Bioactive Substances of Some Herbals and Their Effectiveness as Antioxidant, Antibacteria and Antifungi

Sinurat AP, Wina E, Rakhmani SIW, Wardhani T, Haryati T, Purwadaria T. 2018. Bioactive substances of some herbals and their effectiveness as antioxidant, antibacteria and antifungi. JITV 23(1): 18-27. DOI: http://dx.doi.org/10.14334/jitv.v23i1.1660 A study was conducted to explore the bioactive substances of some local plants in order to find their effectiveness as antioxidant, antibacteria and antifungi to be used as feed additives. Twelve plants material were used in this study. The total phenol, tannin and saponin contents in the plant extract were assayed. The extracts were also assayed on their antioxidant activities and on their ability to depress in vitro gas production of microbes obtained from chicken’s guts, their ability to inhibit growth of bacteria (E. coli and Salmonella enteridis) and fungi (A. niger). The results showed that the highest total phenol and total tannin contents were found in clove leaf extract, while the highest saponin content was found in Sapindus rarak fruit pericarp. The highest antioxidant activity was found in the leaffruit extract. Gas produced by microorganims was reduced to the level similar to antibiotic addition were found with addition of hexaneextract of leaffruit, kapok seed or methanolextract of mangosteen fruit rind pulp or clove leaves. The best inhibitory effect on E. coli growth (measured by clearing zone) was found in methanol extract of S. rarak fruit. However, the most effective growth inhibitor for both E. coli and Salmonella was the liquid smoke of cashew nut shell. The best growth inhibitor for fungal growth was found in extract of clove leaves. Therefore, clove leaves extract (anti fungi), liquid smoke of cashew nut shell (antibacteria) and leaffruit (antioxidant) may have potential to produce feed additives to substitute antibiotic growth promoters.


INTRODUCTION
Feed additives are commonly used for animal production in aim to improve the productive performance, feed utilization efficiency and quality of animal products.One of the feed additives that widely used is antibiotic, known as antibiotic growth promoters (AGP).The AGP has been used since 1940's (Dibner & Richards 2005) and legally recognized as feed additives in all over the world until 1970's when the Swann Reports 1969 showed the negative impact of the AGP usage such as the occurrence of microorganism resistant to antibiotics.
Since the Swann report, many countries banned or at least restricted the use of AGP.Indonesia also bans the use of AGP in animal feed as stated in the legislation Act 18/2009 juncto Act 41/2014.Endorsement of the regulation without precaution may deteriorate the poultry productivity and feed efficiency drastically.Therefore, it is important to find nonantibiotic feed additives to substitute for the AGP.On the other hand, many reports and opinion conclude that the use of AGP promote the occurrence of antibiotic resistant microorganisms which is harmful for the human.
Some efforts have been done in search of materials to replace the AGP which are "friendly" for environments and human health.Among them are enzymes, acidifiers, probiotic, prebiotic, plant bioactives and the combination of those products.As a tropical country, Indonesia has abundant of plants as source of plant bioactives.Some plants or herbs have been used traditionally as "healthy drinks" and for medications for human in Indonesia and elsewhere.
Bioactive compounds in plants are defined as compounds produced by plants having pharmacological or toxicological effects in man and animals (Bernhoft 2010).The typical bioactive compounds in plants are produced as secondary metabolites.Plant bioactives have been reported to have functions as antibiotic, anti fungi, antioxidant and immuno modulator.Some researches have been conducted in order to proof the beneficial effect of plant bioactives to replace AGP such as Aloe vera (Purwadaria et al. 2001;Bintang et al. 2001;Sinurat et al. 2003;Sinurat et al. 2004;Sinurat 2013), Curcuma longa and Curcuma xanthorrhiza (Samarasinghe et al. 2003;Sinurat et al. 2009), and guava leaves (Hoque et al. 2007;Kidaha et al. 2013).Most of the researches studied the effectivity of the bioactives obtained from single ingredient and the results have not been applied in the livestock industry commercially due to some factors such: variability on the effectivity, high production cost or too expensive when compared to the commercial AGP and when the use of AGP is still allowed in the country.
Synthetic antioxidants such as ethoxyquin, BHA (butylated hydroxyanisole) and BHT (butylated hydroxytoluene) are also used as feed additives in order to improve the healthyness (immunity of the animals).The plant antioxidant can induce the phase 2 enzymes or cytoprotective proteins in the cell and act to neutralize toxic agents when they appear (Blomhoff 2010).Farahat et al. (2016) reported that natural antioxidant obtained from decaffeinated green tea extract are effectively to enhance the immune response and vaccination potency in broilers without adversely affected the growth performance of broilers.The addition of antioxidant in feed may help the animal to 'fight' pathogen microorganisms.
In order to find an alternative to AGP, this research was focused on the searching of plants that contain bioactives effective for inhibiting bacteria growth, fungus growth and have high antioxidant activity.The combination of three bioactives obtained from plants are expected to form a feed additive that can replace or even better than antibiotic growth promoters.2011).9. Mindi (Melia azedarach L.) leaves contain terpenoid and azadirachtin (Meziane and Goumri, 2015) which are effective as antibacteria, antivirus, insectiside and as antioxidant (Khan et al. 2011).

