INTRODUCTION:

Indonesia is the 4th largest coffee producer in the world (Indonesia News, 2018), and it supplies the domestic consumption and export demand. There are many types of coffee in Indonesia, including Sumatra coffee, Sidikalang coffee, Arang coffee, Lanang coffee, preanger coffee, Toraja coffee, Aceh Gayo coffee, Bali Kintamani coffee, Wamena Papua coffee, Flores Bajawa coffee, Javanese coffee, Robusta, and Arabica (Garuda Mas Papua, 2018), but the best one is Lampung Robusta coffee.

One of the reasons to drink coffee is for its antioxidant quality because it contains phenolic (chlorogenic acid), alkaloids, carbohydrates, fats, and high amount of caffeine (Affonso et al., 2016). Meanwhile, balanced diet contributes to sustain animal’s health as in protecting them against infectious and non-infectious diseases and maintain the animal’s productivity, and among all active ingredients, antioxidant-rich feed is the key factor (Pathak et al., 2017). In addition, coffee can serve as a hepatoprotective, hypoglycemic and antimicrobial, and antispasmodic agent (Farah and Donangelo, 2006).

Research on the use of coffee on chickens, especially during the starter phase of laying hens, has not yet existed. Adding coffee to chicken feed is expected to improve the chicken’s health. Polyphenols in coffee can improve the immune system and avoid chronic inflammation. Chlorogenic acid (CGA) can inhibit pro-inflammatory cytokines in human endothelial cells. The ferulic acid, caffeic, and n-coumaric acids, gallic acid, cyanine, caffeic acid, vanillin, epicatehin and ferulicid acid can be used as antioxidants that work through inhibition of oxidation (hydroxyl free radicals) so as to inhibit the occurrence of fat peroxides (Yashin et al., 2013; Kreicbergs et al., 2011). Through the analysis, the researchers aim to explore the health benefits of coffee for chickens.

MATERIAL AND METHODS:

Ethics:

This study used the ISA brown strain chicken layer and had obtained letter of Ethical Clearance No 773-KEP-UB.

Experimental Design:

This research used 4 treatments and 6 replications consisting of a negative control (healthy chicken, fed without coffee), P1 (fed with 500mg coffee/kg bw), P2 (fed with 1000mg coffee/kg bw) and P3 (fed with 1,500 mg coffee/kg bw). Vitamins were given when the chickens were 1, 5 and 11 days old to prevent stress. The chickens were vaccinated with live ND-IB at 4 days old and killed ND G7B and AI subtype H5N1 at 10 days old. The coffee extract was inserted using gastric tube to 3-16 day old chickens. The chickens were not fed for 10 hours prior to this treatment.

Birds, housing and feeding:

The chickens were placed in cages and fed and watered ad libitum. Standard commercial feed was used.

Coffee extraction:

The coffee extraction was carried out at UPT Materia Medika, Batu, Indonesia using 90% ethanol. 414 grams of Lampung robusta coffee was soaked with 1500ml 90% ethanol in a jar. The jar was sealed and then shaken at 50rpm. The liquid extract was filtered and left to evaporate using Rotatory evaporator. The brown extract from this process was used for the experiment.

Measurement of MDA Concentration (Malondyaldehyde):

MDA concentration was measured with the preparation of Standard Curve 1, 1, 3, 3-tetrametoksipropana (TMP), reacted with 1.0mL TCA 20% and 1.0mL 1% TBA in 50% glacial acetic acid solvent. All solutions were incubated for 45 minutes at 95^oC and then centrifuged at 1000rpm for 15 minutes. The supernatant in the upper layer was measured by its absorbance using a spectrophotometer at a wavelength of 532nm. The sample concentration was obtained by plotting the absorbance data of the sample into the standard curve (Momuat et al., 2011). Blood for plasma production was collected when the chickens were 16 days old through the brachial vein.

