Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 568528, 8 pages
http://dx.doi.org/10.1155/2013/568528
Research Article
Application of Scutellariae radix, Gardeniae
fructus, and
Probiotics to Prevent Salmonella enterica
Serovar Choleraesuis
Infection in Swine
Chiung-Hung Chang,
1,2Yueh-Sheng Chen,
1Ming-Tang Chiou,
3Chiu-Hsian Su,
4Daniel S.
Chen,
5Chin-En Tsai,
3Bi Yu,
6and Yuan-Man Hsu
41 School of Chinese Medicine, China Medical University, Taichung 404, Taiwan 2Department of Traditional Chinese Medicine, Taichung Veterans General Hospital,
Taichung 407, Taiwan
3Department of Veterinary Medicine, National Pingtung University of Science and
Technology, Neipu, Pingtung 912, Taiwan
4Department of Biological Science and Technology, College of Life Sciences, China Medical
University, 91, Hsueh-Shih Road, Taichung 404, Taiwan
5Department of Biochemistry, College of Agriculture and Life Sciences, University of
Wisconsin, Madison, WI 53715, USA
6Department of Animal Science, College of Agriculture andNatural Resources,National
ChungHsingUniversity, Taichung 402, Taiwan
Correspondence should be addressed to Yuan-Man Hsu; yuanmh@mail.cmu.edu.tw Received 29 June 2012; Revised 14 January 2013; Accepted 21 January 2013 Academic Editor: Youn Chul Kim
Copyright c 2013 Chiung-Hung Chang et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Salmonella enterica serovar Choleraesuis, a host-adapted pathogen of swine, usually
causes septicemia. Lactic acid bacteria (LAB)
strains have been widely studied in recent years for their probiotic properties. In this study, a mouse infection model first screened
for potential agents against infection, then a pig infection model evaluated effects of LAB strains and herbal plants against infection.
Scutellariae radix (SR) and Gardeniae fructus (GF) showed abilities to reduce bacteria
shedding and suppressing serumlevel of
TNF-� induced by infection in swine. Bioactivities of SR and GF were enhanced by combining with LAB strains, which alone could speed
up the bacteria elimination time in feces and boost immunity of infected pigs. Baicalein and genipin exhibited stronger cytotoxicity
than baicalin and geniposide did, as well as prevent Salmonella from invading macrophages. Our study suggests LAB strains as
exhibiting multiple functions: preventing infection, enhancing immunity to prepare host defenses against further infection, and
adjusting intestinal microbes’ enzymatic activity in order to convert herbal compounds to active compounds. The SR/GF-LAB
strain mixture holds potential infection-prevention agents supplied as feed additives.
1. Introduction
Salmonella infection of humans and animals is still a severe
health problem worldwide [1], S. enterica a key gastrointestinal pathogen responsible for food-borne diseases. In particular,
S. enterica serovar Choleraesuis, a host-adapted pathogen
of swine, sometimes causes necrotizing enterocolitis as well as septicemia characterized by hepatitis, pneumonia, and cerebral vasculitis [2]. S. Choleraesuis also produces systemic infection in humans. It is a major serovar for salmonellosis transmitted from animals to humans [3]. Yet antibiotic treatments have served to eliminate Salmonella infection. As with other bacterial pathogens, antibiotic resistance to
Salmonella is a growing problem for eradication. Finding safe
and efficient prevention to alleviate the need or even replace antibiotics for cleaning infection looms is a vital task.
Lactic acid bacteria (LAB) strains have been widely
studied in recent years for their probiotic properties and are used as feed supplements [4, 5]. A number of studies show
LAB strains bolstering immunity and warding off infection by gastrointestinal pathogens [4–7]. They can also act as immunomodulators or adjust composition and activity of intestinal microbes [8]. Intestinal microbial community can also play a role as probiotics [9]. Prior studies indicated that intestinalmicroflora could transformherbal components 2 Evidence-Based Complementary and Alternative Medicine Table 1: Concentrations of indicator compounds in herbal extracts used in this study.
