INTRODUCTION:

Diabetes mellitus (DM) is a metabolic disease characterized by impaired metabolism of carbohydrates, and fats due to interference with activity, insulin secretion, or both (Federico et al., 2016; Hajiaghaalipour et al., 2015). DM sufferers will experience an increase from 171 million (2.8%) to 366 million (4, 4%) until 2020, and tend to experience an increase in prevalence across countries (Wild et al., 2004)). DM is associated with high-risk chronic diseases such as heart attacks, blindness, and kidney failure (Sharma et al., 2016; Hackett and Jacques, 2009). Diabetes affects the decrease in effector immune cells and regulator T cells.

The inability of β-pancreatic cells to produce insulin result in regulatory disturbances from glucose uptake signals, namely GLUT-4 protein, which causes cells to secrete proinflammatory cytokines such as TNF-α, and Il-6 which cause tissue damage (Hajiaghaalipour et al., 2015). DM causes an imbalance of microbiota in the intestine that will increase the number of opportunistic pathogens and oxidative stress (Tai et al., 2015).

Management of DM to date uses anti-diabetes drugs and insulin to maintain quality of life and prevent ongoing damage due to impaired circulation of blood vessels. The use of anti-diabetes drugs continuously and for a long time will affect the damage to the liver and kidneys. Patients tend to choose herbal medicines that have fewer side effects, as a functional food to prevent and treat diabetes and its complications. In this study, Coriander seeds (Coriandrum sativum L.) will be tested on its effect of diabetes mellitus particularly on cellular immunity and the production of insulin.

MATERIAL AND METHODS:

This research used 20 mice Balb-C strain, a model of experimental animals in testing hyperglycemia. The mice were given streptozotosin before treatment of different levels of coriander seed extract. There were five (5) treatment groups and 4 replications each. The use and maintenance of experimental animals have received an ethic-worthy certificate no 1109-KEP-UB from Brawijaya University.

The Coriander Fruit Extraction Method Using Ethanol as a Solvent:

Coriander seeds were mashed using a blender until smooth into powder and were sieved using a 200 mesh sieve. 100 grams of coriander powder was mixed in 1000 ml of 96% ethanol solvent, stirred and soaked for 24 hours. The extract was filtered to separate the precipitate (pulp) and the supernatant (solution), it was repeated 3 times. The liquid extract is evaporated into a rotary evaporator at 50°C and dried using an oven at a temperature of 40°C to produce a viscous extract. The method of Sogara et al. (2014) was adapted with modification.

Treatment on Experimental Animals:

The mice were kept and adapted to temperature, the area and their feed for 7 days. The study used Balb-C strain mice, aged 2.5-3 months, with five (5) treatment groups which were given STZ at a dose of 145mg/kg bw on the 8th day intra-peritoneally (IP), then coriander extract was added with successive doses of 25 (T1), 50 (T2) and 100mg/kg bw (T3), negative control (healthy) (T4) and positive control (STZ) (T5) through the administration of intraperitoneally. Glucose level measurements were performed before administration of STZ and after administration of streptozotosin on days 13.18 and 23, and 28 in all treatment groups. Weighing was done on days 14, 18, 23 and 28 to determine whether there was an effect of weight loss in mice after administration of STZ and coriander extract.

Giving of Streptozotosin and Measurement of Blood Sugar Levels:

Mice were injected with a single intraperitoneal dose of Streptozotocin (STZ) (145mg/kg bw) (Biolegend®) initially dissolved in 0.01 M sodium citrate, with pH 4.5 (Wang-Fischer and Tina, 2018). Blood glucose levels were measured after 5 days of STZ injection. Mice with fasting blood glucose levels of more than 220mg/dl were considered positive for diabetes as showed by Deepa and Anuradha (2011) and Furman (2015). Blood sugar was measured using a Glucostick digital blood glucose level (Gluco-Dr.®) device on the 5th day after it was administered with STZ (Qosimah et al., 2019).

Necropsy of Animal:

Necropsy was carried on the 29th day of the experiment where the spleens and pancreas were collected for further observation and study.

