Decreasing Carbon Tetrachloride Toxicity using Date-seed (Phoenix dactylifera L.) Steeping in Rats

Many toxic compounds in foods cause liver damage and disturbance of bodily function. Inflammation will precede liver damage as an initial response to poisoning. The inflammatory response depends heavily on the strength of the body’s immune system. Many foods, drugs, and other compounds can decrease the immune system, but few serve as immunostimulants. This study aims to prove the decreasing of carbon tetrachloride toxicity using date-seed (Phoenix dactylifera L.) steeping to improve rat immunity. This was an experiment with pre- and posttest with a control group design. Wistar white rats were grouped into 6 groups, healthy control (HC), negative control (NC), positive control (PC), treatment dose 1 g/ kg (T1), treatment dose 3 g/kg (T3), and treatment dose 5 g/kg (T5). All of the groups were induced by CCL4 before treatment except the HC group. The observed data were interleukin-6 (IL-6), lymphocyte count, and C-reactive protein (CRP). Data from the groups were compared with an ANOVA test and followed by a post hoc test if a significant result was found. The results showed that there were significant differences between IL-6, lymphocyte count, and CRP between HC and other groups that CCL4-induced. After the delivery of date-seed steeping, levels of IL-6 and CRP decreased, and the lymphocyte count increased significantly. The group with the 5 g/kg treatment dose was the most effective group for inhibiting the increase of IL-6 and CRP, but a dose of 3 g/kg was the most effective to increase lymphocyte count. Date-seed steeping suppresses pro-inflammation mediators; it has a potency which improves cytokine pro-inflammation by inhibiting the inflammation process. Thus, date seed can be used as a cheap source of anti-inflammation that can be considered as a health opportunity for developing countries.


Introduction
Liver damage often occurs due to toxic compounds exposure in food. One of them is carbon tetrachloride 1 from the industry on household appliances, which can enter into the human body. Carbon tetrachloride as free radicals will cause inflammation of the liver to cause damage 2 . Inflammation is a basic protective response to increase survival in humans 3 . The inflammation process will lead to increased capillary permeability resulting in increased exudate formation, interstitial edema, no angiogenesis, increased pain, and other signs. This is due to increased inflammatory mediators of interleukin-6 (IL-6), lymphocytes, and C-reactive protein (CRP). Excess exudate will cause maceration of the wound and inhibit granulation and tissue epithelization. The healing wound will result in significant increases in neutrophils, cytokines, IL-8, and VEGF 4 . Chronic inflammation increases the secretion of chemical mediators to form exudates that inhibit wound granulation. One way to accelerate the inflammation time is to improve the immune system, and one means of doing this is to give date-seed steeping.
Date seed contains many flavonoid, phenolic, glycoside, oleic, and procyanidine compounds 5,6 . The content of phenolic acid as an antioxidant in date seed can lower free radicals [7][8][9][10][11][12] . Date-seed steeping has been shown to increase antioxidant status and decrease oxidative stress in premenopausal women 13 . In addition, date seed was also shown to increase T and B lymphocytes synthesis 14 . Date seed contains bioactive components such as polyphenols and fiber in large quantities, which are protective for health 15,16 . The results have also shown that administration of 1 g/kg of date palm extract daily for two weeks has a hepato-protective effect 17 and is anti-genotoxic 18 . Extract of dates has shown activity as anti-inflammatory 19 , but the effect of the steeping of date seed is unknown. Medicinal plants were proven to accelerate wound healing and act as an anti-inflammatory 20 .
The role of IL-6, lymphocytes, and CRP in inflammatory reaction to fight antigens in the wound-healing process is unknown and interesting to investigate. This study aims to prove the influence of the date-seed steeping on immune status in the white rat model.

