Patients infected with the dengue virus will develop thrombocytopenia which can cause bleeding and complications. One of the materials that contain antioxidants and have potential as a functional food is red dragon fruit peel. This peel can be processed into yogurt as a way to increase antioxidant function which ultimately supports the immune system of its users. This study analyzed the effect of red dragon fruit peel yogurt on the platelet levels of thrombocytopenic Wistar rats. It used a pre-post-test control group design. Male Wistar rats were randomly assigned into seven groups: K-; K+; and five treatment groups that received dragon fruit peel yogurt at doses of 5% (K1); 10% (K2); 15% (K3); 20% (K4); and 25% (K5). Thrombocytopenia was induced by 0.1 mL.100g-1 BW of heparin for 3 days. The intervention was carried out for 28 days. The result showed that all groups had significant differences before and after the intervention (
Dengue virus infection is one of the infections caused by mosquitoes. It is spread over all geographic areas and causes 100 million dengue infections every year. Deaths due to infectious diseases decreased from 2005 to 2015, but deaths due to the dengue virus increased by 48% (
Dengue virus infection (DENV) occurs through the transmission of the DENV serotype (DENV 1 – 4) by the
Studies show that there are haematological changes in patients with dengue. These include thrombocytopenia, leukopenia, lymphocytosis, and lymphocytopenia (
Food intake plays a role in maintaining immunity for people with dengue infection. Food and fluids are nonpharmacological therapies to prevent and improve various risk factors associated with infectious manifestations due to dengue. Nutritional therapy related to changes in haematological conditions can be an alternative to prevent worsening of the condition of people with dengue infection.
A symbiotic product can be classed as a functional food due to its content of probiotic and prebiotic bacteria. Intestinal microflora can be affected by food products containing probiotics in appropriate doses so that they can act as antioxidants. Microbiota, both facultative and strict anaerobes including streptococci, bacteroides, lactobacilli, and yeasts, can be found from the mouth to the colon. Prebiotics can act as a growth stimulator and support probiotic activity such as by having biofunctionality in improving intestinal health, improving immunity, and even reducing the risk of inflammation (
One of the fruits that contain antioxidants and have potential as a functional food is red dragon fruit (
Red dragon fruit peel can be processed into yogurt as a way to increase its economic value (Figure
Red dragon fruit peel yogurt procedure.
Red dragon fruit peel yogurt can improve haemoglobin, haematocrit, and platelet levels in heparin-induced thrombocytopenia (HIT) in Wistar rats.
This study used male Wistar rats, weighing 160 – 200 g and aged 12 – 16 weeks. A total of 42 Wistar rats were randomly divided into seven groups.
Thrombocytopenia in this study used Heparin ammonium salt, Merck, Sigma-Aldrich, form powder (biological source from porcine intestinal mucosa).
This study used
The isolated culture for the red dragon fruit peel are
Hematology Analyzer, Sysmex KX-21, Kobe, Japan.
The automated hematology analyzer (AHA) was used to measure hemoglobin, hematocrit, and platelet levels (
Facilities and handling of the experimental animals during the study were managed based on the Guidelines for Care and Use of Laboratory Animals of CFNS, Universitas Gadjah Mada, and were approved by the Committee on the Ethics of Health, Universitas Negeri Semarang (permission number: 077/KEPK/EC/2020). The experimental animals were obtained from the House of Experimental Rats, CFNS, Universitas Gadjah Mada, Yogyakarta, Indonesia. This study used male Wistar rats, weighing 160 – 200 g and aged 12 – 16 weeks. A total of 42 Wistar rats were randomly divided into seven groups consisting of six rats per group: negative control (K-); positive control (K+); and five treatment groups that received dragon fruit peel yogurt at doses of 5% (K1), 10% (K2), 15% (K3), 20% (K4) and 25% (K5) respectively. The cage environment had a light : dark cycle of 12 : 12 h, room temperature of 25 ±1 °C and humidity that was always maintained. Cage cleanliness and sanitation were also maintained to reduce stress on the experimental animals during treatment. Rats were placed in individual cages made of stainless steel. Each Wistar rat received 20 g per day standard feed and ad libitum water. The experimental animals were acclimatized for 7 days before the treatment.
The red dragon fruit peel yogurt; made based on the research conducted by Mardiana and Putriningtyas (
The rats’ body weight was monitored weekly. The rats were euthanized by cervical decapitation after 28 days of intervention, then burned using an incinerator. Blood samples were collected from rats before and after the intervention.
Parameters analyzed. Blood samples were collected from the retroorbital vein and measurements of haemoglobin, haematocrit, and platelets were done using whole blood. Whole blood was obtained from each animal and stored with anticoagulant for the measurement of the haemoglobin, haematocrit, and total platelets. The parameters were measured using a automated haematology analyzer. The experiment was done in duplicate in two biological replication.
