Effect of Mango Leaf Shoot Extract ( Mangifera indica L.) on Zebra Fish ( Danio rerio ) Cell Regeneration Induced by Hyperglycemia

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Volume 5, No. 2, October 2021 | 44
insulin production and performance, abnormality of lipid and protein metabolism that involved in formation of free radicals (Alza, 2013;Permatasari, et al., 2018). If that condition is not treated properly, that conditions can cause several disorders such as damage to the eyes, kidneys, nerves and difficulty in wound healing (García, 2017;Hakim et al., 2010).
One of the ways to prevent diabetic wounds is generally used antiseptic. However, the continuous use of antiseptic can cause several effects. For generations, Indonesian people have used plants as alternative medicine. Advantages of using plants such as ingredients used are easy to obtain, have useful compounds for health problems and affordable price (Mustofa et al., 2012;Permata & Khoirunnisa, 2020).
Mango plant has benefits as a medicinal plant and has been widely used as traditional medicine (Luqyana & Husni, 2019). Mango plants have potential to help wound healing process of diabetics (Risa et al., 2018). In Indonesia, mango leaf shoot are consumed as salad. While in India, young leaves are used as a diabetic medicine (Prommajak et al., 2014).
Based on research by Permatasari et al. (2018), mango leaf extract of Cengkir cultivar can reduce blood glucose levels in fructose-induced mice, with an optimal dose 105 mg/kg/BW. Risa et al. (2018) also proved that there was a significant effect on given mango leaf extract of Manalagi cultivar on wound healing in mice with optimal concentration 20%. This is also supported by Ayuningtyas (2020) (Utami, 2018). Therefore, this study was conducted to determine effect of mango leaf shoot extract on reducing blood glucose levels and regeneration caudal fin of hyperglycemic zebrafish. It also as information for further utilization of mango leaf shoot and as alternative treatment in assisting diabetic wound healing process.

Sampling and Mango Leaf Shoot Extract Making
Mango leaf shoot sample is taken were light green colors, light green brown, pink, purplish and reddish brown. Samples were cleaned, then air-dried for 5 to 7 days. Dry sample was mashed with a blender, then filtered. The method used to make mango leaf shoot extract is maseration method by immersing sample in methanol solvent. The advantage of using this method is that it doesn't use heating which may damage thermolabile compounds. Resulting filtrate is then evaporated to produce a thicker extract.

Identification of Secondary Metabolic Compounds
Identification of secondary metabolites using a simple descriptive method. Identified secondary metabolites are alkaloids, flavonoids, saponins and tannins which are thought to play a role in wound healing process (Hakim et al., 2010

Hyperglycemia Preliminary Test
Preliminary test was performed by comparing two treatment groups. They are control group by immersing fish in UV water and hyperglycemic group with modification based on Hayati et al. (2017) by immersion fish in solution of 400 mg of alloxan in 1 liter of distilled water for 30 minutes then immersion it in solution of 2 grams of glucose in 1 liter of distilled water for 24 hours. Alloxan and glucose inducted for three days. On the fourth day, blood glucose level of fish from each treatment was measured.

Treatment Hyperglycemia and Amputation
Fish group with weights ranging from 0.1-0.4 grams made hyperglycemia by induction of alloxan and glucose for 3 days. On the fourth day, the fish's blood glucose level was measured.
After reaching a hyperglycemic condition, the fish were anesthetized by immersion in water with a temperature lowered from 17-12°C. Then, caudal fin was amputated vertically with a length 1/2 of fin or 0.3 cm from the outer tip of caudal fin. Then, allowed fish to regenerate in normal temperatures (Poss et al., 2000).

Treatment of Metformin and Mango Leaf Shoot Extract
After amputation, the fish were divided into 4 different treatment groups, they are hyperglycemia group, metformin 0.75 w/v, mango leaf shoot extract 10 µg/ml and 20 µg/ml. This treatment was performed for 5 days.

Observation of Caudal Fin Regeneration
Observation of caudal fin regeneration was performed on sixth day after immersion using metformin 0.75 w/v, extract 10 µg/ml and extract 20 µg/ml. Observations were made from base of fin to the tip of outer caudal fin.

