Effects of Angelica gigas Nakai on the production of decursin- and decursinol angelate-enriched eggs
Giselle Ann D. Fontamillas1,*, Si Won Kim1,*, Hoy-Ung Kim1, Sung-Jo Kim2, Jong Geun Kim1,3, Tae Sub Park1,3, Byung-Chul Park1,3,§
1Institute of Green-Bio Science and Technology, and 3Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang-gun, Gangwon-do 25354, Korea
2Division of Cosmetics and Biotechnology, Hoseo University, Asan-si, Chungnam, 31499, Korea
*Giselle Ann D. Fontamillas and Si Won Kim contributed equally in this work.
Author’s e-mail;
Giselle Ann D. Fontamillas: [email protected] Si Won Kim: [email protected]
Hoy-Ung Kim: [email protected] Sung-Jo Kim: [email protected] Tae Sub Park: [email protected] Jong Geun Kim: [email protected] Byung-Chul Park: [email protected]
§Corresponding Authors:
Byung-Chul Park Tel: +82-33-339-5792, e-mail: [email protected]
Running title: Production of decursin and decursinol angelate-enriched eggs
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jsfa.9526
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Abstract
BACKGROUND: The livestock industry requires high-quality products as well as improved productivity. There have been many studies regarding the utilization of feed additives to increase productivity, enhance immune functions, and prevent infectious diseases in livestock. Biofunctional feed additives would be beneficial not only for animal health, but also for consumers. In this study, we utilized root and byproduct (stem and leaf) powders of Angelica gigas Nakai (AGN, Korean Danggui) as feed additives and examined the deposition of biofunctional compounds, such as decursin and decursinol angelate, into egg white and yolk.
RESULTS: We optimized the detection system for decursin and decursinol angelate, and determined the amounts of decursin and decursinol angelate derived from AGN byproducts (stem and leaf) as well as root. In Experiment I, laying hens were fed with the dried AGN root powder and the effective compounds were detected in egg white and yolk. Subsequently, in Experiment II, we examined AGN byproducts as an alternative feeding supplement. Additionally, biochemical parameters were analyzed to evaluate changes in the health of the hens by feeding AGN root powder. The results of this study indicated that decursin and decursinol angelate were stably transferred into egg white and yolk by feeding AGN byproducts as well as root. Intriguingly, plasma cholesterol levels were significantly decreased in a dose- dependent manner, and those of interleukin-1β, as an immune-related biomarker, were considerably increased in the treated hens.
CONCLUSION: These results indicated that AGN root and byproducts (stem and leaf) could be utilized for production of value-added eggs and to improve the health of hens in the poultry industry.
Keywords: Angelica gigas Nakai, decursin, decursinol angelate, designer egg, chicken
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Introduction
Angelica gigas is a traditional medicinal plant that has been used in Korea, Japan, and China to prevent and treat various human diseases1. Since ancient times, its root has been used as a sedative to reduce pain2, treat anemia3, and replenish the blood4. The use of its root as an herbal remedy was first recorded in a Chinese book on agriculture and medicinal plants known as Shennong Ben Cao Jing (The Classic of Herbal Medicine), as early as around 100 BC4. Coumarin derivatives, such as decursin and its isomer, decursinol angelate, are the primary compounds that can be isolated from the plant, and have attracted considerable attention because of their numerous pharmacological effects1,5,6. Anti- inflammatory5,7 and antioxidant activities8 of these compounds were reported in humans, and they also exhibited inhibitory effects against cell proliferation and activation of apoptosis in cervical cancer3.
Angelica gigas Nakai (AGN), the most common strain in Korea and also popularly known as Cham-Dang-Gui (Korean Danggui), has been widely used for the treatment of dysmenorrhea, amenorrhea, menopause, pain, injuries, infection, migraine headaches, and articular rheumatism2,9. Its dosage form is usually prepared by boiling the dried root in water to extract the effective ingredients2. In addition to its potential use as a pharmaceutical agent, AGN is also marketed globally as a functional food product9,10 and AGN extract or AGN-containing herbal mixtures are sold as dietary supplements for relief of post- menopausal symptoms, as a memory enhancer, and as a pain killer2. AGN contains several active ingredients, including essential oils and polyacetylenes in addition to coumarins (decursin and decursinol angelate); these are its major effective components6,10,11. Extensive studies have been conducted on these substances, and particularly on decursin and decursinol angelate, and numerous reports have been published regarding their various pharmaceutical effects on human health6.