Results
Based on the qualitative analysis of phytochemical examination, the steaming of date seed powder contains flavonoids (+++), tannin (+++), saponine (++) and alkaloid (+). While quantitatively, the steeped powder of date seed contains polyphenol and flavonoid levels as high as 8.44 mg/mL and 20.68 mg/mL, respectively.
The first study examined utilization of date-seed steeping to reduce pro-inflammatory indicators such as IL-6, lymphocyte count, and CRP. Levels of IL-6 after CCL 4 was induced increased, and within 14 days, the increase in IL-6 was higher (seen from the negative control group). There was no significant difference in IL-6 levels between CCL-induced groups before treatment, unlike the healthy group ( Figure 1). Administration of anti-inflammatory agents in the form of dexamethasone (in the PC group) and the steeping of date seed (in the treatment group) resulted in a significant increase in IL-6 levels (p<0.05). The treatment group dose of 5 g/kg had the lowest IL-6 levels after being given the date-seed steeping ( Table 1).
The lymphocyte count after CCL 4 induction decreased and further decreased on the 14th day along with the inflammatory rate. In the PC group, administration of dexamethasone can stimulate lymphocyte synthesis so the resulting lymphocyte count can be increased ( Figure 2). Giving of date-seed steeping can inhibit the decrease of lymphocytes, and the biggest inhibition occurred in groups with a dose of 3 g/kg. However, there were no significant differences in inhibition of lymphocyte count decreasing between the PC group and dose 3 g/kg on treatment group (Table 2).
CRP levels increased during inflammation, and there was no significant difference in CRP levels between groups after CCL 4 -induction ( Figure 3). Administration of dexamethasone in the PC group may inhibit the  Figure 1. IL-6 levels before and after treatment of date seed steeping. Interleukin-6 levels before treatment showed no significant difference between all groups, except with the healthy control group (HC). The NC group had the highest IL-6 level and T5 had the lowest. There is a significant difference in IL-6 after treatment in every group (p<0.05). HC: health control, NC, negative control, PC: positive control, T1: treatment with 1 g/kg date seed steeping, T3: treatment with 3 g/kg, T5: treatment with 5 g/kg. There is a significant difference between groups after treatment (p<0.05). HC: health control, NC, negative control, PC: positive control, T1: treatment with 1 g/kg date seed steeping, T3: treatment with 3 g/kg, T5: treatment with 5 g/kg.
CRP increase, along with the delivery of date-seed steeping. There was a significant difference in the CRP inhibition levels between treatment groups (p<0.00). The highest level of inhibition occurred in the group that was given the 5 g/kg date-seed steeping ( Table 3).
The hepatocyte cell damage can be occurring due to the toxicity caused by CCl 4 . This will have an impact on the increase of ALT after CCl 4 induction ( Figure 4). However, there was a significant difference in ALT levels before and after giving of dates seeds (p<0.05). After CCl 4 is induced, ALT levels increase, as is seen from the mean of ALT in negative control group. But after giving date-seed steeping, the ALT level significantly decreased. The group that was given 5 g/kg of date seed steeping has the highest ALT inhibition.