Data analysis
The results of data collection on blood glucose levels and length of caudal fin were processed using IBM SPSS Statistics version 26 (Prayoto dan Nugroho, 2021). Data from observations of decreasing blood glucose levels and increasing length of caudal fin were processed usinf ANOVA to determine whether there was an average difference between treatment groups. If data doesn't meet assumptions of parametric test, then data is processed with a non-parametric alternative test Kruskal Wallis. In this study, extraction is performed by maceration method. This method was chosen because it's often used and is fairly simple method (Susanty & Bachmid, 2016). The principle of maceration method is secondary metabolites in cytoplasm can dissolve in solvent because cell walls and cell membranes are broken. It happened because pressure differences from inside and outside cell (Firdaus et al., 2015).

RESULT AND DISCUSSION
Filtrate as an extraction result were evaporated using a rotary vacuum evaporator until obtaining a thicker extract. Futhermore, identification of secondary metabolites was performed to determine compounds in extract which could be use as parameters related to pharmacological effect. The result of identification secondary metabolites in mango leaf shoot extract can be seen in

Hyperglycemia Preliminary Test
Preliminary test to determine appropriate method for animal test to achieve hyperglycemic conditions. This test consisted of two groups, they are control group and hyperglycemic group with alloxan and glucose induction. Induction of hyperglycemia was performed for 3 days.
Glucose level of fish was measured from each treatment in fourth day. The results of measuring fish blood glucose levels after 3 days of alloxan and glucose induction can be seen in Figure 1. According to Heckler and Kroll (2017), blood glucose level of zebrafish in hyperglycemic conditions can reach 310 mg/dL. It's mean induction of alloxan and glucose for 3 days can significantly increase blood glucose levels.

Effect of Mango
Alloxan is an unstable hydrophilic compound that acts as a diabetogenic. Its unstable can be causes alloxan to easily redox reactions. Alloxan reduction will produce dialuric acid while dialuric acid reoxidation will become alloxan (Hayati et al., 2017). In this process, alloxan radicals are released which will produce superoxide radicals which will dismutation into hydrogen peroxide (H2O2) which has potential to become hydroxyl radicals (Szkudelski, 2001;Ighodaro et al., 2017).

The Effect of Inducing Mango Leaf Shoot (Mangifera indica L.) Extract Arumanis cultivar on Blood Glucose Levels
Alloxan and glucose induction were performed for three days, then blood glucose level of fish was measured. The results of measuring fish blood glucose levels after three days induction can be seen in Figure 2. Hyperglycemic group then was anesthetized and their caudal fin was amputated. After that, the fish were treated by metformin 0.75 w/v, extract 10 µg/ml and extract 20 µg/ml for 5 days. On day 6, blood glucose level was measured. The results of measuring fish blood glucose levels after 5 days of exposure to mango leaf extract can be seen in Figure 3.  Metformin is an oral antihyperglycemic or antidiabetic medicine that has main function of lowering blood glucose levels (Gumantara & Oktarlina, 2017). Metformin works by repairing and increasing insulin receptor sensitivity and inhibiting gluconeogenesis in liver. Giving extract can reduce blood glucose levels in animal test because compounds contained in mango leaf shoot extract have potential as antidiabetic.

Adisty
The compounds in extract include alkaloids, flavonoids, saponins and tannins which are thought to have hypoglycemic activity or can reduce high blood glucose levels. These compounds play a role in repair of pancreatic cells, stimulate insulin release, restore insulin receptor sensitivity, increase glucose absorption into cells so that glucose in blood decreases, inhibit disaccharide activation, activate glycogen synthesis, inhibit gluconeogenesis, inhibit glucosidase activity and prey on free radicals (Miten & Setiasih, 2014;Syaputri, 2014;Elza et al, 2016;Amiraragab et al, 2017;Haryoto & Devi, 2018;Herlina et al, 2020;Indrayani & Mustarichie, 2020;).

