The effect of variations in coffee and tiwai plants as drink products on blood lipid profiles and hematological indices

Plants or spices can be processed into herbal drinks as an alternative to traditional medicine. Using existing local resources, it is possible to produce functional food in the form of drinks. Tiwai coffee (TC) is a drink made by mixing Tiwai onion powder (TP) with coffee powder (CP), sugar, and salt. Therefore, this research aims to determine the effect of administering TC, ground coffee, green coffee (GC), TP, and instant Tiwai (IT) on lipid profiles of cholesterol, LDL, HDL, and triglycerides, hematological indices of Hb, erythrocytes (RBC), leukocytes (WBC), and hematocrit (HCT), glucose, heart rate, and body temperature in mice. Furthermore, a post-test-only control group design was applied with six treatment types and seven replications. The data were analysed using ANOVA and once a significant difference was discovered, the Tukey’s with a 5% level was subsequently conducted. The administered products had a significant effect only on HDL, TG, WBCs, and body temperature, excluding other parameters. In general, it is best to give green coffee. In general, the best treatment is giving green coffee, especially when increasing immunity as indicated by the lowest WBCs value.


Introduction
East Kalimantan is an area rich in plant diversity with the potential to be used as herbs which are often applied as medicinal ingredients.The use of wild plants is very important in maintaining local wisdom and a sustainable environment.One of them is the tiwai plant so that it can be cultivated well in the future.Functional food production is currently growing a lot because the community's need for foods consisting of highly active components that are beneficial to health is increasing [1].
Functional food can be processed into any form once made with the right composition to ensure the health effects are felt by the body.Tiwai onion is one plant type that has functional food potential (Eleutherine americana Merr).Moreover, it is a medicinal plant typically found in Kalimantan and is used by the Dayak community to help cure several degenerative diseases and lower glucose levels in diabetics.Tiwai onion contains steroids (-), saponins (-), flavonoids (+++), polyphenols (+++) and alkaloids (+) [2].Phytochemical compounds have an influence on cholesterol, LDL, and hematological indices [3][4].The FTIR results obtained after mixing CP and TP showed one indentation or absorption curve at a wavelength of 2,936.09nm.Meanwhile, roasted coffee previously had two indentations at 2,800 nm to 3,000 nm, containing antioxidant components mainly from the absorption of O-H bonds, which form flavonoid compounds.The antioxidant activity was determined from the highest to the lowest successively from GC, roasted ground coffee, TP, roasted CP mixed with TP, to roasted CP mixed with IT [5].
The high demand for coffee-based drinks and people's lifestyles requires innovation in the manufacture of functional foods in the form of various coffee drinks.Besides, the TC product in this research was a mixture of ground coffee, roasted TP, brown sugar, and salt.Diversification of processed coffee and tiwai products can increase farmers' income.Based on the description above, there is a need to determine the effect of administering TC, TP, ground coffee, GC, and IT drinks on lipid profiles, hematological indices, glucose, heart rate, and body temperature in mice.

Materials and Methods
The material used was 9 months old Tiwai obtained from the garden in, Samarinda.Furthermore, arabica coffee beans were purchased from farmers in Teluk Pandan Village, East Kutai.Brown sugar, salt, mineral water, 70% alcohol, and mice feed in the form of pellets (Comfeed Broiler II) were obtained from an animal feed shop in Samarinda.The feed composition included a mixture of ingredients in the form of corn, bran, soybean meal, coconut meal, meat, and bone meal with a nutritional energy content of 4,100 Kcal/kg, 19% protein, 3-8% fat, 0.9-1.1% calcium, and 0.6-0.9%phosphorus.The experimental animals were procured from the warehouse in Samarinda, was 3 months old, healthy, male white mice (Mus musculus), weighing 20-30 grams.

Tiwai coffee processing procedure
2.1.1.Tiwai coffee (TC).Up to 10 g of the CP was mixed with 20 g of IT in a 1:2 ratio, then blended using a Kitchen Cook Mixer LJ70001 with a rotation speed of 1 for 30 seconds.

Tiwai powder (TP)
. Tiwai onions were washed using running water to remove impurities, then drained.Afterward, they were cut into smaller sizes of ± 1 mm, which were air-dried for two days.These pieces were roasted (using Teflon) until blackish brown, cooled at room temperature, and refined using a blender, then sieved with 80 mesh.

