A study of chemical compounds and antimicrobial activities of Vernonia amygdanlina extract from different areas and the age of leaves as animals’ feed additives

Herbal plants have been used as an alternative to replace the use of antibiotics in animal production. African leaves (Vernonia amygdalina) are herbal plants that have medicinal properties. This study was carried out with the purpose of evaluating the biochemical and antimicrobial compounds of African leaf extract as a phytogenic supplement in animal feed. African leaves (V. amygdalina) which grow massively in Aceh were extracted using 96% ethanol solvent in 1:3 ratio. There were 4 African leaf samples differentiated according to young and old leaves that grow in the highlands and lowlands used in this study. Biochemical compound testing was carried out using a qualitative phytochemical screening method and antimicrobial testing (Salmonella and Escherichia coli) using the Kirby Bauer Disc Diffusion method using Salmonella enterica sv Typhimurium ATCC 14028 and E. coli ATCC 25922 bacteria with a Mc Farland standard of 0.5. Phytochemical test results show that African leaves contain alkaloids, steroids, flavonoids and phenolics. Meanwhile, the results of antimicrobial testing did not show inhibition against Salmonella and Escherichia coli bacteria. This research concluded that the extracted leaves of African plants had the potential to be administrated as a phytogenic additional feed.


Introduction
Antibiotics utilization in feed to stimulate livestock growth has been used for a very long period of time.These initiatives are carried out because they are thought to be very beneficial (boosting livestock growth, livestock health, and feed efficiency).Apart from being a growth trigger, antibiotics are also used to treat Escherichia coli and Salmonella spp contaminations in poultry production that can cause severe problems, because the effects of colibacillosis and salmonellosis may cause substantial mortality in poultry as little as three weeks old.[1].Environmental conditions including high temperatures and humidity negatively affect livestock health and performance by causing oxidative stress.Providing antibiotics as animal feed without any control has a negative effect because it leaves residue to animal products such as milk, meat and eggs.Antibiotic residues in livestock products can cause resistance to pathogenic bacteria in both animals and humans as consumers [2][3].Therefore, since 1st January 2018 the government of Indonesia has banned the addition of antibiotics to feed and switched to the use of phytogenics as alternative to antibiotics.Phytogenics, commonly referred to phytobiotics or botanicals, are additive substances which are plant-extracted containing valuable nutrition, non-nutritive or anti-nutritional substances added to dietary meal or water to promote livestock productivity by enhancing feed characteristics, improving digestive health by decreasing pathogenic bacteria, boosting productivity, and improving livestock product quality [4][5].The bioactive compounds consisted in plants that can be utilized as phytogenic feed additives are flavonoids, phenols, saponins, tannins, alkaloids, essential oils, etc [6][7][8].These compounds have the effect of anti-fungal, anti-bacterial, antioxidant and functional features that can alter animal digestion to improve performance and health [9].Research using herbal plants as phytogenic feed ingredients instead of antibiotics has been widely carried out.[10] examined the effect of lempuyang (Zingiber zerumbet) extract as feed additive on the qualities of carcass, performance and Escherichia coli bacteria inhibition in broiler chickens.Other research also examines the use of phytogenic herbal ingredients such as Syzygium cumini extract as an additive on the performance and hematological parameters of broiler chickens, performance, carcass and offal, as well as characteristics of broiler chickens, the potential of few local phytogenic feed additives as a source of antioxidants and antimicrobials for non-ruminant animals [11][12][13][14].
One of the herbal plants that grows abundantly in Indonesia and starting to be widely used as traditional medicine is African leaves.Therefore, it is crucial to conduct a study regarding the potential of African leaves as an alternative antibiotic derived from phytogenic herb.

Sample collection and ethanol extraction preparation
African leaves are collected from the highlands and lowlands and are differentiated based on old and young leaves.Then dried using an oven at 60 ºC for 2 days until it reaches 15% dry weight.Samples were ground with a hammer mill, then soaked in 96% ethanol in a ratio of 1:3 for 2 days.The extraction process is carried out using an evaporator to produce a paste for testing the chemical content and antibacterial compounds.

Testing chemical and antibacterial compounds
Chemical content evaluation of African leaf conducted using qualitative phytochemical screening methods and antimicrobial test (Salmonella and Escherichia coli) using the Kirby Bauer disk diffusion method [15] using Salmonella enterica sv Typhimurium ATCC 14028 and Escherichia coli ATCC 25922 with a Mc Farland standard of 0.5 with 100% dose of African leaf extract.

Data analysis
Data collected from chemistry compound test analyzed using descriptive manner, and antibacterial test analyzed using ANOVA [16].Data collected based on average results of 2 replication.

