animals
DMBA-induced male rats were rats of the positive control treatment group (K+), treatment groups P1, P2, P3 and P4.The positive control group (K+), P1, P2, P3 and P4 were induced by DMBA at a dose of 10 mg/kgBB dissolved in corn oil and injected subcutaneously into the nape of rats every 2 days 10 times for 3 weeks.DMBA injected every 2 days as much as 10 times with a dose of 10 mg / kg BB causes damage at the cellular level, namely hydropic and parenchymatous degeneration, dilated sinusoids, necrosis and congestion (blood clots in the central vein) [23].

Durian Skin Extract
Durian peel extract was given to rats of groups P2, P3 and P4 after DMBA induction.The extract was administered orally using a sonde daily for 3 weeks.Durian peel extract was given to the P2 group with a dose concentration of 150 mg / kg BB, in the P3 group with a dose concentration of 300 mg / kg BB and in the P4 group with a dose concentration of 450 mg / kg BB.

Organ Harvesting and Preparation
According to [24], the process of making preparations until they are ready to be observed, can begin with the stages of fixation, dehydration, purification, dealcoholization, infiltration, EmbeddingCutting Affixing, deparafinization, staining and Mounting.

Rat Lung Weight
Data on lung weight and the results of its analysis can be seen in appendix 3. ANOVA results showed that administration of durian bark extract had a significant effect on lung weight (p<0.05).Analysis results post hoc Duncan presented in histogram form in Figure 3.1.
Based on the results of the post hoc test in figure 4.1, it can be seen that the DMBA-induced K(+) treatment group has a higher average lung weight of 5.39 g, while the methotrexate-induced treatment group and durian bark extract doses of 150 mg / kg BB, 300 mg / kg BB and 450 mg / kg BB decreased with an average lung weight of P1 (5.19 g), P2 (4.88 g), P3 (4.69 g), P4 (4.37 g) and the K(-) treatment group had the lowest average lung weight of 3.56 g.Increased lung weight in K(+) treatment group rats can occur because the DMBA carcinogen has entered, accumulated, accumulated and is continuously toxic to lung organs.According to [25], toxic materials can inhibit oxygen exchange, reduce the surface area of the lungs and cause wrinkles in part or all of the lung lobes.

Rat Lung Volume
Lung volume data and the results of their analysis can be seen in appendix 4. ANOVA results showed that the administration of durian bark extract had a significant effect on lung volume (p < 0.05).Analysis results post hoc Duncan presented in histogram form in Figure 3  Based on the results of the post hoc test in figure 3.2, it can be seen that the DMBA-induced K(+) treatment group had a higher average lung volume of 7.67 ml, while the methotrexate-induced treatment group and durian bark extract doses of 150 mg/kgBB, 300 mg/kgBB and 450 mg/kgBB decreased with an average lung volume, namely P1 (7 ml), P2 (6.62 ml), P3 (6.17 ml) and P4 (5.75 ml).The K(-) treatment group had the lowest average lung volume of 5.15 ml.The increase in lung volume in the K(+) treatment group occurred because the alveolus had swollen (enlarged) and filled with blood.Swelling of the alveolus due to DMBA induction can cause turbidity and tissue density that inhibits circulatory function in the lungs of mice.According to [26], alveolar damage due to DMBA induction results in the formation of atypical alveolar hyperplasia and is characterized by lung organs having an increasing diameter and weight.

Color and Texture Morphology of Rat Lungs
The results of observations on the morphology of color and surface texture of rat lung surfaces (Rattus norvegicus) induced 7,12-Dimethylbenz (α) Anthracene (DMBA) and have been given durian bark extract from the entire treatment group can be seen in Table 3 3.1, normal values were obtained in the K(-) treatment group with pink lungs and chewy texture.The K(+) group has the highest abnormal values and the lungs are blackish-red with a more pronounced texture.Groups P1, P2 and P3 have abnormally lower values, the lungs are pale red, partially blackish-red with a supple and slightly spotted texture.In the P4 group, the lowest abnormal value was obtained and close to normal values with a chewy and spotless texture.This is because the substances contained in durian bark extract play a very good role in improving the morphology, color, and texture of the surface of the lungs of mice that have been damaged.According to [1], morphological characteristics in normal mouse lungs are pink, smooth texture (chewy), shiny and spotless.