ND and AI antibody testing using the HI test (Hemaglutination Inhibition):

The HI test was carried out in accordance with Qosimah et al., 2018, using the antigen ND and AI 4 HA units per well and 1% chicken erythrocytes. HI titers showed the highest dilution of specific antibodies that could inhibit viral antigens agglutinate erythrocytes, shown as log 2. Each sample testing was repeated twice. Serum ND and AI samplings were performed when the chickens were 18 days old.

Histological examination:

Necropsy was performed on 18-day-old chickens to collect the jejunum organs. The jejunum was rinsed with PBS (phosphate Buffer Saline) and then put in 10% formalin. The samples for histopathology were prepared in a series of stages which included graded alcohol dehydration, clearing, xylene treatment and embedding in paraffin. The sections were colored with haematoxylin and eosin (Gerzilov et al., 2015).

Statistical analyses:

Data on Antibody titers and MDA levels (the quantitative data of average and standard errors) were analyzed using one-way analysis of variance (ANOVA) at a confidence level of 95%, while the histopathological data were analyzed qualitatively to describe jejunum damage.

RESULTS:

Bioactive test results:

After the phytochemical screening using 70% ethanol solvent, tannin and alkaloid were found in the samples, while no flavonoids were detected. Based on LC-MS (Liquid Chromatography with Mass Spectrometer) test, the samples contained CGA.

Antibody examination results for ND virus and AI virus:

The blood serum layer was taken at the age of 18 days. Then antibody was measured using an HI (hemaglutination inhibition) test to measure the humoral immune response. The results showed that the ND antibody titers in the chickens not given green coffee (negative or healthy control, only vaccinated with ND vaccine) were higher than those treated with green coffee (Graph 1). On the other hand, the antibody titers against AI virus showed titers 0 in all treatments (with or without coffee extract - data not shown). Lower ND antibody titer was found in the treatment group given higher dose of coffee extract, which was shown in P3 treated chickens (given 1500 mg coffee extract/ kg bw). The negative control group indicated a protective titer of the ND virus of 2^6.

The ND antibodies appeared in the expected time, which was 14 days after ND-IB live vaccine was given and 8 days after the ND G7B and AI subtype H5N1 killed vaccines were given. However, the antibody titers decreased as the increasing doses of coffee extract were given. AI antibody titer analysis was carried out earlier, 8 days after the AI kill vaccination was administered, so antibody titers against the AI virus had not been formed (titer 2°).

Graph 1. Robusta Coffee versus Antibody Titer of Newcastle Disease Virus

(The different letters in each column show statistically significant differences (p< 0.05).)

The research findings showed that the administration of coffee extract can lead to decrease in MDA (Malondyaldehyde) levels in all treatment groups, especially in P1, but the MDA level was still higher than that in the normal or negative control group. The MDA level of P2 was higher than that in P1 and P3.

Graph 2. Robusta Coffee versus MDA Level

(The different letters in each column indicate statistically significant differences (p< 0.05)).

Histopathology of the Jejunum Intestine:

In the negative control group showed normal villi which appeared intact, slender and coated with cuboid layer epithelium (Fig. 1). This condition was also found in P1 and P3. In contrast, histopathology of P2 displayed intestinal erosion, infiltration of inflammatory cells and epithelial vacuolization, which could be caused by the coffee extract (Fig. 2,3 and 4).

Fig.1a negative control: The villi appeared intact, slender and coated with cuboid epithelium (black arrow), 100x magnification

Fig 1b. negative control: Epithelial arrangement with a regular cuboid, no erosion (black arrow) detected, 400x magnification

Fig. 2a. P1: Villi were intact, elongated (black arrow), 100x magnification

Fig. 2b. P1: Accumulation of Epithelial cell nuclei, showing epithelial proliferation (black Arrow), 400x magnification

Fig. 3a. P2: villi were intact and elongated but erosion was detected (black arrow), 100x magnification

Fig 3b. P2: vacuolization epithelium (a), lamina propria contained inflammatory cell infiltration (b), 400x magnification

Fig.4a. P3: villi were elongated and intact (black arrow), 100x magnification

Fig.4b. P3: epithelial proliferation, cell nuclei accumulated (black arrow), 400x magnification.