Herbal extract Indicator compound mg/g
Scutellariae radix Baicalin 89.74 Gardeniae fructus Geniposide 40.15 Houttuyniae herba Quercetin 0.07 Taraxaci herba Chlorogenic acid 0.18 Glycyrrhizae radix Glycyrrhizic acid 27.91 Puerariae radix Puerarin 29.40
Rhizoma dioscoreae∗ None — ∗no indicator compound available.
into bioactive compounds, for example, convert ginsenoside Rb1 to compound K, which is more active and subsequently absorbed [10–13]. We first screened seven herbs via mice infectionmodel for potential agents, that is, Scutellariae radix (SR), Gardeniae fructus (GF), Houttuyniae herba, Taraxaci
herba, Glycyrrhizae radix, Puerariae radix, and Rhizoma dioscoreae. LAB strains and potential herbal materials as pig
feed supplements allowed the study of their preventive effects against Salmonella infection, thus offering an alternative way to improve immunity and digestive abilities of animals by using combinations of LAB strains and herbs as feed additives.
2. Materials and Methods
2.1. Preparation of Herbal Extracts. All herbal materials
were purchased from Ko Da Pharmaceutical Co., Ltd. at Taoyuan, Taiwan: Scutellariae radix (SR), Gardeniae fructus (GF), Taraxaci herba, Houttuyniae herba, Glycyrrhizae radix,
Puerariae radix, andRhizoma dioscoreae. Plantmaterialswere
finely powdered and extracted using distilled water at 100∘C
removed by filtration, filtrate was concentrated in vacuum and lyophilized to yield residue. Percentages of indicator compounds in each original herbal plant were confirmed by high-performance liquid chromatogram (HPLC) by Ko Da Pharmaceutical Co., Ltd. and listed in Table 1.Water extracts were used for mice infection model; plant materials of SR and GF pulverized to fine powder and passed through an 80-mesh sieve. Finely powered plant materials were used for pig infection model. For in vitro tests, baicalin and baicalein were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA); geniposide and genipin were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and Challenge Bioproducts Co., Ltd. (Taichung, Taiwan), respectively.
2.2. Bacterial Strains and Culture Conditions. S. Choleraesuis
(ATCC 13312) reference strain was obtained from the American Type Culture Collection (ATCC). Strain SC37 was collected from a carrier pig to be used as inocula for infecting animals; is serotype identification applied as follows.
Antiserum to detect O and H antigens was purchased from S&A Reagents Lab Ltd. (Bangkok,Thailand) and from Denka Seiken Co., Ltd. (Tokyo, Japan), respectively. Isolate was analyzed according to Kaufmann-White scheme and serotyping
protocols developed by Centers for Disease Control and Prevention (Atlanta, GA, USA) [14]. Bacterial inocula were grown in Luria Bertani (LB) broth to log stationary phase at 250 rpm and at 37∘C for 8hr to an OD600nm of 0.8. After
centrifugation at 8,000 ×g, bacterial pellet was resuspended in phosphate-buffered saline (PBS) and adjusted to final concentration of 1010 colony forming units (CFUs)/mL in
PBS. Two lactic acid bacteria (LAB) strains, Lactobacillus
acidophilus strain LAP5 [4] and L. reuteri Pg4 [15], were
isolated from the gastrointestinal tract of swine and broilers, respectively, as described previously. LAB strains were grown in deMan-Rogosa-Sharpe (MRS) broth (Merck, Darmstadt, Germany) for 24 hr at 37∘C. Whole cells were obtained by
centrifugation at 6000 ×g for 10min and washed twice with sterilized PBS (pH 7.2).Mix-LAB strains LAP5 and Pg4 were combined in a ratio of 1 : 1 used for swine experiments. LAB
culture preparation was then lyophilized and stored at 4∘C
until later required. The bacterial count of the LAB strain powder was approximately 109 CFU/g.
