INTRODUCTION:

Stunting is one of the manifestations of malnutrition. Stunting is a linear growth disorder caused by malnutrition and chronic infectious diseases. Stunting is a long-term reflection of the inadequate quality and quantity of food consumed, and often suffering from infectious diseases in childhood. The problem of stunting is a nutritional problem that needs attention because it can affect the quality of human resources^1,2.

Nutritional quality emphasizes food diversity. The more diverse and balanced the composition of the food consumed, the better the nutritional quality because, in essence, there is no single type of food that has complete and sufficient nutritional content both in quantity and type³. Overcoming malnutrition can be done by fulfilling nutritional quality. Incorrect or inappropriate nutritional intake will cause health problems, including a lack of protein energy⁴. Some nutrients needed to prevent malnutrition are carbohydrates, fats, proteins, vitamins, calcium, zinc, and other minerals. Lack of fat in children can also reduce hemoglobin levels, while albumin levels are closely related to hemoglobin levels and body weight. Total protein can also be used as a marker for the diagnosis of malnutrition in adults. Therefore the profile of body weight, albumin, total protein, and hemoglobin can be a parameter of nutritional status^5–7.

Fatty acids such as EPA and DHA are quite important for the human body and cannot be made naturally in the body, so they must be obtained from food. EPA and DHA deficiency can cause various problems, one of which causes growth disorders. EPA and DHA are types of omega-3 fatty acids that play an essential role in brain development⁸. In addition, linoleic acid and oleic acid are also crucial for the body. Linoleic acid plays a role in growth because it is a substrate for eicosanoids such as prostaglandins which play a role in cell growth. Intake of essential fatty acids is reflected in their levels in the blood, so adequate intake of essential fatty acids can affect fatty acid levels in the blood and reduce the incidence of stunting⁹.

Natural supplements containing linolenic acid, oleic acid, and linoleic acid that have the potential to improve nutritional status are tengkawang, VCO, and astaxanthin. Tengkawang fruit that is spread in Indonesia is only found on the islands of Kalimantan and Sumatra. While VCO and astaxanthin have been widely used as medicines. So it is expected to overcome malnutrition¹⁰. This is the background of the purpose of this study, which is to determine the effect of giving tengkawang fruit, VCO, and astaxanthin on body weight profiles, albumin, total protein, and hemoglobin in male white rats (Rattus norvegicus L.) who are malnourished.

MATERIALS AND METHODS:

Materials:

The tools used in this study were as follows: analytical balance, micropipette, oral sonde, eppendorf, centrifugation, manual feed grinder, and spectrophotometer. The materials used in this study were the Shorea stenoptera fruit ethanol extract, hemoglobin kit, albumin kit, total protein kit, sugar cane, cornstarch, vegetable oil, CP551 feed, astaxanthin, VCO, and EDTA.

Methods of Preparation of Tengkawang Extract Test Preparation.

Tengkawang (Shorea stenoptera Burck.) fruit were collected from the Bengkayang area, West Kalimantan Province, Indonesia, and determined in the Herbarium Bandungense, School of Life Sciences and Technology, Bandung Institute of Technology Bandung, Indonesia. The TFE was washed to remove the materials left on the fruit and dried for 2 days. Dry sorting was done to remove unwanted materials. Next, the fruits were pulverized using a blender and stored in a dry, well-ventilated place away from direct sunlight. TFE was extracted by continuous extraction using a Soxhlet apparatus with 96% ethanol. Ethanol was used because it is considered a universal solvent that can draw potential compounds from the evaluated plants, and this solvent is suitable for continuous extraction using a Soxhlet apparatus as well. Compared to other solvents, 96% ethanol is safer to use. After complete extraction, the collected extract was dried under reduced pressure using rotavapor and dried in a water bath. Tengkawang (Shorea stenoptera Burck.) fruit extract was weighed according to the dose. Weighing dose adjusted to the conversion of 100mg/kgBW and 300mg/kgBW doses, VCO was administered at 10mL/kgBW and astaxanthin was administered a dose of 1,8mg/kg BW.

Production of Low Protein feed:

Low protein feed is produced by weighing and mixing cornstarch, bagasse powder, and cooking oil with a 12:1:9 ratio. The feed mixture is formed using a feed grinder. The feed that has been formed is then baked in an oven at 50°C and left for 24hours. After drying for 24 hours, the dried feed is removed, then stored in a tightly closed jar, and the feed will be given to the rats.