Source of plant bioactives
10. Mangosteen (Garcinia mangostana) fruit pericarp is rich in xanthones which are known as antioxidant, anti inflamation, antibacteria and antifungi (Palakawong et al. 2010).The ethanol extract have been reported effective to inhibit growth of pathogen bacteria that usually found in gastro intestinal tract such as Salmonella typhi, Shigella dysentriae, E. coli, Klebsiella pneumoniae, V. cholerae, Pseudo-monas aeruginosa and Staphylococcus aureus (Geetha et al. 2011).11.Liquid smoke obtained from Cashew nut (Anacardium occidetale) shell contains phenols which are effective as antifungal and anti-microbial (Parasa et al. 2011).The liquid smoke has a low pH which may be effective to be used as an antimicrobial in feed (Saenab et al. 2016).12. Gripeweed or leaffruit (Phyllanthus urinaria) plants commonly called chamber bitter or leafflower, is a herb species in the family Phyllanthaceae.The bioactives were reported as high antioxidant activities and effective as antibacteria (Kumaran & Karanukaran 2007;Eldeen et al. 2010).

Preparation of plant crude extract
Samples of plant materials were dried and ground prior to extract.The samples were extracted by methanol for polar fraction or by hexane for non-polar fraction.Each 5 g of the sample was soaked in 10 ml methanol solution.The soaking was performed with sonicator for 30 minutes.The solutions were then centrifuged for 15 minutes (2500 rpm) and the supernatant was collected as a polar fraction.
Similar procedures were performed to obtain the extraction of the non-polar fraction by using hexane instead of methanol.

Total phenol and tannin
Phenol levels in the samples were measured by Folin-Ciocalteu method.Analyses were performed twice, i.e. before and after addition of polyvinyl polypyrrolidone (PVPP).The methods have been described by Makkar ( 2013)

Analysis of antioxidant potential
The antioxidant content of plant extraxts were determined following method as described by Shekar & Anju (2014) The procedure in brief was as follows: 0.5 g of dried samples were diluted with 10 mL methanol.The solution (1 mL) were made in some different concentrations and each was reacted with 2 mL 2,2 diphenyl-1-1-picrylhydrazil (DPPH) 0.2 mM.Then, they were incubated at room temperatures for 30 minutes.The assay was performed in dark room and the absorbance of the solution was measured at 517 nm.Methanol was used as the blank (1 mL methanol added with 2 mL DPPH 0.2 mM).Concentration to inhibit 50% oxidation, ng/mL was calculated in each curve of extract concentration towards oxidative inhibitions.

Effectiveness to inhibit microorganism growth
Two methods were performed in order to identify the effectiveness of the plant bioactives to inhibit the growth of microorganisms.The first method was the in vitro fermentation method or by measuring the total gas production.In this method, 0.1 g pepsin digested corn meal as substrate in the 15 ml glass test tube, was added with 0.5 ml plant extract and 10 mL artificial saliva buffer (McDougall buffer) which has been mixed with the chicken digesta as inoculum with ratio 4:1 to mimic gut's condition during fermentation.
The CO 2 gas was ran for 15-20 minutes into the digesta-buffer prior to addition of the samples.After mixed homogenously and flushed with the CO 2 gas, the tubes were tightly sealed and incubated at 37-40ºC for 24 hours.Samples without plant extract were performed as negative control.The gas produced was measured every hour till 9 hours of fermentation and at the end of fermentation (24h).Total gas production was calculated as the sum of gas produced during 24 h of incubation.The amount difference between total gas production in the treated tubes and the control (without plant extract) were considered as the indicator on the effectiveness of the extract to inhibit microorganism growth.The higher the difference (or the less total gas production), the more effective the plant extract to inhibit the microorganism growth.Each treatment was replicated 4 (four) times.
The second method was by observing the growth of E. coli and Salmonella enteritidis in petri dish as described by Rollins & Joseph (2000).Assays were performed by placing paper on the agar media inoculated with E. coli or S. enteritidis sp.The diameter of clear zone was measured as an indicator of effectiveness of the plant extract to inhibit the bacteria growth.