2.3. Antimicrobial Activity. Minimum inhibitory concentration
(MIC) of extract or compound was tested by twofold
serial dilution. Test extracts were first dissolved in PBS, then incorporated into Muller-Hinton broth to obtain concentration of 100mg/mL and serially diluted to attain 50, 25,
12.5, 6.25, or 3.125mg/mL. Test compounds were dissolved in dimethyl sulfoxide (DMSO) to obtain concentration of 20mM and serially diluted to achieve 10, 5, 2.5, 1.25, and 0.625mM. A volume of 100 �L of bacterial suspension
adjusted previously at concentration 106 CFU/mL was added
to 100 �L of the herbal extracts or compounds, then an additional tube containing broth only used as a negative control was added. All test tubes and control were incubated at 37∘C for 18–24hr, afterwhich the tube containing the lowest
concentration of extract showing no visible growth was considered as MIC. Further, concentrations showing total inhibition of visual bacteria growth were identified. 50 �L of each culture broth transferred onto agar plates and incubated for 18–24 hr at 37∘C.Complete absence of growth of bacterial
colonies on the agar surface in the lowest concentration of sample was defined as minimum bactericidal concentration (MBC). Each assay in this experiment was replicated three times.
2.4.Mice InfectionModel. Male BALB/c mice weighed about
20–22 g, obtained from the National Laboratory for Animal Breeding and Research Center, were maintained in specific pathogen-free (SPF) condition and used at 8–10 weeks of age. They were housed in an air-conditioned room at 25 ± 2∘C
with relative humidity 40–70% and 12-hour light/dark cycle and were fed with tap water and standard laboratory rodent diet. All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of China Medical University (approval no.: 99-156-B); experiments were performed according to the institutional ethical rules and laws. Mice were fed basal diet for a week before being
Evidence-Based Complementary and Alternative Medicine 3 used in the study. On treatment day 0, mice were randomly allocated into nine groups of ten mice. Herbal extract was dissolved in water and given orally at a dose of 5mg/mice for seven days via stomach tube. Control and water groups were given the basal diet and were fed by water instead. After the last dose, herbal treated and watered mice were challenged orally with 1010CFU of strain SC37 permouse.Clinical signs
were monitored, body weight measured, and feces of each individual were collected daily for four days after challenge. Sera, intestines, livers, and spleens were collected from all groups on day 4 after challenge; all mice were humanely euthanized.
2.5. Pig Infection Model. In all, 35 crossbreds weaned pigs
weighing about 8–10 kg were obtained from the Animal Technology Institute Taiwan, maintained in SPF condition, and used at 28 days of age. All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of National Pingtung University of Science and Technology (approval no.: NPUST-IACUC-100-004); experiments were performed according to the institutional ethical
rules and laws. SPF pigs were housed in an environmentally controlled isolation facility with filtered air and supplied with sterilized food and water. They were fed basal diet for three days before study. On treatment day 0, pigs were randomly allocated into seven of five groups (control, water, Mix-LAB, SR, SR +Mix-LAB, GF, and GF +Mix-LAB groups). Herbs were mixed in a diet with concentration 0.1%, and was Mix-LAB added to final amount of 106 CFU per gram.The dosage
of herbs andMix-LAB supplied in diet was based on previous studies [16, 17]. Both control and water groups were given basal diet and were fed by water instead. After 10 days of experimental diet, treated and water groups were challenged orally with 1010 CFU strain SC37 per pig. Clinical signs were
monitored for six days after challenge. During this time, pigs were fed with experimental diets. Body weight, body temperature, and feces of each pig weremeasured daily for six days. Sera and intestines were collected from all pigs on the
sixth day after challenge; pigswere then humanely euthanized by electrocution.
2.6. Shedding of Challenge Bacteria from Feces. Fecal samples
were collected at 1–6 days in pig trials after administering strain SC37, and numbers of Salmonella per gram in feces were determined. Aliquots (100 �L) of fecal suspensions were serially diluted in PBS, plated on duplicate
Salmonella-Shigella (SS) agar plates (Difco, NJ, USA), and incubated
overnight at 37∘C. Typical colonies were counted for plates
containing between 30 and 300 colonies. S. Choleraesuis was confirmed by PCR assay, as described in a previous report [18].