Discussion
Inflammation is the body's defense response to fight pathogen infection, but if the infection lasts a long time, it will inhibit the healing process. Administration of carbon tetrachloride will induce free radical and oxidative stress in hepatocyte cells that will produce pro-inflammatory cytokines in the form of IL-6. In chronic inflammation, the increase in IL-6 stimulates the Kupffer cells to produce CRP. IL-6 serves to induce acute-phase inflammation. Oxidative stress will trigger macrophages and neutrophils to produce pro-inflammatory cytokines, resulting in increased expression of pro-inflammatory cytokines, IL-1, IL-6, and TNF-α.
The results showed that IL-6 and CRP had increased while the lymphocyte count decreased after giving the date-seed steeping. This evidence supports that the anti-inflammatory action of date seed is related to the inhibition of one or more intracellular signaling pathways involved in the effects of some chemical mediators of inflammation. Giving date-seed steeping suppresses the body's response in lymphocyte production during inflammation. Date seed contains more flavonoid and phenolic compounds. Some flavonoid and phenolic compounds have been proven to suppress inflammation through several mechanisms 21 . Different ways of date seed drying will differentiate on total  Figure 3. CRP levels before and after treatment. CRP levels before treatment showed no significant differences between treatment groups (CCL 4 -induced), but were significantly different in the healthy control group (HC). The highest date seed steeping, the lowest CRP level. Figure 4. ALT levels before and after treatment. After induction of CCl 4 , ALT levels were still low in the blood, but at the 14 th day after oxidation damage, ALT levels increased. Provision of date seeds can repair hepatocyte cells so that levels of ALT decreased again. The lowest levels of ALT were in the treatment group that was given a date-seed dose of 5 g/kg, lower than the positive control (given dexamethasone). phenolic content, flavonoid and antioxidant activity. The higher the phenolic level, the more antioxidant activity increases. Flavonoids can work as an anti-inflammatory through antioxidant activity, radical capture, and modulation of enzyme activity in the arachidonic acid metabolism (phospholipase A2, cyclooxygenase and lipoxygenase and nitric oxide synthase) 22 .
Decreasing pro-inflammatory mediator production is considered as an alternative to prevent or treat complications associated with inflammatory diseases. In this study, the inhibitory effect on inflammation using date-seed steeping was compared with the drug dexamethasone as the gold standard. The results showed that the dose of 5 g/kg is more effective than dexamethasone. Steroidal anti-inflammatory drugs are commonly used as a first choice, but these have many harmful side effects such as fluid retention, disrupting hematopoiesis, osteoporosis, and others 23 . Medicinal plants are very useful as a logical alternative treatment to inhibit inflammation. The extract of date seed contains proanthocyanidin, and this has recently been shown to increase recovery in animals from toxicity that is induced by free radicals 24 .
The active substances in date seeds can work as anti-inflammatories. They may be related to the high total phenolic content of date seeds (about 18 times the GAA/100 g gallic acid equivalent) and have antioxidant activity (about 4-7 times more Trolox equivalent/ g) than its flesh 25 . Phenolic compounds have many health benefits; they are a bioactive phytochemical that act as an antioxidant agent that can prevent free radical disease 3,26,27 . The anti-inflammatory effects of phenolic compounds stem from their ability to regulate pro-inflammatory gene expressions such as NOS, cyclooxygenase, and lipoxygenase, and also work via nuclear factor (NF-kB) and mitogen-activated protein kinase 28,29 . Activation of NF-kB will increase the expression of several genes involved in inflammatory processes such as IL-1, IL-6, IL-8, COX2 and TNF-α. Therefore, inhibition of NF-kB activity may decrease the expression of pro-inflammatory mediators including lymphocytes 30 .
Free radicals play a key role in the inflammatory process. The destruction of cell membranes will trigger the production of reactive oxygen substances that will increase inflammation. Various active compounds in date seeds have potential as herbal anti-inflammatory medicine 31,32 . The age of the date tree influences the antioxidant levels in the seeds 33 . Secondary metabolites of herbal plants such as flavonoids, alkaloids, glycosides, tannins, steroids, and others proved to have an anti-inflammatory effect [34][35][36][37][38] .
Several previous studies have shown that flavonoids can increase the body's antioxidant capacity by increas-ing superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) levels. High antioxidant capacity can reduce oxidative stress, thus accelerating the loss of inflammatory processes that occur.
The oxidized hepatocyte cells will be inflamed and become damaged. Cell membranes are easily oxidized by free radicals from CCl 4 induction. The liver enzyme Alanine aminotransferase (ALT) will come out of the cell and increase in the bloodstream. After giving the date seed steeping, antioxidants in the date seed will inhibit the oxidation of liver cells and repair damaged cells. Flavonoid as an active compound in the date seed steeping works like antioxidant 39 . Therefore, the ALT liver enzyme decreases back to normal levels. So, the steeping of date seed can repair hepatocyte cell inflammation. Based on this indicator, steeped date seed is safe to be consumed.
CRP measures overall inflammatory levels in the body due to infection 40 . In the event of a bacterial infection or inflammation, the leukocytes will be activated and then release cytokine into the bloodstream. Cytokines will stimulate liver cells (hepatocytes) to produce CRP. CRP concentrations will begin to increase at 4-6 hours after surgery and reach the highest levels at 48-72 hours after surgery. CRP levels will return to normal after 7 days postoperatively. However, if after surgery an inflammation or sepsis develops, the level of CRP in the blood will continue to increase. Many compounds with high polyphenol content are shown to have antiinflammatory activity 41 . These compounds contribute to the decrease of CRP.
Roasting can cause chemical and biological activity changes, and form the Maillard reaction, due to the process of degradation of reducing sugars and amino acids 42,43 . Roasting date seed can be controlled by regulating the temperature and duration of the roasting process. The results of roasting can be qualitatively known by comparing the colour of roasted beans likes brown, city roast, and dark 43 . The longer the roasting time and temperature, the darker brown colour. If the temperature and duration of roasting are increased beyond the tolerance limit, an unwanted flavour will be obtained. Roasting is the key of the stage in the process of formation of date-seed steeping. The roasting process with a temperature of 200°C for 12 minutes will produce a good roasted coffee powder. Roasting will produce the aroma and taste of coffee that comes from heat treatment. When compared to the green bean date, date seed roasting has less antioxidant activity than green bean dates, but higher than dried by oven and under the sun. Date seed roasted is also more delicious in aroma and flavor so it can increase acceptability on humans.
The implications of this research are that this com-pound can be used for people who are exposed to CCL 4 from industrial or food contaminated with CCL 4 or other hepatotoxic agents. Date-seed steeping can be applied as a natural anti-inflammatory agent. This study has a limitation due to its inflammatory processes only being seen from cytokines pro-inflammation, and not yet being seen from inflammatory products such as arachidonic acid, prostaglandin, and prostacyclin.