Phytochemical content
The phytochemical content of plants is conducted to detect compounds that can be used as feed additives, one of which is flavonoids.Table 1 reveal the results of phytochemical testing of African leaves using the phytochemical screening method.

Description: +) yes, -) none
Tests on the ethanol extract of African leaves confirmed the presence of alkaloids, steroids, as well as flavonoids and phenolics which are classified as antioxidant compounds, but terpenoids, saponins and tannins were not detected.Meanwhile, deep the review to a number of results researches by [17] concluded that, African leaves contains variety of bioactive substances such as flavonoids, saponins, alkaloids, tannins, phenolics, terpenes, steroids, glycosides, triterpenoids, and some types sesquiterpene lactone.Bioactive compounds have distinctive pharmacological effect such as antimicrobial, antimalarial, antithrombotic, antioxidant, antidiabetic, laxative, hypoglycemic, antihelminthic, anti-inflammatory, cathartic, anticancer, antifertility, antifungal, and antibacterial.The existence of different phytochemical content between the same type of plant, but living in different areas is the result of environmental factors such as weather conditions, season, soil type, altitude, and plant genetic or epigenetic variability [18].[11] tested the antibacterial and antioxidant activity of various herbal plants as feed additives revealed that the antioxidant activity of African leaves was very strong with an IC50 value at a concentration of 23,922 ppm.[19] reported the results of antioxidant activity tests on African leaves using the DPPH method with methanol solvent, which obtained an average IC50 of 37.69 mg/ml and the total flavonoid content extracted with various concentrations and different solvents, the highest results were obtained in methanol solvent 14.02 -44.35 mg QE/g), followed by ethyl acetate (11.79 -42.52 mg QE/g) and Petroleum ether (2.44 -15.16 mg GAE/g) at concentrations around 20 -100 mg/ml.[20] concluded that African leaves have the potential to be used as actioxidants because they contain flavonoid compounds with an IC 50 value of 87,992 ppm.

Antimicrobial activity
Promising outcome of the antimicrobial test are determined by the development of a clear zone near the disc with the identified extract known as inhibitory ability.Antibacterial activity test result for phytogenic feed additives using the Kirby-Bauer disk diffusion method [15] shown in   2, the ethanol extract of African leaves did not show inhibitory power against the bacteria Eschericia coli ATCC 25922 and Salmonella enterica sv Typhimurium ATCC 14028, which means that there was no antibacterial response to the two test bacteria.The results of testing the inhibitory activity of African leaves are presented in Figure 2.This result the same was reported by [19] with various extraction methods (methanol, ethyl acetate, petroleum ether) and different doses of African leaves (20 mg/l, 40 mg/l, 60 mg/l, 80 mg/l, and 10 mg/l) did not shows inhibition or resistance.African leaf ethanol extract with concentrations of 100%, 75%, 50% and 25% was also reported not to form an inhibitory zone against Escherichia coli ATCC 25922 [22], thought to be caused by several factors, including the weak diffusing power of African leaf extract, extraction solvent, inappropriate extraction time and method, and low concentration of active ingredients so that African leaf extract cannot inhibit bacterial growth properly.Different results were obtained in the research of [11], who tested the antibacterial activity of various herbal plants, found that the African leaf shoots (young leaves) and old leaves at various concentrations were able to form inhibitory power against Escherichia coli and Salmonella spp bacteria, with the highest inhibitory power of 8.60 mm and 7.31 mm respectively at a concentration of 750 ppm on Eschericia coli bacteria and 10.12 mm and 7.05 mm at a concentration of 750 ppm on Salmonella spp.[23] also founded that the ethanol extract of African leaves formed an inhibitory zone against Escherichia coli and Salmonella typhi of 23 and 13 mm respectively, with a minimum inhibitory concentration (MIC) of 500 mg/ml.Dissimilar antimicrobial content of plant extracts might caused by how old the plant is, the freshness of the plant material, external factors (temperature, water light), field microbial contamination, inappropriate preparation and dosage, and the astringent properties of the plant extract.[24] in his research on African leaves as an alternative antibiotic for nosocomial infections caused by Pseudomonas aeruginosa bacteria concluded that African leaves are one of the herbal plants that can be used as an alternative to antibiotics due to its antibiotic effect.[25], who reviewed the literature on the potential of African leaf extract as an antibacterial against grampositive and negative bacteria, concluded that African leaf extract was more efficient on gramnegative bacteria, namely Pseudomonas aeruginosa.

Conclusions
Based on the results of phytochemical and antimicrobial tests on the ethanol extract of African leaves, it was resolved that the phytogenic extract in this study could be used as a feed additive as an antibiotics alternative.

Table 1 .
Phytochemical content of African leaves using the phytochemical screening method.

Table 2 .
Results of antibacterial activity test of African leaves.