2.7.Measurement of Bacteria Load in Blood andOrgans. After
the animals were sacrificed, their livers, spleens, intestines were aseptically removed, and blood was collected to determine bacteria load. Organs were homogenized in 1.0 mL of
sterile saline with the aid of a tissue homogenizer maintained at 4∘C, aliquots of homogenate processed for CFU counts.
2.8. Enzyme-Linked Immunosorbent Assay (ELISA) for Evaluation of Cytokine Levels. Tumor necrosis factor-� (TNF-�),
interferon-� (IFN-�), and interleukin-8 (IL-8) in peripheral blood were detected with an ELISA kit (BioLegend, Inc., San Diego, CA, USA) according to instructions of manuals.
2.9. Cell Viability Assay. The murine monocyte/macrophage
cell line RAW 264.7 (ATCC TIB-71) was obtained from ATCC; RAW264.7 cells were seeded onto 24-well plates at a density of 1 × 105 cells/well for 24 hr. Indicated concentrations
of baicalin, baicalein, geniposide, or genipin were then added to cells, while only adding 0.1% DMSO in the control group, and grown at 37∘C for 24 hr. To ascertain cell viability, trypan
blue exclusion protocol was used. Briefly, about 10 �L of cell suspension in PBS (pH 7.4) was mixed with 10 �L of trypan blue; stained (dead cells) and unstained (live cells) were counted by a hemocytometer. Viability represented the percentage of cell survival after treatment.
2.10. Invasion Assay. RAW264.7 cells were treated with
baicalin, baicalein, geniposide, or genipin for 0.5 hr before infection in antibiotic-free Dulbecco’s Modified Eagle
Medium (DMEM) supplemented by 10% fetal bovine serum (FBS), then cocultured in PBS-resuspended SC37 atmultiples of infection (MOI) 10. Designated concentration of each indicator compound was then added to cells, while only adding 0.1% DMSO in the infection group. Cell-associated bacteria were quantified 0.5 hr after infection, then treated by 100 �g/mL of gentamicin for 1.5 hr. Cell culture supernatants were removed gently; cells were washed with PBS, and osmotic lysis was performed to quantify total bacteria. For this purpose, sterile water was added to infected cells after washing, and cell lysates were resuspended with PBS, and bacterial numbers were derived by plating serial dilution on SS agar plates. Invasion activity was calculated as triplicate mean, and results were expressed as percent relative invasion of RAW264.7 cells were compared with infection group.
2.11. Statistical Analysis. Differences between treated and
control groups in mean values were rated by Student’s t-test, using SPSS software (SPSS, Inc., Chicago, IL, USA).
3. Results
3.1. Antibacterial Activity of Seven Herbal Extracts. In order
to screen for the herbs with bactericidal property against �. Choleraesuis infection, seven herbal plants were tested. Table 2 summarizes their MBCs against �. Choleraesuis, ATCC 13312, and SC37; only SR displayed antibacterial
activity against Salmonella strains tested. MBCs against reference strain and SC37 were 6.25 and 25mg/mL, respectively,
but bactericidal activities of other herbal extracts
exceeded 50mg/mL. SR showedmoderate antibacterial activities against �. Choleraesuis, and the reference strain was
more susceptible to SR than to clinical isolate.
3.2. Effects of Herbal Extracts on Preventing S. Choleraesuis Infection in Mice. Supplemental dosage of herbal medicine
4 Evidence-Based Complementary and Alternative Medicine Table 2: MBCs of herbal extracts and compounds against S. Choleraesuis strains.