Flowcytometer Procedure:

The spleen and pancreas of mice were observed using flowcytometer to calculate the relative cell percentage of effector cells and insulin. The procedure of Rachmawati and Rifa'i, (2014) was adapted. The antibodies used recorded were: CD4 FITC, Insulin per Cp, and CD11 by FITC-IL6PEcy5.

Data analysis:

Diabetes data collected were quantitative: blood sugar levels, relative amounts (CD11b cell-IL6, insulin and CD4 + T cells). The data were analyzed using the One-Way Analysis of Variance (ANOVA) test with a 95% confidence level to determine the effect of the treatment of coriander extract treatment on mice induced by STZ.

RESULTS:

Measurement of Blood Sugar Levels:

Blood sugar levels in all treatment groups before being induced with STZ showed normal (average range of 103-156mg/dl). Observation of sugar levels after STZ induced on the 13th day, followed by administration of coriander extracts on the 19th, 24th and 28th days of which there was a decrease in sugar levels in a row namely T1 treatment (327.5; 190.5; 177.5; 138.5) mg/dl, while the treatment of T2 and T3 decreases in sugar levels were found only at the last administration of the extract that is the 28th day namely T2 (362; 244.5; 248.5; 186.25)mg/dl and T3 (446; 322.25; 295.5; 178.8) mg / dl. The positive control group (STZ) showed an increase in blood sugar levels (436.75; 405; 326; 307.75) mg/dl while the negative control did not have an increase in blood sugar levels or normal blood sugar levels respectively (96.5; 120,75; 124,25; 126,25) mg/dl.

Weight Measurement:

The results of weight measurements of all treatment groups when given therapy for 14 days and observed on days 14, 19, 24 and 28 showed that in the T1 and negative control groups there was an increase in body weight while in T2, T3 and positive control there was a decrease in body weight (table 1).

Table 1: Observation of body weight in the treatment after administration of STZ and coriander extract

The treatments	Observation of Average Weight (gram)
	Early prior to treatment (Day 8)	After Giving STZ (Day 13)	After administration of Coriander extract therapy
	Early prior to treatment (Day 8)	After Giving STZ (Day 13)	Day 14	Day 19	Day 24	Day 28
T1	20	20,5	21,5	22	22,5	22.75
T2	23	18,5	24,75	22,25	20,5	20,25
T3	20	22	19,25	18,5	18,25	16,5
Positive control	20	18,5	20,25	19,75	18,5	17
Negative control	23	24,5	25,5	25,75	26	26,25

The Relative Amount of Insulin:

The relative amount of insulin in the negative (healthy) control group was higher than the positive control group (STZ-induced mice). In the therapy group there was an increase in the relative amount of insulin in mice in the T1 and T2 groups that were not significantly different from the negative controls (Figure 1). A decrease in the relative amount of insulin was found in the positive control and T3 treatment found both showed no significant effect. This shows an increase in the number of insulin occur in the coriander extract with a small dose, 25mg/kg body weight.

Figure 1: Notation a, b shows the difference between treatment groups (P<0.05) using the One way ANOVA test followed by the tukey test

Relative amount of CD4⁺ T cells:

The results showed that the activation and proliferation of the adaptive cellular immune system CD4 + T cells in mice in the negative (healthy) control group was higher than the positive control. The group of mice T1 treatment had an increase in CD4 + T cells that were not significantly different from negative control, while the decrease in the number of relative CD4 + T cells occurred in group T2, T3 which was not significantly different from positive control treatments. This suggests that activation of CD4 + T cells occurs at doses lower coriander extract is 25 mg / kg bw (Figure 2).

Figure 2: Notation a, b showed a significant difference between treatment groups on CD4 + T cells (P<0,05)

Relatif number of CD11b cell-IL6:

The results showed that the relative number of macrophage cells that produce proinflammatory cytokines (CD11b cell-IL6) of the positive control (STZ-induced mice only) higher compared with negative control group (healthy or normal). The treatment group were given extracts of coriander therapy showed a decrease in CD11b cell-IL6 significantly in the treatment of T1, T3 T2 and while the treatment is still an increase in the number of relatively CD11b cell-IL6 were not significantly different from the positive control (Figure 3). In the group of T1, coriander dose given is able to repair the pancreas through increasing insulin levels, decreased blood sugar levels, weight gain, proliferation and activation of CD4 + T cells and decrease inflammation, CD11b cell-IL6 on the tissue.