Animal Grouping:

Tests on male Wistar rats were carried out in the normal control group given CP551 and the malnourished group given a low-protein diet. After the rats given the low-protein diet were malnourished, they were regrouped into four randomly selected groups, and each group consisted of 5 rats with a body weight variation of not more than 20% of the average body weight. These groups are: (1) NC: The normal control group was not induced by low protein feed and without administering the test preparation; (2) VCO: The VCO group, induced by low protein feed, was then given VCO (Virgin Coconut Oil) a dose of 10mL/kg BW; (3) Astaxanthin: The astaxanthin group, induced by low protein feed, was then given astaxanthin a dose of 1,8mg/kg BW. (4) TFE 100: The group induced by low protein feed was then given tengkawang fruit extract at a dose of 100mg/kg BW. (5) TFE 300: The group induced by low protein feed was then given tengkawang fruit extract at a dose of 300mg/kg BW.

Body Weight Parameters Adjustments Made to Animal Models:

Body weight was observed by weighing the test animals immediately before treatment, during treatment until the day of termination of the test animals. Animals are weighed at least once a day.

Biochemistry Parameters:

Observation of biochemical blood levels consisted of albumin levels, total protein levels, and hemoglobin levels. Albumin and total protein levels were observed by taking rat blood through the tail vein and heart and stored in Eppendorf without giving anticoagulants. Blood collection on day 0, after malnutrition, and after termination. The blood taken is then centrifuged for 10 minutes at a speed of 1000rpm to separate the serum and blood cells. The serum is taken for measurement. In contrast, the observation of hemoglobin levels was carried out by placing rat blood into Eppendorf, which had been given the anticoagulant EDTA and not centrifuged.

Albumin levels were measured using a kit from Reiged Diagnostics. Albumin levels were examined in rat blood using BCG (Bromcresol Green). Blood serum was taken as much as 0.5ml, then added 2.5ml of 0.01% BCG reagent and left for 10-15 minutes. Then analyzed with a spectrophotometer with a wavelength of 546nm, the measurement results are read in units of g/dL. Total protein levels were measured with a kit from Reiged Diagnostics. 20μL of blood serum obtained was pipetted, then 1000μL of reagent was added to the microtube and incubated for 15minutes. The solution was analyzed using photometry at a wavelength of 595-610nm.

Measurement of hemoglobin levels using the cyanmethemoglobin method. Pipette exactly 2.5ml of Drabkin's reagent and put it in the 1st and 2nd test tubes. Add 10µl of blood to the 2nd test tube containing Drabkin using a micropipette. Mix the solution in the tube well, then leave it at room temperature for at least 10minutes to allow the cyanmethemoglobin to form properly. Take a solution reading using a calorimeter or spectrophotometer at a wavelength of 540nm.

Analisis Data:

The statistical analysis of the data is conducted utilizing a one-way analysis of variance (ANOVA), with each group's data expressed as mean±standard deviation (SD). At a p-value of 5% (p<0.05), all analyses and comparisons were evaluated. Using an ethical clearance number of 805/UN22.9/PG/2023, the protocol was approved following peer review by the Faculty of Medicine Ethical Clearance Committee of the University of Tanjungpura with regards to animal welfare in medical health research.

RESULT:

Low-Protein Induction Feeding to Create Models of Malnourished Animals:

The animal model of malnourished body weight dramatically decreased, decreasing by 32% in rats administered low-protein feed for a 24-day interval, according to Figure 1's findings concerning the percentage change in body weight in rats throughout that time. The group also experienced physical changes, like hair loss. In contrast, the normal control group that consumed CP551 as a standardized diet experienced a 38% increase in body weight.

Figure 1. Percentage of change in test animals' body weight regarding malnutrition on days 15 and 24

In addition to body weight, biochemical blood parameters such as hemoglobin, total protein, and albumin levels are utilized for measuring malnutrition. Before consuming a low-protein feed on day 0, biochemical levels were examined. These measurements were subsequently repeated on days 15 and 24 following the low-protein feed. Figure 2 presents a diagram of the blood biochemical level analyses. The average albumin levels identified in the low-protein feed group after 24 days demonstrated an extremely slight decrease in albumin levels in comparison with normal controls, as confirmed by the graph in Figure 2A.