Herbal extract ATCC 13312 SC37 isolate
Scutellariae radix 6.25mg/mL 25mg/mL Gardeniae fructus >50mg/mL >50mg/mL
Houttuyniae herba >50mg/mL >50mg/mL Taraxaci herba >50mg/mL >50mg/mL Glycyrrhizae radix >50mg/mL >50mg/mL Puerariae radix >50mg/mL >50mg/mL Rhizoma dioscoreae >50mg/mL >50mg/mL Baicalin 5mM 5mM Baicalein 5mM 5mM Geniposide >10mM >10mM Genipin 10mM 10mM 0 0.5 1 1.5 2
Blood Liver Spleen Intestine Fold to water group
Control Water SR GF ∗ ∗ ∗ ∗ ∗
Figure 1: Effects of SR and GF supplements on Salmonella load in blood, livers, spleens, and intestines on the fourth day after challenge. Bacterial load in organs of water group was set as 100%; results were expressed as mean ± standard deviation (10 mice per group). Asterisks indicate significant difference from water group (∗
� < 0.05).
used in this study was 5mg/mouse, that is, 0.1% of daily regimen for each mouse. Clinical signs were observed, and body weight was measured. SC37 infection did not cause significant symptoms and did not affect body weight in BALB/c mice (data not shown). Yet no herbal plant treatment could reduce level of Salmonella shedding in feces
could eliminate �. Choleraesuis in organs of infected mice and significantly decrease Salmonella load in spleens. GF treatment reduced amount of Salmonella in both spleen and intestine (Figure 1). No bacteria were harvested from blood and livers of infectedmice. IFN-� levels were alsomonitored. GF could significantly decrease �.Choleraesuis-induced IFN-� expression in serum (Figure 2), but not the other six herbs. Hence, GF could suppress inflammation induced by
Salmonella infection. Given the above, SR and GFmight offer
an alternative way to prevent infection.
0 0.5 1 1.5 2 2.5 Control Water SR GF Fold to control ∗ ∗
Figure 2: Effects of SR and GF supplements on SC37-induced � expression in serum on the fourth day after challenge. IFN-� level of control group set as 100%. Results are expressed as mean ± standard deviation (10 mice per group). Asterisks indicate significant difference from water group (∗
� < 0.05).
3.3. Effects of SR, GF, and Mix-LAB on Preventing S. Choleraesuis Infection in Pigs. Based on data in mice model, SR
and GF had the best bioactivities in eliminating bacteria and suppressing inflammation induced by infection. Bioactivities of SR and GF were further evaluated by pig infection
model; probiotics as feed supplements are commonly used in livestock diets.This study applied two LABstrains (Mix-LAB) for investigating their probiotic property along with herbs. After a 10-day supplement, pigs were challenged with the clinical isolate SC37. This study was designed to show the interaction and bioactivity of herbs and probiotics as feed supplements in preventing Salmonella infection.We were not
intent on producing server sickness in pigs. The infection did not affect the body weight or cause fever, but only induced diarrhea and mild inflammatory responses in pigs. Mix-LAB, SR, and GF supplements could all reduce the levels of diarrhea caused by infection. Furthermore, SR, SR +Mix-LAB, GF, and GF +Mix-LAB groups could also dramatically reduce the Salmonella shedding in feces (Figure 3(a)) and intestines (Figure 3(b)). The amount of Salmonella fecal shedding in the group fed with SR LAB and GF +Mix-LAB was even lower by 68% and 80% than that in the groups fed with SR and GF alone. SR, SR +Mix-LAB, GF, and GF+Mix-LAB could all decrease SC37-induced IL-8 levels in serum. And SR +Mix-LAB andGF +Mix-LABcould reduce both IL-8 and TNF-� expressions in serum, closer to the levels of control groups. Duration of Salmonella fecal shedding in pigs fed with Mix-LAB was shorter than that in the infection group. Still, as of day 6 after challenge, bacteria load in intestines of theMix-LAB group tallied the lowest and was reduced by 97% comparedwith the infection group.Mix-LAB supplement elevated immune abilities in swine. Serum levels of TNF-� and IL-8 were still high in Mix-LAB group, even with most Salmonella shed by that time (Figure 4).