Conclusion
Inflammation due to free radicals of carbon trichloromethyl from carbon tetrachloride exposure will lead to increased IL-6 and subsequently stimulate CRP synthesis, whereas the lymphocyte count decreases. Giving date-seed steeping can inhibit the rate of IL-6 and CRP increases, so it can be inferred that it inhibits inflammation. The lymphocyte count that decreases after CCL 4 -induction can be increased again by giving dateseed steeping. Date-seed steeping can be used as an anti-inflammatory agent.

Preparation of Dates
Deglet Noor date seed was selected and separated from the flesh. Seeds were washed with running water and dried under the sun for 1 day. The dried seeds were roasted with medium heat, crushed with a blender, and filtered to obtain a fine powder. Date seeds were roasted at a medium temperature for 5-7 minutes to produce the best aroma and taste. Date seed powder was weighed according to dosage, brewed with boiling water, and given when cold (room temperature) for 2 weeks.

Study Design
The research was conducted using a laboratory experiment with pre-and post-test with a control group design approach. The samples used were male Wistar rats aged about 2-3 months with weight range 150-200 g. The number of samples used as many as 30 rats consisting of 6 tails per group. Rats were grouped into 6 groups, healthy control (HC), negative control (NC), positive control (PC), treatment dose 1 g/kg (T1), treatment dose 3 g/kg (T3), and treatment dose 5 g/kg (T5). Each group was replicated five times. Rats were acclimatized for 7 days before the experiment and were divided into cages based on group. All animals in the experiment were given standard food as much as 10% of body weight, and drinking water ad libitum every day. Food was given every morning and afternoon. The re-search was conducted according to animal research protocols.
All of the groups, with the exception of the HC group, were induced by a CCL 4 dose of 0.25 mL/200 g in a single dose before treatment: The carbon tetrachloride was dissolved in olive oil with a ratio of 1 : 1. Quantitative IL-6 plasma measurements were taken with enzyme-linked immunosorbent assays using commercially available kits (BT Laboratories, Shanghai) according to the instructions available from the suppliers. A blood analyzer (Sysmex [TOA Medical Electronics, Kobe, Japan]) was used to test all venous blood samples for the determination of the complete blood cell counts and the leukocyte differential counts. A GIEMSA staining was used to test the leukocyte differential counts. ALT was measured by spectrophotometer.

Statistical Analysis
Normal distribution of variables was tested using the Shapiro-Wilk test. The obtained data will be analyzed by one-way ANOVA tests to know the differences between groups. If there is a significant difference, the data analysis will be continued with the LSD post hoc test. The test results are said to be significant if p< 0.05. The value is presented in the text as the mean± standard deviation.