Figure 2. Average blood biochemical levels on day 0, 15 & 24 on albumin (A), total protein (B), hemoglobin (C)

In test animals administered low protein feed after day 24, Figure 2B showed a decrease in total protein levels by 5.679g/dL below the normal limit, which is 6g/dL. In contrast, the total protein level in the normal control is still within the normal range. After 24 days, test animals fed low-protein feed showed a decrease in average hemoglobin levels of 10.256g/dL, which is less within the range of normal hemoglobin levels, which is 11.6 g/dL (Figure 2C).

Treatment of Tengkawang Fruit Extract (TFE), Virgin Coconut Oil (VCO), and Astaxanthin to Malnourished Animals:

Treatment of Tengkawang Fruit Extract (TFE), Virgin Coconut Oil (VCO), and astaxanthin on malnourished animals was conducted on the 24th day until the 55th day after the animal model was declared malnourished. The malnutrition model animals were divided into four groups, namely normal control, VCO, astaxanthin, and two groups of tengkawang fruit extract doses of 100 mg/kgBW and 300mg/kgBW, with the parameters measured being body weight, albumin levels, total protein levels, and hemoglobin levels. Figure 3 shows that there was an increase in body weight on day 55, which was significantly different after the administration of VCO, Astaxanthin, and 2 doses of Tengkawang fruit extract, which was previously malnourished compared to the normal control group (p<0.05). The highest increase in body weight was in the astaxanthin group, which amounted to 93%, and the VCO group and Tengkawang fruit extract doses of 100mg/kgBB and 300 mg/kg BB for 30 days of treatment were 71%, 63%, and 61%, respectively. As for the normal group, the percentage of body weight increase was lower than the treatment group by 27%. However, there was no significant difference between the two TFE groups when compared to the VCO (70.73%) and astaxanthin (93.33%) (p>0.05).

Figure 3. Body weight change percentage of each group after 55 days on malnourished animals and 30 days of treatment compared to day 0 is a significant difference between all treatment groups compared to the normal group (* p <0.05).

Biochemical blood levels, including hemoglobin, total protein, and albumin profile, were examined in malnourished rats that were given the examination treatment for 30 days. The 24th day following low-protein feeding (malnutrition conditions) and the 55th day following treatment of the examination preparations were used to measure biochemical levels. The albumin, total protein, and hemoglobin levels in the groups receiving normal control, VCO, astaxanthin, and TFE at doses of 100mg/kgBW and 300mg/kgBW are shown in Figure 4, Figure 5 and Figure 6.

Figure 4. Value (A) and percentage (%) (B) of albumin level. The data represent the mean ± SD (n = 5). VCO, Astaxanthin, and TFE doses of 100 and 300 mg/kgbw were not significantly different (* p < 0.05) by the ANOVA test.

As presented in Figure 4, the astaxanthin group exhibited the highest albumin levels (38%), whereas the normal group had the lowest albumin levels (14%; p<0.05). In comparison, the groups provided VCO, EBT 100, and 300mg/kgBW in addition had percentage increases in albumin levels of 27%, 26%, and 30%; the results of these groups were not significantly different from the astaxanthin group (P>0.05). All treatments have the potential to increase albumin levels during treatment for a duration of 30 days.

Figure 5. Value (A) and percentage (%) (B) of total protein levels. The data represent the mean ± SD (n = 5). Normal, VCO, Astaxanthin, and TFE doses of 100 mg/kgbw were not significantly different (* p < 0.05) by the ANOVA test.

As evidenced by the results shown in Figure 5, the EBT group, which administered a dose of 300 mg/kgBW, had the greatest percentage of total protein levels after 30 days of treatment by 34%, whereas the VCO group had the lowest total protein levels by 20%. Regarding the normal group, the increases in total protein levels by astaxanthin and TFE 100 mg/kgBW are 27%, 29%, and 22%, respectively. These variances are not significantly different (P > 0.05). Studies suggest that, adhering to 30 days of treatment, the TFE 300 mg/kgBW group can increase total protein levels to the largest potential compared to other groups that differ significantly (p<0.05) from the normal group.

Figure 6. Value (A) and percentage (%) (B) of hemoglobin levels. The data represent the mean ± SD (n = 5). Normal, VCO, Astaxanthin, and TFE doses of 100 mg/kgbw were not significantly different (* p < 0.05) by the ANOVA test.