The mean ± standard deviation (SD) was calculated for each group of six rats. As all data were normally distributed, the significance of differences before and after treatments was determined using the pair t-test. The significance of differences between the groups were assessed by one-way analysis of variance (ANOVA), calculated by the SPSS version 20 program, with a significance level of
Table
Nutritional content of red dragon fruit peel yogurt.
Materials | Nutritional content | |||
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Vitamin C (mg.100g-1) | Total Flavonoid (mg.100g-1) | |||
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22.379 | 38.519 | |||
10.612 | 48.485 | |||
6.515 | 22.209 | |||
11.024 | 42.349 | |||
20.072 | 43.277 |
Lactic acid bacteria can grow and carry out fermentation activities maximally by utilizing sugars or carbohydrates in the media to form lactic acid and cause a decrease in pH (
The static bodyweight of the rats was assessed weekly (Biosep-In Vivo Research Instrument, USA) to determine rats’ development during the study (Table
Mean body weight of Wistar rats.
Group | Body weight (g) | ||||
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Pre-injection | Pre | Post | Δ |
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|
|
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184.17 ±4.49 | 189.83 ±4.58 | 214.67 ±4.97 | 24.83 ±0.75b,c,d | 0.001 | |
186.50 ±5.61 | 190.83 ±5.38 | 206.00 ±5.51 | 15.17 ±0.75a,c,d,e,f,g | 0.001 | |
190.00 ±4.90 | 194.17 ±4.71 | 214.17 ±4.79 | 20.00 ±0.63a,b,e,f,g | 0.001 | |
187.83 ±2.86 | 191.50 ±3.27 | 211.50 ±3.62 | 20.00 ±0.89a,b,e,f,g | 0.001 | |
184.17 ±6.97 | 188.17 ±7.06 | 212.50 ±7.31 | 24.33 ±0.82b,c,d | 0.001 | |
185.17 ±3.82 | 189.67 ±3.78 | 213.83 ±3.76 | 24.17 ±0.75b,c,d | 0.001 | |
186.00 ±4.78 | 190.17 ±4.96 | 214.33 ±5.24 | 24.17 ±0.98b,c,d | 0.001 |
Note: Sampling was done 3 days after induction of thrombocytopenia and 28 days after the start of the treatment. K-: negative control; K+: thrombocytopenia; K1: thrombocytopenia treated with 5% red dragon fruit peel yogurt; K2: thrombocytopenia treated with 10% red dragon fruit peel yogurt; K3: thrombocytopenia treated with 15% red dragon fruit peel yogurt; K4: thrombocytopenia treated with 20% red dragon fruit peel yogurt; K5: thrombocytopenia treated with 25% red dragon fruit peel yogurt. Values represent the mean ± SD for observations made on six rats in each group. Statistical analysis: *paired t-test, significant difference (
aStatistically significant difference (
bStatistically significant difference (
cStatistically significant difference (
dStatistically significant difference (
eStatistically significant difference (
fStatistically significant difference (
gStatistically significant difference (
The result of the data analysis shows that there were significant differences in each group before and after the intervention (
Weight gain in the treatment groups was probably due to the consumption of red dragon fruit peel yogurt. Fermentation using
Thrombocytopenia is a complication caused by heparin, commonly referred to as HIT. HIT can be characterized by venous and arterial thrombosis, decreased platelets, and platelet aggregation. Heparin can function to bind antithrombin as the main inhibiting protein for the coagulation process (
Table
Mean levels of haemoglobin, haematocrit and platelets in Wistar rats.
Group | Parameters tested | ||
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Hemoglobin (g.dL-1) | Hematocrit (mL %) | Platelets (× 103. µL-1) | |
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14.92 ±0.50 | 47.82 ±0.21 | 321.12 ±3.73 | |
14.73 ±0.47 | 46.93 ±0.26 | 312.54 ±3.45 | |
-0.19 ±0.10b,c,d,e,f,g | -0.88 ±0.06 | -8.58 ±0.64 | |
0.001* | 0.005* | 0.001* | |
9.07 ±0.20 | 30.32 ±0.72 | 97.13 ±3.96 | |
8.95 ±0.21 | 29.40 ±0.71 | 96.03 ±4.06 | |
-0.12 ±0.04b,c,d,e,f,g | -0.93 ±0.02 | -1.11 ±0.60 | |
0.001* | 0.001* | 0.001* | |
9.00 ±0.22 | 30.24 ±0.49 | 99.21 ±0.48 | |
10.87 ±0.15 | 36.05 ±0.49 | 133.37 ±0.90 | |
1.87 ±0.07a,b,c,d,e,f,g | 5.82 ±0.01a,b | 34.16 ±0.46a,b | |
0.001* | 0.001* | 0.001* | |
9.13 ±0.36 | 29.20 ±0.90 | 97.95 ±0.41 | |
12.24 ±0.34 | 39.05 ±0.91 | 195.34 ±2.20 | |
3.12 ±0.05a,b,c | 9.85 ±0.04a,b,c | 97.39 ±2.14a,b,c,e,f,g | |
0.001* | 0.001* | 0.001* | |
8.67 ±0.13 | 29.81 ±0.66 | 98.12 ±0.45 | |
13.61 ±0.13 | 40.60 ±0.62 | 238.06 ±3.92 | |
4.94 ±0.05a,b,c,d | 10.78 ±0.08a,b,c,d | 139.94 ±3.95a,b,c,d,f,g | |
0.001* | 0.001* | 0.001* | |
8.56 ±0.20 | 29.80 ±0.34 | 97.95 ±0.43 | |
13.92 ±0.20 | 42.61 ±0.44 | 279.24 ±5.08 | |