Coffee powder (CP).
Dried coffee beans were roasted until blackish brown, then cooled at room temperature, refined using a blender, and sieved with 80 mesh.

Green coffee powder (GC).
To make the green coffee powder, coffee beans were dried without roasting and mashed with a blender, then filtered using 80 mesh.

Instant tiwai (IT).
An extract was prepared from the onions that had been sorted, cleaned, reduced in size, mashed with a blender at a Tiwai to water ratio of 1:2 (w/v), and then filtered.This was put into a frying pan and brown sugar was added at a ratio of 1:3, then heated over low heat for the brown sugar to melt.After being slightly thick, the fire was turned off and continuous stirring was done to produce instant brown sugar granules.

Research design and procedure
A post-test-only control group design with six treatment types and seven replications requiring 41 mice was employed in this research.The mice drink was prepared by brewing 15 g of TC, IT, ground coffee, GC, and TP each, using 150 mL of water, for the control of 40 ml of mineral water, then put in a 40 ml drinking bottle, and replaced every day.The feed used was standard (Comfeed Broiler II).
The mice were maintained using plastic cages covered with wire, and a sand mat (Tatapet) was provided to facilitate the cleaning process.Adaptation was carried out for five days before administering feed and treatment ad libitum for another 14 days.The feed and drink were replaced each morning, and parameter analysis was performed on the 15th day.

Research ethics
This research was carried out on mice and humans by referring to the ethical feasibility letter No.83/KEPK-FK/IX/2021 received from the Health Research Ethics Commission (KEPK) Faculty of Medicine, Mulawarman University.

Data analysis
2.4.1.Weight.The weight of mice was measured with a digital scale (electronic SF-400).

Body temperature.
The body temperature was also measured on day 15 using a thermogun (AICare).

Mice heartbeat.
The heart rate and respiration of mice in each group were evaluated using pulses and a stopwatch for 15 seconds.

Lipid profile analysis.
On the 15th day, the lateral tail vein of the mice was cut, but the first blood drop was discarded, and the next drop was touched to the sensor gap at the end of the test strip that had been installed on a digital detector (SD Lipido Care tool) for lipid profile results to be read.

Analysis of blood glucose and
Hb. Blood was collected from the lateral tail vein, then added to the sensor of Accu Check Active to measure glucose level, while hemoglobin (Hb) was tested using MultiCheck 2 (Nesco).

RBC, Htc and WBC analysis.
The blood for WBC, RBC, and Htc analysis conducted in the Fisheries Laboratory of Mulawarman University, was obtained from the orbital sinus of the eye with a micro-hematocrit.The erythrocytes (RBC), leukocytes (WBC), and Hematocrit (Htc) were evaluated using the manual counting room method, Thoma pipette, and standard micro-hematocrit method, respectively [6].

Statistical analysis.
The data that showed a significant effect during the ANOVA were then subjected to the Tukey's test with a 0.05 level.

Mice' body temperature
The research results show that the GC and control treatment were significantly different, while those of TC, ground coffee, TP, and IT were not significantly different.Table 1 shows the average body temperature of mice in all treatments and the results varied.During maintenance and testing, the room temperature was regulated to become lower and cooler using an air conditioner.However, the food or drink consumed could be a factor that affected the mice's body temperature.In this research, Tiwai onions and coffee contain flavonoids that caused a reduction in the mice' body temperature.Mice as living beings also have the ability to adapt and maintain a stable body temperature despite fluctuations due to changes in environmental temperature.Once the environment is cold, the metabolism increases for the body to become warmer than the environment or vice versa [7].x The data in the same column followed by the same letter indicate that there is no significant difference in the HSD test with a 5% level.y Tiwai Coffee (TC), Coffee Powder (CP), Green Coffee Powder (GC), Tiwai Powder (TP), and Instant Tiwai (IT).

Heartbeat
After being treated for 14 days, the number of heartbeats produced by mice was calculated within 15 seconds using a time-measuring device in a stopwatch form.Heart rate testing was conducted by placing the index finger on the mice's hearts and counting the number of heartbeats.The variance results showed no significant effect on the heart rate in mice.The average heart rate measured while carrying out this research on the TC, ground coffee, GC, TP, IT, and control groups, was 35.20/15 seconds, 35.25/15 seconds, 27.83/15 seconds, 35.83/15 seconds, 30.28/15 seconds, and 29.16/15 seconds, respectively.It was discovered that the administration of different drinks to mice had no significant effect on heart rate.