3.4. Bioactivities of Baicalin, Baicalein, Geniposide, and Genipin In Vitro. Based on the data above, SR and GF were
bioactive, eliminating bacteria in intestines and suppressing inflammation induced by infection. Since both have shown aglycones as metabolites wrought by intestinal microbes Evidence-Based Complementary and Alternative Medicine 5
0 2 4 6 8 10
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Water
SR GF
106 CFU/g of feces Mix-LAB SR + Mix-LAB GF + Mix-LAB (a) 0 2000 4000 6000 8000 10000 12000 Water SR GF CFU/g of tissue ∗ ∗ ∗ ∗ ∗ Mix-LAB SR + Mix-LAB GF + Mix-LAB (b)
Figure 3: Effects of SR, SR +Mix-LAB, GF, and GF +Mix-LAB supplements on bacteria load in (a) feces during six-day period after challenge
and (b) intestines on the sixth day after challenge. Results are expressed as mean ± standard deviation (five pigs per group). Asterisks indicate
significant difference from water group (∗ � < 0.05). 0 2 4 6 8 Control Water SR GF Fold to control ∗ ∗
∗ ∗ ∗ ∗ ∗ ∗ ∗ Mix-LAB SR + Mix-LAB GF + Mix-LAB IL-8 TNF-�
Figure 4: Effects of Mix-LAB, SR, SR +Mix-LAB, GF, and GF +Mix-LAB supplements on SC37-induced IL-8 and TNF-� expressions in serum on day 6 after challenge. IL-8 and TNF-� levels of control group set as 100%. Results are expressed as mean ± standard deviation (five pigs per group). Asterisks indicate significant difference from water group (∗
� < 0.05).
[19, 20], we evaluated bioactivities of baicalin, baicalein, geniposide, and genipin. MBCs of baicalin and baicalein against �. Choleraesuis strains were both 5mM, whereas MBCs of genipin against test strains were both 10mM; those of geniposide against �. Choleraesuis strains were above 10mM(Table 2). Baicalin and baicalein showed stronger
anti-S.Choleraesuis activity than geniposide and genipin. Furthermore,
RAW264.7 viability was inhibited by baicalin with IC50
of 475 �Mand was unaffected by geniposide at concentrations less than 3mM. By contrast, IC50 of baicalein and genipin
dropped as low as 155 �M and 1.42�M, respectively. Their aglycones exhibited stronger cytotoxicity.
Abilities of these compounds to prevent SC37 frominvadingmacrophages were scrutinized. Neither baicalin nor geniposide
could inhibit Salmonella invasion of macrophages, even at concentration of 200 �M. However, 100 �M of
baicalein and genipin could prevent 52% and 44% of bacteria
invading cells, respectively (Figure 5), this decrease in a dosedependentmanner. Data also suggest aglycones with stronger
ability to impede �. Choleraesuis invasion.
4. Discussion
Lactobacillus spp., Bifidobacterium spp., Enterococcus spp.,
and many other intestinal microbes are proposed as probiotics. LAB strains show beneficial effects in health promotion
for a host [21]. Some LAB strains as feed supplements can prevent gastrointestinal infection [22–24]. Mechanisms considered for such probiotic function include enhancing immune response of the host [21] and protecting colonization by pathogens for attachment [5]. Several studies have indicated that probiotics could play immunomodulatory roles
in reducing Salmonella colonization in mice and chickens [4, 7, 23, 24]. In addition, intestinal microflora offers ability to convert and then alter bioactivity of herbal compounds [25, 26].
This study aimed for a combination of LAB and herbal
plants as feed additives to prevent �. Choleraesuis infection in swine.Themouse infection model screened for infection preventive agents; pig infection model evaluated effects of target
herbs and LAB strains against infection. Seven herbal plants served to evaluate their efficacy in preventing Salmonella infection. They grow locally in Taiwan and hold antibacterial potential, seeing wide use in folk medicine. SR alone showed antibacterial activity in vitro against �. Choleraesuis.Mice are not common models to establish such infection. However, it is a relatively accurate way to evaluate bioactivity of herbs in
vivo in order to screen for preventive agents. We managed
to monitor several useful indicators affected by challenge, for example, Salmonella shedding in feces, Salmonella load in organs, and levels of serum IFN-�. In mouse infection model, both SR and GF could eliminate �. Choleraesuis in spleens. GF could also decrease the bacteria load in 6 Evidence-Based Complementary and Alternative Medicine
0 0.5 1 1.5 2
Infection Fold to infection Baicalin Baicalein * * ∗ ∗ 50 �M 100 �M 200 �M (a) 0 0.5 1 1.5 2 Infection Fold to infection Geniposide Genipin ∗ ∗ 50 �M 100 �M 200 �M (b)
Figure 5: Effects of (a) baicalin and baicalein and (b) geniposide and genipin on SC37 invasion in RAW264.7 cells. Results are expressed as mean ± standard deviation from three independent trials. Asterisks indicate significant difference from infection group (∗
� < 0.05).