In the livestock industry, phytogenics derived from medicinal plants are utilized as feed additives to improve animal health, welfare, and performance, and are also considered potential alternatives to antibiotics3,12,13. Studies on herbal plants known to have medicinal properties and other natural products
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such as spices (e.g., cayenne pepper, turmeric, and ginger) and essential oils, have been reported to be beneficial to improve productivity and health performance including gastrointestinal nutrient absorption and barrier functions in the swine and poultry sectors12,13,14. The potential utilization of AGN as a feed additive should be explored further. In addition, root of AGN has been widely utilized for various purposes as representing its main economic value, whereas byproducts of AGN (stem and leaf) are discarded imposing cost for waste disposal and environmental pollution. This study was therefore conducted to investigate whether byproducts and root of AGN as feed supplements could have benefits in laying hens, and whether bioactive substances of them, particularly decursin and decursinol angelate, could be transferred into eggs.
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Materials and Methods
Experimental animal care and utilization of Angelica gigas Nakai as a feed additive
Chickens were maintained according to the standard management program at the University Animal Research Farm, Pyeongchang Campus, Seoul National University, Korea. The procedures for animal management adhered to the standard operating protocols of our laboratory.
The root and byproducts (stem and leaf) of AGN (Korean Danggui) were harvested and dried at 65°C for 48 hours. The moisture content in the dried components of AGN was approximately 15%. Subsequently, the dried root and byproducts of AGN were ground and sieved to achieve a consistent particle size of 1 – 2 mm. Finally, the ground powders were added to commercial feed (0.3% or 0.6% for dried root and 1% or 3% for dried byproducts, w/w). Based on the preliminary study that AGN byproducts of stem and leaf contained one-third of the effective components (decursin and decursinol angelate) compared to AGN root, we designed the experimental treatments with 0.3% or 0.6% for dried root (Experiment I) and 1% or 3% for dried byproducts (Experiment II).
Thirty-week-old Hy-Line layers were fed with control diet, a diet containing 0.3% or 0.6% of the dried root (Experiment I), or a diet containing 1% or 3% of the dried byproducts (Experiment II). Ten layer hens per each group were fed with control diet or diets with the dried root or byproducts for 30 days. Each cage separately contained one layer hen in each treatment group as well as control group during the study period. In all experimental treatments, 120g of a diet with or without AGN (root and stem/leaf) were individually fed for one layer hen per a day, and there was ad libitum access of fresh drinking water at any point of the day throughout the study period. Hen day egg production and average egg weights of all treatments were measured and compared after feeding with 0.3% or 0.6% of the dried root.
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Sample pretreatment for extraction and detection of decursin and decursinol angelate
For sample preparation of root and byproducts (stem and leaf), each AGN component was dried in a
forced-air drying oven at 65°C for 48 hours. After drying, each component was finely ground using a
lab-scale miller. To prepare egg white and yolk samples, after separating and weighing, egg white and
yolk were freeze-dried in a refrigerator for 5 days.
Samples of 1.0 g of the freeze-dried and ground products (components of AGN, egg white, and yolk) were dissolved in 25 mL of methanol (99% high performance liquid chromatography [HPLC] grade; Sigma-Aldrich, St. Louis, MO) and incubated in a water bath at 70°C for 2 hours followed by stirring at 20-minute intervals. The samples were filtered with Whatman paper No. 40 (Whatman PCL, Maidstone, UK). Using an evaporator device (HyperVAP HV-300; Hanil Scientific Inc., Gimpo, Korea), the samples were evaporated at a temperature of 40°C and pressure of 30 psi. Subsequently, the evaporated samples were dissolved in 5 mL of methanol (99% HPLC grade; Sigma-Aldrich) and then centrifuged at 4°C, 3,000 rpm, for 10 minutes. Finally, after filtering through 0.45-μl nylon membrane filters, the samples were aliquoted and stored in a refrigerator at –20°C for lipid chromatographic analysis.
Ultra-performance liquid chromatography (UPLC)
A UPLC system (Xevo® TQ MS ACQUITY UPLC® System; Waters, Milford, MA, USA) was used to analyze the preprocessed samples. Aliquots of 0.5 ml of standard or plant samples were directly injected into a column (ACQUITY UPLC® BEH C18 1.7 mm, 2.1 mm × 50 mm; Waters) using a gradient water-acetonitrile solvent system. Then, 0.75 ml of the pretreated samples of egg white and yolk was subjected to UPLC analysis with a flow rate of 0.64 ml min-1. Decursin and decursinol angelate were detected at UV 330 nm, and MassLinx V4 software (Waters) was used for data analysis. The purified
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decursin and decursinol angelate (as a standard marker) were purchased from Natural Product Biotechnology, Co. (Daejeon, Korea).