According to Figure 5's hemoglobin levels, the EBT group, at a dose of 300 mg/kgBW, has the highest hemoglobin levels; in addition, the VCO group has the lowest (p > 0.05). Hemoglobin levels increased by 49% in the EBT group and 19% in the VCO group at a dose of 300 mg/kgBB. Hemoglobin levels can increase by 19%, 22%, 19%, and 24%, respectively, in the VCO, astaxantin, and TFE 100 mg/BW groups without being significantly different from the normal group (P > 0.05). Compared to other treatment groups, this indicates that the TFE 300 mg/kg BW group can increase hemoglobin levels to the greatest potential.

DISCUSSION:

This study used male Wistar rats (Rattus norvegicus), and the ethical protocol has been approved for ethical review by the Faculty of Medicine Ethical Clearance Committee of the University of Tanjungpura dengan an ethical clearance number of 805/UN22.9/PG/2023. The selection of animals in the form of rats is because they have advantages compared to other species, such as being easy to obtain, easy to care for, fast metabolic capabilities, low maintenance costs, and similarity to humans in terms of organs, physiology, and anatomy^11,12. Rats before being administrated fruit extract test preparations of tengkawang (Shorea stenoptera Burck.) were conditioned in a state of malnutrition beforehand, except for the normal control group. Malnourished rats were obtained from low-protein feed induction.

The incidence of malnutrition is closely related to weight loss to becoming underweight. Factors such as food intake can influence increasing and decreasing body weight¹³. Body weight was observed by weighing the rats' body weight every day during 55 days. This observation was carried out to see changes in body weight in rats after being given a low-protein diet and compared with normal controls.

The body weight of the rats was affected by feed consumption. High-protein feed acts as a source of energy for the growth of rats. Protein is an essential nutrient for supporting optimal growth, development, weight management, and human health. According to Anggraeny's (2016) research, changes in protein intake will greatly affect changes in body weight¹⁴.

The test animals that had experienced malnutrition were then divided into four groups, namely the VCO group, which was given VCO; the astaxanthin group, which was given astaxanthin; and the two test preparation groups, which were given 100mg/kgBW and 300 mg/kgBW of tengkawang fruit extract, respectively.

Astaxanthin from the ASTRIA® comes from the green microalga Haematococcus pluvialis. Astaxanthin (3,3′-dihydroxy-ß-carotene-4,4′-dione) is a secondary carotenoid pigment belonging to the terpenoid group^15,16. The highest increase was in the administration of astaxanthin, as was the case in the Xiaobin Li (2016) study, which showed that astaxanthin was able to increase body weight in rats significantly¹⁷.

The increase in body weight in the VCO group is thought to be due to the rats experiencing a significant loss of nutrition in a state of malnutrition. This causes the rats to experience very high hunger conditions, thus increasing food intake¹⁸. This results in a very rapid increase in body weight compared to the normal group of rats. In addition, based on Wijaya and Surdijati’s (2020) research, giving VCO supplements can also increase the body weight of rats¹⁹.

The increase in body weight in the TFE group was lower than the VCO group because tengkawang (Shorea stenoptera Burck.) has a higher unsaturated fatty acid content than VCO, which has the most content in the form of saturated fatty acids²⁰. Unsaturated fats do not cause obesity as much as they cause weight gain as much as saturated fats²¹. Tengkawang (Shorea stenoptera Burck.) contains unsaturated fatty acids in the form of oleic acid of 59.60%. Oleic acid is oxidized faster and more, so it does not cause fat accumulation or increase body weight as much as saturated fatty acids^22,23. So that the administration of Tengkawang fruit extract, both doses of 100 and 300mg/kgBW, cannot be said to affect increasing body weight.

Parameters of malnutrition observed besides body weight were biochemical blood levels in the form of albumin profile, total protein, and hemoglobin. Albumin is the main human plasma protein, making up about 60% of the total plasma protein²⁴. Albumin has an important role in maintaining health and fluid balance in the body, so it is important to ensure that albumin levels in the body are sufficient. A deficiency of albumin levels can be categorized as a condition of hypoalbuminemia. Hypoalbuminemia is caused by an inadequate supply of amino acids from proteins, thereby interfering with the synthesis of albumin and other proteins by the liver^25,26.

The total protein is all types of protein found in serum or plasma, consisting of albumin and globulin. Albumin and globulin concentrations affect decreases and increases in total protein levels^27,28. Kwashiorkor itself is a form of severe protein-energy deficiency (PEM) caused by a lack of adequate protein intake with sufficient energy intake. Lack of energy and protein can affect the immune system¹⁴. So that the total protein profile can be used as an indicator of symptoms of malnutrition.

