Introduction
Many phytogenic extracts are being studied as additives. Phytogenic extracts can improve antibacterial and antioxidant activities and inflammatory responses (Go et al., 2021; Song et al., 2021). In addition, phytogenic extracts can increase growth performances of broilers. They are also easy to use because there is no residual effect (Hashemi and Davoodi, 2010). Houttuynia cordata Thunb (HC) is a perennial herb belonging to the family Trifoliumaceae. It is a plant that grows wild in the southeastern part of Asia and the central region of Korea. Leaves and flowers of HC contain quercitrin, quercetin, rutin, hyperin, isoquercetin, aristolactams, and decanoyl acetaldehyde. These functional materials have been proven to have antibacterial action against Gram-negative bacteria. They also possess sterilization and bacteriostatic action against E. coli, paratyphoid group, gonorrhea, and so on (Kang et al., 1997; Kim et al., 1997; Cho et al., 2008). HC fermented with lactic acid bacteria has excellent antioxidant capacity (Kim et al., 2016). In east Asia, HC is also used for tea, cosmetic, and medicinal ingredient (Choi, 2016). Li et al. (2005) have suggested that water extract of HC might be useful for treating of mast cell-mediated anaphylactic reactions. Seul et al. (2007) have reported that a dietary mixture of oldenlandiae herba and HC could mitigate the incidence of diarrhea in calves. However, studies on the effect of supplement HCE on intestinal microflora using animal models are lacking.
Therefore, the purpose of this study was to determine the safety of HCE and its effect on growth performance, colony count, and optimal concentration in Institute of Cancer Research (ICR) mice.
Materials and Method
Ethics
The experimental protocol was approved (CBNUA-1643-21-01) by the Institutional Animal Care and Use Committee of Chungbuk National University, Cheongju, Korea.
Houttuynia cordata Thunb extract (HCE)
HCE was provided by Dronic (Dronic Co., Sungnam, Korea). It mainly contained flavonoids and it was consisted of rutin (95.61%), isoquercetin (1.98%), quercetin (0.89%), quercitrin (1.47%), and afzelin (0.06).
Animals and experimental design
Five-week-old male ICR mice were obtained from DBL (Incheon, Korea). A total of 60 ICR mice with an average initial body weight (BW) of 27.24 ± 0.44 g were used in 4 weeks. The mice were reared in plastic cages in a temperature-controlled room with a 12 h light/12 h dark cycle. In 5 repetitions with 4 treatment groups, mice were randomly assigned to 3 mice per cage. Treatment groups were as follows: the first group (CON) was fed normal distilled water; the second group (T1) was fed normal distilled water with 0.05% HCE; the third group (T2) was fed normal distilled water with 0.1% HCE; the fourth group (T3) was fed normal distilled water with 0.2% HCE. Commercial feed and HCE containing drinking water were provided ad libitum for an experimental period.
Sample analysis and measurements
The body weight gain (BWG) was calculated by weighing each participant on the 1st, 14th, and 28th day of the experiment. When measuring body weight, feed intake (FI) was calculated by subtracting the remaining amount from the feeding amount, and the feed conversion ratio (FCR) was calculated by dividing the BWG by the FI. Similarly, drink is measured by subtracting the remaining amount from the serving amount.
Samples for colony count were collected form the large intestine and feces at the end of the experiment. After homogenization by suspending in aseptic distilled water, samples were used for measuring the number of viable microorganisms by serial dilution from 10-2 to 10-6. In order to measure the colony count of Lactobacillus and E. coli, de MAN, ROGOSA and SHARPE (MRS agar) for Lactobacillus, and MacConkey agar for E. coli were used, and E. coli was cultured at 37℃ for 20 hours, and Lactobacillus was cultured for 48 hours.
Statistical analysis
All data were analyzed of variance (ANOVA) for a completely random design using the general linear model (GLM) procedure of SAS software (SAS Institute, Cary, NC, USA). Duncan's multiple range test identified significant differences (p < 0.05) among treatments. All statistical analysis differences were taken to be significant at p < 0.05, and p < 0.01 were considered clearly significant.