intestines and infection-induced IFN-� expression in serum. However, SR did not suppress the load of �. Choleraesuis in intestines; therefore, the serum levels of IFN-� were not suppressed in serum significantly. Biotransformation in mice’s intestines might not be active enough to process SR into active compounds, therefore, affecting the elimination of bacteria from intestines.
SR sees wide use in traditional Chinese medicine [19, 27, 28], for example, antiallergy [29–31], antioxidation [32, 33], -inflammation [28, 33], and anticancer [34, 35] treatments.
It has shown strong antimicrobial effect in vitro [27, 36, 37]. GF functions as herbal remedy to treat liver and gall bladder disorders such as hepatitis and acute jaundice, as well as inflammation and fever in Chinese medicine for many years [38]. GF has the effective biological actions, including protective activity against oxidative damage [39], as well as cytotoxic [40], anti-inflammatory [41, 42], and fibrinolytic activities [43]. However, no study refers to its anti-Salmonella activity.
SR and GF were proved to be able to reduce bacteria shedding and decrease inflammation induced by challenge in swine in a significant manner. Likewise, bioactivities of SR and GF were enhanced combining withMix-LAB. In our pig infection experiments, Mix-LAB did boost immunity of infected pigs, even with �. Choleraesuis already eliminated from the body. Results concurred with previous findings [4, 7] and indicated LAB strains adjusting immunity to prepare host defense against further infection. Mix-LAB could also speed up bacteria elimination time. Maximum bacterial shedding in the group treated with Mix-LAB was two days earlier than in the infection group, suggesting LAB strains adhere to gastrointestinal epithelium and providing a barrier against pathogen colonization [21, 22, 24].
For further identification of active compounds in herbs, bioactivities of indicator compounds baicalin and geniposide and their aglycones baicalein and genipin were studied in
vitro.The aglycones revealed significant effects in bactericidal
activity and cytotoxicity. Prior study demonstrated similar
phenomena. It was reported that antimetastatic or anticarcinogenic activity of ginsenoside Rb1 could not show until
biotransformation by intestinal bacteria [10–12]. Baicalin,
crocin, amygdalin, geniposide, puerarin, ginsenoside Re, glycyrrhizin, hesperidin, and poncirin must be biotransformed
by human fecal microflora in order to manifest cytotoxicity against tumor cells [26].We attained conversion by supplying herbs and LAB mixture in diet and by using an infection model to study the effects. LAB strains could transform compounds in SR and GF, meaning that the amount of
Salmonella fecal shedding and inflammation in the group fed
with probiotics and herbs was even lower than those fed with herbs alone. In our study, LAB strains thus showed multiple functions for preventing infection, enhancing immunity to prepare host defense for infection, and adjusting enzymatic activity of intestinal microbes in order to convert bioactivity of compounds. Since SR and GF herbs grow in Taiwan, this study would also improve economic value of both plants as infection preventives for farm animals.
Authors’ Contribution
C.-H. Chang and Y.-S. Chen contributed equally to this work. B. Yu and Y.-M. Hsu contributed equally to this work.
Acknowledgments
This study was funded by Grants from China Medical University (CMU100-S-20) and Bureau of Animal and Plant Health Inspection and Quarantine and Council of Agriculture, Executive Yuan, Taiwan (99AS-5.3.1-ST-aH and 100AS-5.3.1-ST-aC).
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