Protein intake is influenced by the quality of the protein contained in food. Sources of protein consist of animal and vegetable food ingredients. Proteins derived from animals are complete proteins because they contain all types of essential amino acids. Whereas vegetable protein is an incomplete protein because it does not contain all types of essential amino acids²⁹. This is in accordance with the research conducted. There was a decrease in total protein levels below the normal range in the group given a low-protein diet containing only vegetable protein. In contrast, the normal control was given CP551 feed, which contained animal protein, so the total protein content was still within the normal range.

Hemoglobin is formed due to the involvement of protein and iron. Protein, especially the amino acid glycine and the mineral Fe, is the main component of hemoglobin formation. Hemoglobin is a complex organic compound consisting of four red porphyrin pigments (heme), each containing an iron atom plus globin, a globular protein. So low-protein diets such as cornstarch can reduce albumin, total protein, and hemoglobin levels. The decrease is in the range below normal^30–32.

Research by Rao (2015) and El-baz (2021) found that administering astaxanthin can increase total protein and albumin levels in rats, which are quite high compared to the normal group^33,34. The increase is because astaxanthin shows protection for liver function. So that is consistent with this study: the administration of astaxanthin increased the percentage of total protein and albumin levels³⁵. Even the highest increase in the rate of albumin levels was found in the astaxanthin group that was given the drug astaxanthin. At the same time, the increase in hemoglobin levels in the astaxanthin groups is because astaxanthin is a secondary carotenoid pigment, including the terpenoid group^15,36. Carotene in the body will be converted into vitamin A. Vitamin A plays a role in mobilizing iron reserves to synthesize hemoglobin. Adequate vitamin A will increase the hemoglobin value along with the increase in vitamin A³⁷.

The increase in albumin levels in Tengkawang fruit extract is because tengkawang contains omega-3. According to Rashidi (2020), it shows that intake of omega-3 increases albumin levels in hepatotoxic patients. Serum albumin levels may be reduced by hepatic impairment³⁸. However, administration of omega-3 showed protection against liver function. This increase in albumin levels is associated with improved nutritional parameters, including increased appetite³⁹. However, there was no difference with the group given the VCO because VCO also contains omega-3, although a little. So that VCO can also increase albumin levels. According to El-shemy (2018), administering VCO to rats had higher albumin levels than the normal control group⁴⁰. This is in line with the research conducted. Administration of VCO can increase albumin levels in malnourished rats.

The increase in total protein levels in the group given tengkawang fruit extract is thought to be due to the content of essential fatty acids. This is in line with research by Paradee (2023), which stated that the content of essential fatty acids in Perilla frutescens fruit oil increased the total protein content of the test animals compared to the control group⁴¹. The increase in hemoglobin levels is thought to be due to the omega-3 content in tengkawang fruit (Shorea stenoptera Burck.). According to Salasah (2016), giving omega-3 supplements to pregnant women can increase Hb levels and improve the nutritional status of newborns⁴². According to Shibuyah (2020), acai fruit extract with the same content as tengkawang fruit can also increase hemoglobin levels⁴³. This study also found that tengkawang fruit extract at a dose of 300 mg/kg BW could increase hemoglobin levels higher than malnourished animals given astaxanthin.

The results of processing data on various parameters after administration of tengkawang fruit extract showed that malnourished animals returned to normal conditions with marked increases in average values and percentages and statistical test results using SPSS. It cannot be concluded that tengkawang fruit extract effectively overcome stunting. So it is suggested to do further research with tengkawang fruit extract and to observe other parameters such as total cholesterol in malnourished rats.

CONCLUSION:

Based on this study results, we found that administration of tengkawang fruit extract may improve the body weight profile of malnourished animals. Moreover, it also improves the albumin, total protein, and hemoglobin levels with the best improvement was found in dose of 300mg/kgBW.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

This study received financial support from Tanjungpura University through the DIPA Faculty of Medicine.

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Received on 30.01.2024 Revised on 18.05.2024

Accepted on 12.08.2024 Published on 28.01.2025

Available online from February 27, 2025

Research J. Pharmacy and Technology. 2025;18(2):585-593.

DOI: 10.52711/0974-360X.2025.00087

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