Result and Discussion
Growth performance and water intake
Table 1 shows effects of HCE in drinking water for four weeks on growth performance of ICR mice. The T2 group has significantly higher FCR (p < 0.05) and BWG (p < 0.05) than other treatment groups at 0 - 1 weeks and 0 - 2 weeks. During the entire experimental period, the T2 group showed a significantly improved (p < 0.05) FCR than other treatment groups. In the present study, FCR and BWG showed quadratic effects (Q, p < 0.01) according to the amount of HCE at 0 - 1weeks and 0 - 2 weeks.
HCE did not significantly affect the amount of drinking water intake (Table 2). However, water intake was increased linearly (Lin, p < 0.01) with increasing concentration of HCE.
Cho et al. (2012) have reported that HC can improve the growth performance of weanling-growing pigs. Hah et al. (2010) have reported that when broilers are challenged with aflatoxin B1 toxin, a dietary mixture of activated carcoal and HC could improve feed efficiency by reducing aflatoxin B1 toxin. When 1 g·kg-1 flavonoid rutin additive is used, BW, BWG, and FCR of broilers are improved (Hassan et al., 2019). Flavonoids present in HC exhibit a wide range of biological effects, including anti-inflammatory, anti-allergic, anti-virus, anti-bacteria, and anti-oxidation activities (Jian and Xiao, 1986; Narayana et al., 2001; Havsteen, 2002). Flavones may also promote growth in animals by raising insulin-like growth factor 1 concentrations (Ouyang et al., 2016). It is thought that HC can improve the growth performance of mice due to the above positive effects. However, research about the effect of HC on growth performance of mice is insufficient. Thus, additional research is needed.
Colony count
Tables 3 and 4 show the effect of HCE on E. coli and Lactobacillus counts in the large intestine and feces. At the 2nd and 4th weeks, the T2 group showed significantly decreased (p < 0.05) number of E. coli in the large intestinal compared to other treatment groups. It showed a decreasing (Q, p = 0.01) trend when the amount of HCE added was increased. The T1 group showed a significant increase (p = 0.01) in the number of Lactobacillus in the large intestinal at the 2nd week. At the 4th week the T1 group had the highest number of Lactobacillus (p < 0.05). However, there was no significant difference from the other treatment groups.
In this study, adding HCE to drinking water showed a significant effect on E. coli and Lactobacillus concentration in intestine and feces. Similarly, Wang et al. (2018) have reported that dietary HC supplement in high fat diet could reduce E. coli concentration in mice. In addition, Kim et al. (2010) have suggested that the mechanism of action of E. coli reduction upon HC addition involves a multifaceted process including inhibition of cell wall synthesis, impaired folate synthesis, and alteration of outer membrane permeability through modulation of multiple drug efflux pumps. The production of quercetin from rutin in HC can affect intestinal flora. Decanal, endobornyl acetate, fenchene, decarnoic acid, and decanoyl acetaldehyde show strong antibacterial activities against E. coli (Kang et al., 1997; Tamura et al., 2007; Weng et al., 2017). Quercetin supplementation can also enhance populations of Bacteroides, Bifidobacterium, Lactobacillus, and Clostridia and significantly reduce populations of Fusobacterium and Enterococcus (Lin et al., 2019).
Conclusion
Supplementation of HCE in drinking water showed a positive effect on growth performance and colony count in mice. When looking at the mice FCR and colony count of large intestine, 0.1% HCE supplementation can be seen as the optimal concentration. Through this study, HCE is thought that it will be possible to develop HCE supplements through additional research using other livestock models.
Acknowledgements
Following are results of a study on the "Leaders in Industry-university Cooperation +" Project, supported by the Ministry of Education and National Research Foundation of Korea.
Authors Information
Jae Woo An, https://orcid.org/0000-0002-5602-5499
Sung Bo Cho, https://orcid.org/0000-0002-2593-2758
Ji Hwan Lee, https://orcid.org/0000-0001-8161-4853
Han Jin Oh, https://orcid.org/0000-0002-3396-483X
Yong Ju Kim, https://orcid.org/0000-0002-0960-0884
Se Yeon Chang, https://orcid.org/0000-0002-5238-2982
Young Bin Go, https://orcid.org/0000-0002-5351-6970
Dong Cheol Song, https://orcid.org/0000-0002-5704-603X
Hyun Ah Cho, https://orcid.org/0000-0003-3469-6715
Jin Ho Cho, https://orcid.org/0000-0001-7151-0778