Glucose
Based on the results, average blood glucose levels obtained in the TC, CP, GC, TP, IT, and control groups were 94.80 mg/dL, 68.25 mg/dL, 73.17 mg/dL, 77.33 mg/dL, 75.42 mg/dL, and 92.33 mg/dL.The average glucose after consuming TC, GC, TP, IT, and controls was from 72-126 mg/dL and can be categorized as being in the range of normal glucose levels.This indicated that the mice did not experience hyperglycemia and diabetes.This is caused by the provision of pure ground coffee without sugar addition to obtain a less energy source in the form of glucose and can be compared with the TC and IT treatment containing sugar, to ensure relatively more normal glucose levels.
In this research, drinking had no significant effect on glucose in mice.This is in line with the result reported by Urzua et al [8] that caffeine did not affect blood sugar levels in non-diabetic rats.The blood sugar levels in fasting control rats decreased significantly more than in the caffeine-consuming rats.Based on the highest antioxidant activity to the lowest, Saragih et al [5] classified GC and roasted ground coffee as strong antioxidants, while TP, TP mixture with roasted coffee powder, and IT mixture with roasted coffee powder were in the medium category, and TC was included in the weak category.

Weight
The variance test results presented in Table 2 showed no significant difference in body weight before and after treatment.Based on the results for each replication of the treatment sample, the body weight of mice given TC, CP, and GC decreased from 23.86 -23.29 g, 23.14 -23.00 g, and 21.86 -20.00 g, respectively.Meanwhile, those given TP, IT, and control had increased from 22.71 -24.71 g, 22.00 -24.86 g, and 23.00 -24.00 g or gained an average weight of 1.45, 2.86 g, and 1.00 g, respectively.Hausenblas and Huynh [9] stated that the chlorogenic acid in GC contains no caffeine but it can significantly cause weight loss in obese individuals.The current meta-analysis results support the use of GCE (green coffee extract) supplementation for an increase in the obesity index, with the subgroup analysis highlighting a greater increase in individuals with an initial BMI of 25 kg/m 2 [10].3 showed the treatments had no significant effect on mice cholesterol.The highest average cholesterol was found in the TC treatment at 129.50 mg/dL while the lowest was 112.75 mg/dL in the CP treatment.The increase in total cholesterol levels in the treated group may occur due to the coffee administration effect capable of inducing free fatty acids (FFA) release.The highest HDL average of 42.83 mg/dL was found in the IT treatment, with other treatments.HDL has a strong antiatherogenic effect, hence it is often referred to as good cholesterol.Other research shows that plants consist of abundant phytochemicals that have substantiated with evidence to be defensive against cardiovascular diseases [11] [12].Decreased TG levels in this research can occur due to the influence of flavonoids playing a role in reducing TG levels by increasing lipoprotein lipase enzyme activity through the reduction of lipid peroxidation [13].3 showed that the treatment had a significant effect on mice's HDL levels.The highest HDL average of 42.83 mg/dL was found in the IT treatment, while the lowest was 27.50 mg/dL in TP.The HDL increase in IT compared to other treatments was suspected to be caused by the Tiwai flavonoid compounds which can also lower cholesterol.The results of this research are supported by several research results showing that flavonoids can reduce the risk of high cholesterol in the blood [14] [15].3, the TG levels of mice in the four treatments were lower than those in the control group.The highest average of TG was 119.75 mg/dL in the control, while the lowest was 62.50 mg/dL in the GC treatment.The highest LDL average of 79.40 mg/dL was found in the control treatment and the lowest was 58.30 mg/dL in TC.Decreased TG levels can occur due to the influence of caffeine contained in coffee, which can increase fatty acid levels based on the positive effect of hormone-sensitive lipase and the flavonoids present in Tiwai onions.In contrast, high coffee consumption was related to high blood pressure [16].

Low-Density Lipoprotein (LDL).
The variance test results in Table 3 showed the treatments had no significant effect on mice LDL.The highest LDL value was found in the control and the lowest was in GC.According to Meng et al [17] research on test animals, chlorogenic acid showed activity in glucose and lipid metabolism such as increasing insulin secretion, reducing susceptibility to LDL oxidation, causing hypoglycemia, and producing antidiabetic effects.The amount of chlorogenic acid in coffee can be affected by the manner of processing, for example, during roasting.