DISCUSSION:

According to Doss et al. (2009) and Andayani (2011), secondary metabolisms of CGA, tannins, and alkaloids act as an antibacterial, antiviral, antifungal, antimycosis, immunostimulator and antioxidants. ND and AI vaccines on layer chickens are administered to prevent the outbreak of the disease that will lead to decreased egg production or fatality. The Newcastle disease spread by ND virus can result in economic losses in poultry business. The high transmission of the disease in poultry is due to low antibody titers (Sarcheshmei et al., 2016). ND and AI viruses can attack the respiratory, digestive and nervous systems in chicken. The antibodies are produced by Bursa fabricius (FB), the primary lymphoid organ, where the lymphocyte cells maturate. Bursa fabricius develops since the chicken is in its embryonic phase and hatch (Tîrziu, and Seres, 2010).

HI test or Neutralizing antibodies can be detected in 6-10 days post-vaccination, while antigen-specific cytotoxic T-cells can be detected in 7-10 days. The antibody titer peaks and reaches persistent protection in 5 weeks post-vaccination (Vrdoljak et al., 2018). Live vaccines such as La Sota derived from ND low, lentogenic virulence strains are often used because of their high efficacy and availability. The killed vaccine is used as immunity booster when antibodies are dropping (Khalifeh et al., 2009). ND vaccine is given to accelerate the formation of antibody titers and to release the antibody slowly to the protective level. Failure in vaccination is caused by many factors, namely the presence of immunosuppressant agents that cause chickens unable to respond adequately to the presence of antigens (vaccine) that enter the body; inappropriate vaccines, ailments, poor sanitation management, or the more virulent viruses than the vaccine (Sarcheshmei et al., 2016).

The results of this research showed that an increase in the coffee dose tends to decrease the antibody titers. Until now there is no scientific literature on the bioactive contents of green coffee and on the toxic dose of green coffee in chicken. In mice, on the other hand, the toxic dose of LD50 CGA in coffee is 100mg/kg bw orally, and the dose of LD50 caffeic acid is 400-1500mg/kg bw by IP (intraperitoneal) (Tyce, 1998).

Decreasing antibody titers is caused by several factors: individual health, vaccine administration, age of vaccinated animals, post-vaccination evaluation, and the active contents of herbs that can help activate immune cells. This is contrary to Kwang-Soon (2017) that the bioactive content of coffee, namely crude polysaccharide (CCP-0) isolated from cold-brew coffee can activate the immune system by activating the macrophages and intestine immunity. Macrophages can secrete proinflammatory cytokines for the activation of proliferation of B cells, which will differentiate into plasma cells producing antibodies. According to Horrigan et al. (2006), the caffeine in coffee (in an appropriate dose) may act as an immunomodulator.

MDA is a parameter of oxidative stress caused by exposure to free radicals (peroxide). Peroxide breaks down the phospholipid membrane which can produce MDA (Bhutia et al., 2011), and antioxidants cannot compensate for the presence of free radicals that cause oxidative stress conditions. Free radicals damage membrane cells, which results in tissue damage and necrosis. Oxidative stress affects the immune system so it will be more sensitive to microbial infection. Cellular components are rich in polyunsaturated fatty acids and sensitive to oxidative attacks which cause damage to fat peroxidation. Peroxide products are highly cytotoxic and they affect the cellular immune responses (Venkatalakshmi et al., 2016).