Histological Features of Rat Lungs
The results of observations of DMBA-induced rat lung histology preparations and durian bark extract were obtained through the Olympus CX 23 binocular microscope with the help of Amscope Digital Camera FMA050.The histological picture of the lungs of the rats obtained can be seen in Figure 3 Observational data on lung cells and the results of their analysis can be seen in appendix 6.The results of the analysis with Kruskal Wallis showed that the administration of durian bark extract had a significant effect on lung cells (p < 0.05).The results of Duncan's post hoc analysis of all treatment groups are presented in the form of average scores in Table 3.2.In table 3.2 it can be seen that the K-group did not find significant damage because it was not DMBA induced, while the P1, P2 and P3 groups showed little damage to lung cells, but the P4 group was the treatment that had the lowest histopathology score value and tended to increase better than the P2 and P3 treatment groups.Cell abnormalities in the P2, P3 and P4 treatment groups were still found in relatively small amounts because the double dose of durian bark extract increased its ability to significantly reduce cell abnormalities back to normal conditions compared to the DMBA control group that was not given drugs and extracts.States that flavonoids and polyphenols are able to improve the histopathology of rat lungs.Flavonoids as one of the classes of reducing compounds that accommodate hydroxy radicals and superoxides so as to protect membrane lipids against reactions that damage tissue due to free radicals.Flavonoids have phenolic hydroxyl (OH) groups that are important in their molecular structure.The reaction of OH groups by flavonoids plays an important role in inhibiting the growth and proliferation of cancer cells [27].

Histophatology Scoring of Rat Lungs Alveolus
The results of the analysis with Kruskal Wallis showed that the administration of durian bark extract had a significant effect on the histological picture of the lung alveolus (p < 0.05).The results of Duncan's post hoc analysis of all treatment groups are presented in the form of average scores in Table 3 Changes in lung histology based on table 3.3 show that the K(-) treatment group has a close relationship between alveolus, there is no damage to the membrane and lumen of the alveolus.In contrast after administration of durian bark extract in the P2 and P3 groups which had scores close to the P4 group which had the lowest average lung alveolus score.This happens because flavonoids of durian bark extract have the potential to repair damage and increase the ability of epithelial cells in the basement membrane to protect the alveolus from exposure to the carcinogen DMBA.Flavonoids balance antiproteases with proteases in alveolar tissue, inhibiting the work of proteases so as to improve alveolar lumen dilation, damage to alveolar membranes and relationships between alveolus in rat lung tissue [28].
The results of [29], showed that microscopically normal lungs have a structure characterized by the presence of normal cells in the epithelium that makes up the alveolar membrane, the endothelium contains cells that are clearly visible around the alveolus, the lumen of the alveolus is rounded, the relationship between alveolus is tight and no proliferation is found in the alveolar epithelium.In abnormal lungs, epithelial cells that make up the alveolar membrane are no longer nucleated, endothelial cells are also not found, the lumen of the alveolus appears dilated and the relationship between the alveolus is very tenuous.

Conclussion
The morphology (color, weight, texture and volume) of the lungs of mice induced by 7,12-Dimethylbenz (α) Anthracene (DMBA) improved significantly (p<0.05) after administration of durian fruit peel extract (Durio zibethinus Murr. ) and also Durian (Durio zibethinus Murr.) rind extract with the highest dose, namely 450 mg/kgBW, significantly (p<0.05) can repair damage to the alveolar membrane, alveolar lumen and connections between alveoli and reduce the number of cells experiencing degeneration, congestion, inflammation and necrosis in rat lungs. .2.
.1 Tabel 3.1 Average Morphological Scores of Color and Texture of Rat Lungs