Substrate preparation and source of microbes for inoculum
Substrate was prepared as follows: 10 g corn meal was mixed with 300 ml pepsin solution (0.2% in HCl 0.1 N).The mixture was incubated on water bath shaker at 40°C for 45 minutes followed by centrifugation at 3000 rpm for 10 minutes.Then, the precipitate was washed with distilled water, and again centrifuged at 3000 rpm for 5 minutes, dried and regarded as substrate for in vitro fermentation.
Source of microbes used for in vitro fermentation in this experiment was total microbes that present in the digesta of the chickens and were attacked by antibiotic growth promotors.The digesta were collected by squeezing the contents of the guts of chickens obtained from poultry slaughter house.The digesta were pooled and kept in the freezer and used as inoculum for in vitro fermentation.

RESULTS AND DISCUSSION Identification of bioactive substances of some native plants
Concentration of some bioactive compounds in some plants extract is shown in Table 1.Extract of clove leaves contain the highest total phenol (6.04 %) followed by the mangosteen fruit pericarp extract (4.76 %) as compared to other plant extracts.The kapok seed oil contained the lowest (not detected) total phenol.Similar to the total phenol content, the highest tannin levels was also found in clove leaf extract (3.34%) followed by mangosteen fruit pericarp extract (2.72%) and the lowest (not detected) in kapok seed oil.The tannin level in both clove leaf and mangosteen extract were found more than 50% of the total phenol.The saponin level, however, showed different trend.The highest saponin level was detected in S. rarak fruit pericarp extract (59.71%) followed by liquid smoke of cashew nut shell (50.46%) and the lowest level was found in kapok seed oil (1.19 %).Total phenol, tannin and saponin are compounds that dissolved in polar solvents, therefore their content in kapok seed oil which dissolved in non polar solvent was very low compared to other plant extracts in this study.
Plant phenolic compounds and tannins are well known as potent antioxidant (Ghasemzadeh & Ghasemzadeh 2011) and can serve as protectants against bacterial pathogen (Sytar et al. 2012).Plant saponin was also reported to inhibit sporulation of E. tenella oocysts (Pasaribu et al. 2011).Therefore, it is expected that the highest the content of the total phenol, tannin and saponin in the plants the higher the potential benefit for the consumers (animals).
Analyses on antioxidant activities of the samples were conducted in comparison with vitamin C (as standard) and the concentrations of plant extracts that effectively inhibit 50% oxidation are shown in Table 2.The leaffruit (Phyllanthus urinaria) extract showed the least concentration (124 µg/mL) followed by clove leaves extract (147.9 µg/mL) and cashew nut liquid smoke (156.5 µg/mL).The antioxidant activity of  Binahong (A. cordifolia) leave extract, Mindi (M.azedarach) and Kapok (C.pentandra) seed oil could not be obtained since the green colour of binahong and mindi extract disturbed the color measurement during the analysis, while oil fraction of Kapok seed oil could not mix with the DPPH solution in the analysis.The least the concentration required to inhibit oxidation means the more effective the substances as antioxidant.Therefore, the leaffruit (P.urinaria) extract and clove extract showed the best potential antioxidant activity.Plant phenols are antioxidants by virtue of the hydrogen-donating properties of the phenolic hydroxyl groups (Blomhoff 2010).However, the results showed that the total phenol was not directly related to the antioxidant activity.As shown in Table 1, the highest total phenol was found in clove leaves extract (6.04 %), while the highest antioxidant activity was found in leaffruit extract (Table 2).Although, in general, samples with high antioxidant activities (leaffruit, clove leaves, and mangosteen fruit rind pulp) always contain high total phenols.The phenols have many sturctural component such as phenolic acids, flavonoids, stilbenes and lignans.The effectivity of each compound as antioxidant may be not the same and the level of each compound may different in different plants.
The effectiveness of the plant extracts to inhibit growth of microorganisms (in-vitro) was determined by volume of gas produced by microorganisms obtained from gastro intestinal tract of chickens during 24 h.As shown in Table 3 the addition of antibiotic (50 ppm zinc bacitracin) or the plant extracts significantly (P<0.05)affected the amount of gas produced during the incubation.The gas production was significantly reduced by addition of the antibiotic (50 ppm zinc bacitracin) to 65% as compared to the control without antibiotic.Reduction of gas production to the level similar to antibiotic were found with addition of hexane extract of leaffruit (72% of control), kapok seed oil (69% of control), and methanol extract of mangosteen fruit pericarp (61.0% of control), clove leaves (63% of control), S. polyanthum leaves (72% of control), guava leaves (74% of control) and M. azadirach leaves (77% of control).If the reduction of total gas production can be used as indicator of the effectiveness to inhibit pathogen microorganisms, it could be concluded that these plant extracts are the best candidates to replace antibiotic.However, gas production in this method is the result of all microorganisms (obtained from chicken intestinal) including pathogens and non-pathogens.On the other hand, the purpose of this research was to explore the effectiveness of plant bioactives to inhibit growth of pathogen microorganisms which may be used as alternatives to AGP.Perhaps, gas production may only be used as a preliminary tool to indicate that the plant extract contains substances as anti-microorganism.The conventional method (diffusion test) by measuring the size of clear zone arround the samples place on an agar plate may still the best method to measure the effectiveness of a substance as an antibiotic.
Results of diffusion method to test the effectiveness of the plant extracts to inhibit growth of E. coli and S. enteritidis is presented in Table 4. Negative control did not have a clear zone diameter (0 mm), but addition of antibiotic (pyripen 5.5 mg/10ml) produced Both bacteria are Gram negative and always found in digestive tracts, however, every species including their strains have been recognised differently sensitivity or resistency to antibacteria especially for those not included in broadspectrum.Some of E. coli strains and most S. enteritidis are pathogens.The bacteria used in this experiment was isolated from chicken digestive tract and pathogen, therefore the antibacteria activity detected in the plant extracts may prove the idea to replace the AGP with the plant extracts.Inclusion of zinc bacitracin in the diet alter the microbiota composition in the ileum of broilers (Gong et al. 2008).Similar result was also reported by Engberg et al. (2000) which showed that addition of salinomycin or zinc bacitracin reduced the population of Clostridium perfringens and Lactobacillus salivarius in the intstinal of broilers.
Addition of anti-fungi (ketoconazole) showed effectively inhibit the growth of A. niger with the clear zone diameter 1.13 -2.63 cm.Almost all plant extracts tested were not effective to inhibit growth of fungi (A.niger BPT) as shown in Table 5.However, extract of Clove (S. aromaticum) leaves and liquid smoke of Cashew nut (A.occidetale) shell were effective to inhibit the growth of the fungi.The clove leave meal extract showed the highest anti fungi activities with diameter of clear zone 1.24 cm and the liquid smoke of cashew nut shell with diameter of clear zone 1.18 cm.The bioactive compounds in cloves leaf extract or oil are well known as eugenol (Yitbarek 2015).The eugenol has been used for medication purposes in human.Joseph & Sujatha (2011) reported that the clove crude extract and the clove oil were effective to inhibit of some fungi such as Paeciliomyces, A. flavus, A. niger, Penicillium sp., Rhizopus sp. and Rhizomucor sp.Liquid smoke of cashew nut shell has low pH and contain many simple phenols (Saenab et al. 2016) that have the ability to depress the growth of bacteria and fungi.