Diseases can be prevented by using antioxidants to capture free radicals, to maintain hemostasis at cellular levels in the body of healthy tissue and play a role in molecule signaling. Some cells can produce (O2•−), hydrogen peroxide (H2O2) and nitric oxide (NO) during a tissue damage (Venkatalakshmi et al., 2016). This research showed that low the dose of coffee extract could reduce free radicals, although not as efficiently as in the treatment of negative control group. CGA, an active ingredient in coffee, serves to locally decrease free radicals, mainly in the outer membrane, and it does not induce hemolysis or change the osmotic resistance of erythrocytes. It alters the hydrophilic region of the membrane without changing the fluidity in the hydrophobic region and modifying its properties so as to allow acids to reduce free radicals around the cell and inhibit their diffusion to the inner membrane (Venkatalakshmi et al., 2016; Bonarska-Kujawa et al., 2015). According to Demirtas et al. (20120, another active ingredient, caffeine protects cells against damage caused by radiation and other agents, reduces lipid peroxidation, protects membranes from damage induced by reactive oxygen species, cleanses ROS (reactive oxygen species), and inhibits hydroxyl radical formation and DNA damage.

Intestinal villi contain most of the different absorption cells to increase the surface area of the intestine and its capacity to absorb fluid and nutrients from food. Increased surface area can reduce the average distance traveled by nutrient molecules, thus increasing the effectiveness of the diffusion process (Balbi and Ciarletta, 2013). Intestine as a first line of defense has a wide surface area where the degradation and absorption of food, pathogens and toxins occur. Bowel stressors increase the mucosal immune response to pathogens, causing intestinal damage and poor growth performance. Therefore, feed additives such as antibiotics, probiotics, prebiotics and fitogenic are used increase the health of poultry (Huang and Lee, 2018).

A certain amount of coffee can induce irritations to organs. P2 chickens showed that irritation occurred, and this confirmed the study by Gerzilov et al. (2015) on intestinal histopathology (ileum and cecum), that treatment with herb mix such as 0.05% garlic powder (Allium sativum), 0.3% cinnamon powder (Cinnamomum verum), and 0.03% of each of the following dried herbs: yarrow (Achillea millefolium), rosemary (Rosmarinus officinalis), thyme (Thymus serpyllum), basil (Ocimum basilicum) and oregano (Origanum vulgare)would cause desquamation or erosion of intestinal epithelia and cell inflammation in lamina propria (Gerzilov et al, 2015).

CGA is a phenol that serves as analgesic, antipyretic and anti-inflammatory agent, which works through c-Jun N-Terminal kinase (JNK) and protein 1 (AP-1) activator (Chauhan et al., 2011; Kwang-Soon, 2017). c-Jun N-terminal kinase is associated with inflammatory regulation and immunity to stress (Huang and Lee, 2018). Tannin serves as a lipid peroxide binder, thereby reducing tissue damage, phagocytic cell stimulation, or host-mediated tumor activities, and functioning as a variety of antimicrobials (Venkatalakshmi et al., 2016; Pathak et al., 2017).

When inflammation occurs, the cell will recruit other cells to the local area by releasing more inflammatory cytokines and chemokines. This is a normal process in the body as the host fights infection and repairs damaged tissue. However, prolonged inflammation can cause unnecessary energy expenditure, so preventing inflammation and recovering an animal’s immune level will be beneficial in animal production (Huang and Lee, 2018). The research results demonstrated that the administration of a low dose of Lampung Robusta green coffee extract would increase the antibodies, while a high dose would reduce the MDA level. This suggests that coffee is a potential anti-oxidant, anti-inflammatory and immunomodulatory agent for laying hens.

CONCLUSION:

Lampung Robusta green coffee extract can be used as an immunosuppressor for the humoral, antioxidant and anti-inflammatory responses in laying hens.

ACKNOWLEDGEMENTS:

Thank you to Universitas Brawijaya, which funded this research through its Excellence Higher Education Institution Research grant in 2018.

AUTHOR CONTRIBUTIONS:

DR received research funding, Research concept and design; Collection and/or assembly of data. LER Writing the article, Critical revision of the article. DQS data analysis Data analysis and interpretation, Writing the article, Final approval of article. IAA and DQS conducted research. All team members wrote and revised this article.

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Received on 08.09.2019 Modified on 18.11.2019

Research J. Pharm. and Tech. 2021; 14(5):2779-2784.

DOI: 10.52711/0974-360X.2021.00490