Hematological Indices
After being treated for 14 days, the number of heartbeats produced by mice was calculated within 15 seconds using a time-measuring device in a stopwatch form.Heart rate testing was conducted by placing the index finger on the mice's hearts and counting the number of heartbeats.The variance results showed no significant effect on the heart rate in mice.

3.6.1.
Hemoglobin.The variance test results showed that for the administration of different drinks in mice had no significant effect on hemoglobin levels.Based on Table 4, the average hemoglobin levels obtained in the TC, ground coffee, GC, TP, TI, and control groups were 15.25 g/dL, 16.77 g/ dL, 14.77 g/dL, 15.88 g/dL, 13.75 g/dL and 12.16 g/dL, respectively.Table 4 shows the average Hb of mice in all treatments ranging from 14-16 g/dL which is in the normal range.In humans, normal Hb levels range from 13-17 g/dL for males, and 12-15 g/dL for females [18].a Data in the same column followed by the same letter indicate that there is no significant difference in the HSD test at α 5% level.

Hematocrit (Htc).
The results showed no significant difference in the hematocrit of mice in TC, CP, GC, TP, IT, and control groups.Hct levels in all treatments were in the range of 28-32.5%,but the control treatment had 32.5%.This is thought due to the effect of TC containing brown sugar, coffee, and TP.An increase or decrease in mice' Hb levels affects the number of Hct, RBCs, and WBC.Hct examination describes the ratio of the percentage of RBCs, WBCs, and platelets to whole blood volume or the erythrocytes' concentration (%) in 100mL/dL of whole blood.[20].Hct levels in all treatments were in the range of 28-32.5%.An extremely high or low Hct level can indicate a blood disorder, dehydration, or another medical condition.An abnormally low Hct may suggest anemia, namely a decrease in the total number of RBCs, while an abnormally high hematocrit is called polycythemia.The highest RBCs were found in IT and the lowest was in GC but not significant difference.RBCs in humans or other animals that do not have a nucleus, known as red blood cells, hematids, erythroid cells, or erythrocytes, are the most common type of blood cell and the primary means by which vertebrates deliver oxygen (O2) to the body tissues through the bloodstream in the circulatory system.
3.6.4.Leukocytes (Blood Cells/WBCs).The results showed significant differences in mice WBCs between the GC, TC, CP, TP, IT, and control groups with levels ranging from 3,075 cells/mm3 to 6,775 cells/mm3, where the highest was in IT and the lowest was in GC.WBCs in the control treatment were 4,350 cells/mm3.Meanwhile, the TC had 4,500 cells/mm3.Compared with the control, TC administration created no significant difference in the WBC value of mice.WBCs play an important role in helping the body fight infections or other diseases.A high WBC count can be caused by an infection, but it tends to also indicate a disease [20].

Conclusions
The administration of TC, CP, GC, TP, and IT had a significant effect on HDL, TG, WBCs, and body temperature in mice, however the glucose, heart rate, cholesterol, LDL, Hb, Htc, RBCs, and body weight were not affected.Test results of several mixed products of tiwai, coffee and palm sugar as a diversification effort to produce new products to support farmers' income.

Table 1 .
The effect of administering six different treatment types on glucose, heart rate, and body temperature in mice.

Table 2 .
Differences in body weight of mice pre and post-treatment

Table 3 .
Lipid profile of mice after being given treatment

Table 4 .
Hematological analysis in mice (Mean±SD) [19]yone has a different Hct level, depending on gender and age.The normal Hct values according to age and sex are 40-54% in adult males and 38-46% in adult females.Low and high levels of Hct, Hb and RBCs were associated with vascular smooth muscle dysfunction, and low Hct levels were associated with abnormal vascular structure[19].RBCs in the control treatment were 3.97 cells x106/mm3, while TC had 4.296 cells x106/mm3, which was relatively higher 3.6.3.Erythrocytes (Red Blood Cells/RBCs).The results showed no significant difference in the RBCs of mice in TC, CP, GC, TP, IT, and control groups.RBC levels in all treatments ranged from 2.56 cells x106 cells/mm3 to 5.875 cells x106/mm3.The highest was found in IT and the lowest was in GC.