Figure 3: Notation a, b shows a significant difference between treatment groups with respect to the relative number of CD11b cell-IL6 (P<0.05)

DISCUSSSION:

Weight Gain:

Weight gain was only found in the T1 treatment while T2 and T3 treatments occurred for weight loss. Weight gain at T1 is due to an improvement in the amount of insulin to help maintain carbohydrate metabolism. This study is in accordance with that carried out by Schaschkow et al., 2016, that insulin therapy can increase body weight in female rats with diabetes Lewis strain. Weight loss in mice after being induced with STZ and coriander extract in T2 and T3 treatments according to research conducted by Tian et al., 2010 which states that STZ induction with a dose of 115mg/kg bw in mice causes an increase in fasting blood glucose levels and feed-drinking intakes but lose weight. There was a tendency for weight loss in coriander groups with doses of 50 and 100mg/kg bw because of coriander content, namely linalool which has an appetizer effect (decreases appetite) (Güler et al., 2005).

STZ induction causes hyperglycemia. DM is a chronic condition characterized by relative or absolute deficiency of insulin, which leads to hyperglycemia due to pancreatic β-cell damage (Damasceno et al., 2014). Β cells experience necrosis due to excessive free radicals in the form of glucose levels in the blood (oxygen radicals or nitric oxide) thereby stimulating proinflammatory cytokine cells (Gvazava et al., 2018). STZ is toxic to pancreatic β cells, which causes disruption of insulin production (Wong-Fischer and Tina, 2018), followed by weight loss (Gvazava et al., 2018). Free radicals will mediate activation of the signal transduction cascade and transcription factors that cause increased expression of specific genes that cause tissue damage and diabetes complications. Hyperglycemia causes a decrease in the activity of antioxidant enzymes that play a role in neutralizing free radicals and decreasing the degree of inflammation. Intraperitoneal STZ is often done to induce diabetes because of the 60% success rate compared to aloxan and hypercaloric (20%) (Frederico et al., 2016). STZ is a natural antibiotic, produced by Streptomyces achromogenes actinomycetes. STZ causes a decrease in ATP, thus causing damage to β cells. STZ also causes impaired glucose homeostasis (oxygen consumption and glucose oxidation) and inhibition of biosynthesis and insulin secretion, not directly and directly affecting glucose transport or phosphorylation by glucokinase (Gvazava et al., 2018).

Relative number of Insulin:

Coriander effect can be used as insulin therapy because it can reduce the condition of hyperglycemia, through increased glucose and insulin metabolism (Chizoba, 2015). The coriander extract content β-carotene acts as an immune modulator, by neutralizing and eliminating singlet oxygen or free radical products. This compound can also be broken down enzymatically by lipoxygenase to prevent radical oxidation and photo-oxidation (Kawata et al., 2018). Quercetin secretes insulin and inhibits intestinal starch digestion and hepatic glucose production, increases glucose absorption in skeletal muscle, and protects against pancreatic damage (Hossain et al., 2016). This is the same as research conducted by Federico at al., 2016, that coriander extract can increase insulin levels.

Relative number of CD4+ T cells:

Type 1 DM (T1DM) causes infiltration and accumulation of leukocytes and T lymphocytes around the pancreatic islets, causing a massive destruction of beta cell insulin production found in the positive control treatment group. MHC class II molecules will bind and present antigen peptides in the form of STZ to CD4 T cells via dendritic cells which will then go to the pancreatic lymph nodes for processing (Kakoola et al., 2014; Wang-Fischer and Tina, 2018). T cell activation is mediated by the signaling molecules CD80, CD86 and CD28 originating from dendritic cells (Wangchuk et al, 2018). Activation of Th (T helper) lymphocyte cells will differentiate into Th1, Th17 and Th2. Th1 cells lead to the activation of phagocyte cells for the release of inflammatory cytokines that cause cell damage in metabolic organs such as fat tissue, liver, muscle and pancreas (Xia et al., 2017) and Th2 cells lead to differentiation of Th2 cells into plasma cells that will produce antibodies besides the production of anti-inflammatory cytokines. In the T1 and negative control treatments an increase in CD4⁺ T cells was significantly different from the T2, T3 and positive control treatments. An increase in CD4⁺ T cells is likely to produce anti-inflammatory cytokines that affect tissue repair through a decrease in proinflammatory cytokines. According to Al-Snafi, 2013, that ethanol extract of coriander seeds contained flavonoids such as caffeic acid, chlorogenic, quercetin and routine. Flavonoids function as anti-diabetics through the following mechanisms: improvement in the function of pancreatic β cells and insulin through a decrease in proinflammatory cytokines IL1-β and IL-6; increased presence of antioxidants through reduction of free radicals and lipid peroxidation; regulation of carbohydrate metabolism (decreased gluconeogenesis and increased glycogenesis) and increased insulin sensitivity (Vinayagam and Xu, 2015).