5.36 ±0.27 | 12.81 ±0.52 | 181.29 ±4.87a,b,c,d,e,g | |
0.001* | 0.001* | 0.001* | |
8.63 ±0.19 | 29.90 ±0.41 | 98.03 ±0.43 | |
14.24 ±0.20 | 43.60 ±0.46 | 290.99 ±4.78 | |
5.61 ±0.02 | 13.71 ±0.10 | 192.96 ±4.53a,b,c,de,f | |
0.001* | 0.001* | 0.001* |
Note: Sampling was done 3 days after induction of thrombocytopenia and 28 days after the start of the treatment. K-: negative control; K+: thrombocytopenia; K1: thrombocytopenia treated with 5% red dragon fruit peel yogurt; K2: thrombocytopenia treated with 10% red dragon fruit peel yogurt; K3: thrombocytopenia treated with 15% red dragon fruit peel yogurt; K4: thrombocytopenia treated with 20% red dragon fruit peel yogurt; K5: thrombocytopenia treated with 25% red dragon fruit peel yogurt. Values represent the mean ± SD for observations made on six rats in each group. Statistical analysis: *paired t-test, significant difference (
aStatistically significant difference (
bStatistically significant difference (
cStatistically significant difference (
dStatistically significant difference (
eStatistically significant difference (
fStatistically significant difference (
gStatistically significant difference (
Thrombocytopenia is one of the criteria used by the World Health Organization as a potential indicator for dengue infection. Thrombocytopenia is associated with pro-oxidant conditions in dengue infection.
Oxidative stress can increase when there is an imbalance of oxidants and antioxidants due to internal or external exposure to stressors such as viruses (
Red dragon fruit peel yogurt contains vitamin C, a watersoluble vitamin also known as ascorbic acid which can stimulate immune function and has a role as an antioxidant (
Endothelial damage in DENV is caused by the dengue virus itself. Thrombocytopenia is responsible for bleeding in patients. The condition of thrombocytopenia can be influenced by various factors such as reactive immune response and decreased platelet production, activation, and apoptosis (
Yogurt can release bioactive peptides and bacteriocins. β-Casein-derived peptides released from yogurt can trigger goblet cells to secrete mucin either in vitro or in vivo. The lactic acid bacteria found in yogurt can break down simple carbohydrates to produce lactate, acetate, or propionate (
The increase in haemoglobin, haematocrit, and platelet levels after consumption of red dragon fruit peel yogurt could be due to its phenolic, flavonoid, and anthocyanin content. Phenolics, also called phenols, are found in various tropical plants (
Red dragon fruit peel yogurt is made using
Compounds other than flavonoids contained in red dragon fruit peel are betacyanins and betalains, the content of which in red dragon fruit peel is higher than in the fruit flesh. Betalains are water-soluble polyphenolic pigments composed of yellow betaxanthins and purplish-red betacyanins. Betacyanins consist of betalamic acid and an acyclic amine group which is excellent electron donors which enables them to scavenge free radicals. The red-purple betacyanin pigments in red dragon fruit peel have recently been suggested as a potential betalain source (
The increases in haemoglobin, haematocrit, and platelet levels were directly proportional to the dose of dragon fruit peel given. This mechanism is possible because flavonoids are also known to be able to capture and neutralize free radicals such as ROS or RNS, which are associated with phenolic OH groups so that they can repair damaged tissue or inhibit the inflammatory process (
The flavonoids in red dragon fruit peel are catechin, epicatechin, quercetin, myricetin, and kaempferol, of which catechins have the highest concentration in red dragon fruit peel. Catechin is thought to inhibit DENV infection (
Red dragon fruit peel yogurt is effective for improving haemoglobin, haematocrit, and platelet levels in HIT in rats. Yogurt containing 25% red dragon fruit peel provides an effective dose for improving haemoglobin, haematocrit, and platelet levels in thrombocytopenic rats.
We would like to thank you to Mr. Yuli Yanto for preparing and treating the animal research.
This research was supported by grant Universitas Negeri Semarang No. SP DIPA-023.17.2.677507/2020.
The authors declare that they have no conflict of interest.
The use of animals in this research was approved by the Ethics Committee of Health Universitas Negeri Semarang following the legislation of the Declaration of Helsinki (1964) and CIOMS WHO (2016) (Project identification code: 077/KEPK/EC/2020, Date of approval: 22 June 2020, Name of the Ethics Committee: Ethics Committee of Health, Universitas Negeri Semarang).