Biochemical analysis of blood parameters
To examine the physiological effects of AGN in hens, biochemical analyses of the blood from hens fed mixtures of 0.3% and 0.6% dried AGN root powder were conducted. Blood was obtained from the brachial vein (wing vein) and subjected to biochemical analysis 30 days after feeding in three groups (control, and 0.3% and 0.6% dried AGN root powder-fed). The biochemical parameters examined included packed cell volume (PCV), concentrations of calcium (Ca) and inorganic phosphate (P), and the amounts of albumin (ALB), total protein (TP) and total cholesterol (TC). To evaluate the hepatotoxicity of AGN, aspartate transaminase (AST) and alkaline phosphatase (ALP) levels were analyzed and compared between the treated groups. PCV was measured using a micro hematocrit device (Vision Scientific Co., Seoul, Korea). Biochemical analysis was performed to determine AST, ALP, TP, ALB, TC, Ca, and P using a chemistry analyzer according to the manufacturer’s recommendations (BS-400; Mindray Co., Shenzhen, China). ELISA kits were used for detection of immune-related cytokines, including interleukin-1β (IL-1β), interferon-gamma (IFN-γ), and interleukin-6 (IL-6), according to the manufacturer’s protocol (MyBiosource, San Diego, CA, USA).
Statistical Analysis
Statistical analysis was conducted using the SAS version 9.4 software (SAS Institute, Cary, NC, USA). The significance of differences was analyzed using general linear model procedure and the differences among groups were deemed to be significant when p<0.05.
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Results
Detection of decursin and decursinol angelate using UPLC
We optimized the detection procedure for decursin and decursinol angelate, which are major bioactive components of AGN (Korean Danggui). Using the purified decursin and decursinol angelate as standards, the detection and quantification protocols were optimized for UPLC (Figure 1A and B). Each component could be individually detected even in a mixture of decursin and decursinol angelate (Figure 1C). Next, decursin and decursinol angelate were detected and quantified in the roots, stems, and leaves of AGN (Figure 2A). The amounts of decursin and decursinol angelate in the stem and leaves of AGN were approximately one third of those in the root, indicating that, as byproducts, the stem and leaves could be utilized to provide supplementary bioactive material. Subsequently, decursin and decursinol angelate were detected after mixing the ground root of AGN with egg white and yolk to examine the inhibitory masking effect by egg white and yolk (Figure 2B).
Egg production and detection of decursin and decursinol angelate in eggs after feeding with AGN root
In Experiment I, laying hens were given feed containing 0.3% or 0.6% ground AGN root powder. After 5 days of feeding with the AGN root, the bioactive components (decursin and decursinol angelate) were detected in egg white and yolk at levels ranging from 126.16 mg per egg to 200.89 mg per egg, while these components were not detected in control eggs (Figure 3). In general, there were no significant differences in hen-day egg production or average egg weights between the control and treatment groups given 0.3% and 0.6% AGN root powder during the feeding period (Figure 4).
Biochemical analysis of blood parameters
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To evaluate the physiological healthy effects of AGN on layer hens, we determined and compared the biological parameters of the blood between the treated groups. With the exception of PCV (control; 32.2±1.5%, 0.3%; 32.6±3.0%, 0.6%; 32.4±3.5%), the physiological parameters were significantly decreased by feeding dried AGN root powder in a dose-dependent manner; Ca (control; 28.8±4.4mg dL-1, 0.3%; 24.5±3.6mg dL-1, 0.6%; 19.1±1.7mg dL-1), phosphate (control; 6.9±1.4mg dL-1, 0.3%; 3.5±1.2mg dL-1, 0.6%; 4.9±0.4mg dL-1), ALB (control; 2.1±0.1g dL-1, 0.3%; 1.9±0.1g dL-1, 0.6%; 1.7±0.2g dL-1), TP (control; 6.8±1.4g dL-1, 0.3%; 5.3±1.2g dL-1, 0.6%; 4.9±0.4g dL-1), TC (control; 168.4±43.1mg dL-1,
0.3%; 133.8±21.3mg dL-1, 0.6%; 92.8±13.2mg dL-1) (Figure 5A). the hepatotoxicity of AGN AST in thegroup fed 0.6% AGN was slightly elevated (control; 170.2±15.3U L-1, 0.3%; 158.8±25.4U L-1, 0.6%; 227.6±17.2U L-1), but ALP showed no significant changes (control; 358.4±65.9U L-1, 0.3%; 422.6±366.5U L-1, 0.6%; 322.8±290.6U L-1) (Figure 5B). Immune-related cytokine concentrations were compared between controls and treatment groups fed 0.3% and 0.6% AGN. The expression level of IL-1β in blood was significantly increased in the group fed 0.6% AGN (control; 10.2±5.4pg mL-1, 0.3%; 15.1±2.4pg mL-1, 0.6%; 19.8±7.5pg mL-1), but no differences were observed in levels of IFN-γ (control; 63.5±21.6pg mL-1, 0.3%; 36.4±17.0pg mL-1, 0.6%; 20.4±5.0pg mL-1) or IL-6 (control; 18.5±10.1pg mL-
1, 0.3%; 14.3±3.4pg mL-1, 0.6%; 19.6±11.3pg mL-1) (Figure 5C).