The Effect of Extract of Mango Leaf Shoot (Mangifera indica L.) Arumanis cultivar on Caudal Fin Regeneration of Zebra Fish Hyperglycemia
The regeneration process in zebrafish is characterized by an increase in length of caudal fin.
Metformin 0.75 w/v, mango leaf shoot extract 10 µg/ml and 20 µg/ml were induced for the first five days after amputation. The average increase in the length of zebrafish caudal fin in all treatments can be seen in Figure 4. Based on figure 4, it was found that there was no significant difference between hyperglycemia group and extract 10 µg/ml group. Meanwhile, the metformin 0.75 w/v group did not different significantly from the extract 20 µg/ml group. However, significant results were shown by hyperglycemia control and extract 10 µg/ml group with metformin 0.75 w/v and extract 20 µg/ml group.
The difference is thought because of effect from treatment with immersion animal tests in metformin and extract solution. According to Spampinato et al. (2020), antidiabetic medicine such as metformin has a role in anti-inflammatory and cell proliferation that play a role in wound healing, increase the proliferation of keratinocytes and fibroblasts, angiogenesis and increase the formation of granulation tissue. While the compounds in extract such as alkaloids, saponins, tannins and flavonoids act as antioxidants (Hakim et al., 2010;Laut et al., 2019).
Antioxidants efficiently stopping free radical reactions, are able to reduce superoxide and prevent cell damage, chelate iron ions and slow down oxidation, prevent ROS regeneration and can increase the activity of cellular antioxidant enzymes, as superoxide scavangers, peroxyl radicals and peroxynitrite by transferring H + atoms, prevents the formation of ROS by chelating Fe 2+ and Cu 2+ metals thereby preventing redox reactions that generate free radicals (Syarif et al., 2008;Juniarti, 2011;Hardiningtyas et al, 2014;Fithriani et al., 2015).
The keys of regeneration process is formation of blastema or accumulation of undifferentiated proliferative cells that contain most of cells needed to regenerate structures lost after initial wound closure. Blastema proliferation in zebrafish is regulated by Wnt signaling/βcatenin pathway and Notch pathway which have a role in the regulation of wound area, blastema proliferation, osteoblast maturation, maintaining blastema cells in a plastic and undifferentiated state. Hyperglycemic conditions have a direct impact on regenerative capacity of zebrafish because it can reduce proliferative ability of regenerating limbs (Olsen et al., 2011).
Cells that help in wound healing will produce ROS (Reactive Oxygen Species). In addition, the balance between production and disposal of ROS must be homeostasis. If there is an imbalance, it will cause oxidative stress that can interfere with the wound healing process and can reduce the activity of antioxidant enzymes which causes a decrease in non-enzymatic antioxidants. ROS can be converted rapidly to H2O2 by SOD or superoxide dismutase. H2O2 is then detoxified by catalase enzymes or antioxidant enzymes into H2O and O2. However, if more ROS accumulates then H2O2 will be reduced by Fe 2+ or Cu 2+ to hydroxyl radicals such as OHand OH* which are highly reactive (Djuanda et al., 2012;Paredes et al., 2019).

pH and Water Temperature
The environment parameters measured in this study were pH and water temperature in the treatment aquarium. This measurement was performed to determine changes in temperature and  according to Muchdar et al. (2020), stated that zebrafish live in a temperature range of 24-30°C with the optimal temperature for activity which is around 27°C. Water temperature is maintained at general environmental conditions for zebrafish. according to Darniwa et al. (2020), stated that the higher of water temperature, the stress on zebrafish can have an impact on fish health. While pH of water from each treatment aquarium showed a value of 7. According to Arip Rahman et al. (2012), stated that zebrafish are distributed in fresh waters of the tropics which have pH values ranging from 7-8

Survival Rate
Survival rate is the ratio of the fish number that live at the beginning to the end of study.
Survival refers to the survival rate of a population in a certain period of time. Survival data during the observation can be seen in table 3. 100%. The fish that did not survive were thought to be due to the lack of oxygen intake at the time of observation. Observation of the increase in caudal fin of zebrafish was performed on land with the head of fish given a little water so that the fish could breathe. According to Ismi (2017), stated that one of the main causes of sudden fish death is due to lack of oxygen.