Relative number of CD11b cell-IL6:

The positive control group showed an increase in the relative number of CD11b cell-IL6, which was not significantly different from the T3 group, whereas in the negative control group and the T1 and T2 treatments showed a decrease in CD11b cell-IL6. Type 1 diabetes mellitus (T1D) is an autoimmune disease characterized by beta cell destruction, associated with cellular infiltration and inflammatory responses on the island of Langerhans. The cellular components of this infiltrate include monocytes, macrophages, CD4⁺ and CD8⁺ T cells, and the balance between Th1 and Th2 cells is very important in the pathogenesis of this disease and against antigens and produces inflammatory mediators such as cyclooxygenase-2 (COX2), protein nitric oxide synthase 2 (NOS2) ), free radical nitric oxide (NO), and proinflammatory cytokines such as tumor necrosis factor-alpha (TNFA) and Il-6 (Interleukin 6) that affect the incidence of diseases such as diabetes (Kawata et al., 2018), secreted by T cells and macrophages to stimulate the immune response during inflammation associated with insulin resistance (Gomes, 2017) so that the number of CD11b cell-IL6 increases in diabetes.

Decreasing the relative amount of macrophages (CD11b), which produce Il-6 according to research conducted by Chizoba, 2015, that coriander extract contains polyphenols and essential fatty acids such as linalool, a-pinene, limonene, and camphene, can inhibit macrophages to produce nitric oxide and pro-inflammatory cytokines TNF-α (Nair et al., 2012; Bhat and Kumar, 2014). Decreasing free radicals such as nitrix oxide and hydrogen peroxidase can reduce tissue damage, namely the pancreas. The mechanism of free radical reduction due to polyphenols works by capturing free radicals and increasing antioxidant enzymes and modulation of enzymes involved in glucose metabolism, increased function of pancreatic β cells to produce insulin, and anti-inflammatory. Polyphenols, especially flavonoids, phenolic acids and tannins can inhibit α-glucosidase, interact with glucose absorption from the intestine by inhibiting glucose transporters that are dependent on Na +, SGLT1 and SGLT2, regulate the main pathways of carbohydrate metabolism and liver glucose homeostasis including glycolysis, glycogenesis, and gluconeogenesis, will usually be disturbed in diabetes. Ferulic acid, a hydroxycinnamic acid derivative, effectively suppresses blood glucose by increasing glucokinase activity and glycogen production in the liver and increasing plasma insulin levels in diabetic rats (Bahadoran et al., 2013).

CONCLUSIONS:

Coriander seed ethanol extract serves as a therapy for the hyperglycemia condition that leads to diabetes through an increase in the relative amount of insulin, a decrease in blood sugar levels, cellular immune activation of CD4⁺ T cells and a decrease in inflammation.

DECLARATION OF CONFLICT OF INTEREST:

There was no conflict of interest between the research team.

ACKNOWLEDGEMENT:

Thanks to the Commodity Research funders College in 2019, the Ministry of Research and Technology and the University of Brawijaya.

AUTHOR’S CONTRIBUTIONS:

All teams colalaborate to do research, write and analize data.

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Received on 27.11.2019 Modified on 05.04.2020

Research J. Pharm. and Tech. 2021; 14(7):3689-3694.

DOI: 10.52711/0974-360X.2021.00638