CONCLUSION
Bioactive components such as total phenol, tannin and saponin of plant extract could not be used as an indicator of their effectiveness as antioxidant, antibacterial or antifungi, neither the ability to reduce the in vitro total gas production of microbes obtained from chicken's gut.The conventional method carried out to measure antioxidant activity, growth inhibition (of E. coli and S. enteritidis), and growth inhibition of fungi (A.niger) concluded that leaffruit extract, the liquid smoke of cashew nut shell and the clove leaves extract performed the highest potential as antioxidant, antibiotic and anti-fungi, respectively.Further study is required to prove if they could be used to substitute antibiotic growth promoters in feed.

Table 1 .
Concentrations of total phenol, tannin and saponin in some plant extracts * Percentage in 100 ml liquid smoke

Table 2 .
Antioxidant activity of some plant extracts *The antioxidant activities of some plants were also correlated with antibiofilm activity(Gracia et al. 2017); ND = not detected or no antioxidant activity; NA = not analysed

Table 3 .
In-vitro gas production of digested corn by microbes (obtained from chickens gastrointestinal tract) for 24 h as affected by plant extract addition Different letters in superscripts showed significant different (p<0.05);ND = Not determined; Control was not added with plant extracts. *

Table 4 .
Diameter (mm) of clear area on the culture of E. coli and S. enteritidis as affected by supplementation of plants extract * antibiotic (pyripen 5.5 g/10ml) was added

Table 5 .
Diameter (cm) of clear area on the culture of fungi ( A. niger BPT) as affected by supplementation of plants extract