| Eupatorium odoratum Linn., a perennial herb of Eupatorium in Compositae, grows invasively and intensively in South China. It has been used by Chinese folk as an antipyretic and a detoxicating herbal medicine against bruises, swelling, inflammation and microbial infections. Recent pharmacological studies have found its active components to be mainly flavonoids. However, research on antimicrobial feed additive of E. odoratum has not been reported yet. In the present study, extracts from the E.odoratum were collected and the conditions to extract total flavonoids optimized. The acute toxicity of the total flavonoids from E.odoratum in mice, and the effects on growth performance, immunity and the intestinal microflora in broiler were systemically tested,to determine whether flavonoids from E. odoratum could be used as a new herb-sourced antimicrobial feed additive.1. The extraction yield of the total flavonoids from E. odoratum was taken as the key index in the ultrasonic assisted solvent extraction, and a range of factors like the extraction time, extraction temperature, ratio of liquid to solid, ethanol concentration were optimized through single factor test and orthogonal design. The results showed that the optimized extraction process for the total flavonoids from E. odoratum was with an extraction time of 5 h, extraction temperature of 70℃, liquid to solid ratio of 1:60(g/mL)and the solvent of 70% ethanol(v:v). The percent of flavonoids in the extract was 7.13%.2. The oral acute toxicity assessment of the total flavonoids from E. odoratum in KM mice could not be determined using the classical LD50 test but the maximum tolerated dose was 80 g/kg, which indicated the total flavonoids from E. odoratum were actually non-toxic.3. The effects of the total flavonoids from E. odoratum on the growth performance,immune organ indexes and serum immunoglobulins in AA broilers were studied for 42 days. There was a significant improvement in daily weight gain, the ratio of feed to gain and the ratio of breast muscle(P<0.05) in broilers given feed with added E. odoratum flavonoids in different concentrations and adding 400mg/kg of E. odoratum flavonoids significantly increased the broilers’ thymus index, bursal index, IgG, and SIgA(P<0.05),which meant the E. odoratum flavonoids extract promote broilers growth(meat content)and also improved the immunity.4. In order to probe into the influence of E. odoratum flavonoids on the intestinal microflora in AA broilers at different times, bacterial 16 S r DNA fragments were obtained by Miseq high-throughput sequencing platform and the applications of classification unit cluster analysis, diversity and taxonomic analysis, bioinformatics analysis on the intestinal microflora, diversity and abundance were conducted. The results showed that,at the level of phylum, 95% of the microbes in the intestine could be categorized into Firmicutes and acteroidetes phyla. During the trial period of day 1 to day 21,in A1(controls), A2(low-dose of flavonoids), A3(medium dose of flavonoids), A4(high dose of flavonoids) the percent of the genus Bacaeroides in the microbial community were11.98%, 14.44%, 16.13%, 13.14%, respectively. That is, in groups A2, A3, A4 genus Bacaeroides microbial communities in were higher than that in control group by 2.46%,4.15%, 1.16%. In the trial period of day 22 to day 42, B1(Control), B2(low-dose of flavonoids), B3(medium dose of flavonoids), B4(high dose of flavonoids) the percent of the genus Bacaeroides in the microbial community were 17.63%, 16.64%, 16.86%,32.44%, respectively, and in the B4 group it was significantly higher by 14.81%,compared to the B1 value. Thus, E. odoratum flavonoids could enhance the abundance of intestinal beneficial microbes and thereby improve the gut microflora structure in broiler.In summary, the optimal extraction conditions of the total flavonoids from E.odoratum were an extraction time 5 h, extraction temperature of 70℃, solid-liquid ratio of 1: 60(g/mL) with 70% of ethanol as the solvent. The extraction rate of total flavonoids was 7.13%. The total flavonoids from E. odoratum were relatively non-toxic and therefore very safe for use via oral administration. The flavonoids from E. odoratum promoted growth and meat performance, and alse improved the immunity in broiler as shown by an increase in the thymus index, bursal index, and the level of IgG and SIgA.In addition, the total flavonoids from E. odoratum increased the microbes of the genus Bacteroides, which are regarded as the beneficial intestinal microflora and thereby improved the intestinal microbial composition. |
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