Detection of decursin and decursinol angelate in eggs after feeding with byproducts of AGN
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In the experiment II, the bioactive components (decursin and decursinol angelate) were detected in egg white (90.86 mg/egg white on average) and yolk (106.98 mg/egg yolk on average) after 3 days of feeding the 3% mixture containing the powdered dried AGN byproducts. Decursin and decursinol angelate were detected throughout the whole feeding period. Regardless of the percentages of dried AGN byproduct, 85.00 ± 77.27 mg per egg (18.65–218.31 mg) and 105.34 ± 74.73 mg per egg (15.39–233.51 mg) on average were detected in egg white and egg yolk, respectively. Interestingly, in both the 1% and 3% ground AGN byproduct groups, the amounts of decursin and decursinol angelate transferred to egg white and yolk varied widely among individual hens. Further studies are required to determine the hen egg- transferring pathways of functional metabolites derived from feed additives, to improve the efficiency of transfer into egg white and yolk. These results could be adopted to produce value-added functional eggs and enhance the health of hens by including dried AGN byproducts in their feed.
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Discussion
To exploit the desirable effects of herbs and their byproducts, they can be added to feed in dried forms or as extracts15. In general, the main objectives of livestock production are to ensure good performance of livestock and produce high-quality, safe livestock products. Thus, incorporation of medicinal or herbal plants could play a significant role in both animal nutrition and welfare, as well as human health. Many plants contain one or more active molecules, the secondary metabolites of which are responsible for certain biological effects15. This study focused on the coumarin compounds, decursin and decursinol angelate, from AGN (Korean Danggui). In the initial step, a technical platform for detection of the effective ingredients (decursin and decursinol angelate) was developed by using appropriate solvents for extraction and UPLC. Such molecules were identified in eggs of hens given diets supplemented with byproducts (stem and leaf) and root of AGN (Figures 1-3). Generally, AGN powder supplementation did not affect the productivity of hens, including hen-day egg production and average egg weight (Figure 4).
In this study, we examined the herbal plant as a feed additive and confirmed that the effective components could be transferred into hen’s eggs. However, in the future study, we need to improve the deposition efficiency of biofunctional materials and develop the commercialized protocol for industrial application through the concentration and protective capsulation procedure of bioactive components. Furthermore, the bioactive materials could also be utilized for broiler chickens. To date, many studies on alternative additives for improving growth performance, the plasma fatty acid profile and the immune system in terms of antimicrobial and anti-inflammatory properties, and increasing feed intake and the secretion of digestive fluids in broiler chickens have been condcuted16-19. Additionally, the biofunctional feed additive system could be applicable to other livestock not only to produce the value-added products, but also to enhance the animal health and welfare.
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To confirm whether incorporation of AGN into the diet of hens as a feed additive could play roles in health and nutrition, biochemical analyses were conducted after feeding with dried AGN root powder. Interestingly, TC levels in the blood were significantly decreased in AGN-fed hens (Figure 4A). However, the levels of AST were slightly increased by 0.6% dried AGN root powder, indicating that AGN induced hepatotoxicity at a high dose (Figure 4B). As the effects of herb-derived active components may largely depend on the dosage used, this is an important consideration for future research15. The concentration of IL-1β as an immune biomarker in the blood of hens was elevated by AGN root powder in a dose-dependent manner (Figure 5C). Thus, use of AGN root powder as a feed additive could enhance metabolic and immune activity in hens.
Due to increasing demands for healthier food, there is a great deal of potential for regularly consumed foods to be converted into functional foods by either changing their composition and/or including certain ingredients with beneficial health effects20. Thus, functional foods are now among the fastest-growing sectors of the food industry21. One way to obtain functional foods is to modify the quantities of certain components such that the foods correspond more closely to the recommendations of nutrition experts20. Eggs can be converted into functional foods by modifying the rearing of laying hens or providing the hens with feed supplemented with active ingredients22. For example, to enhance levels of omega-3 in eggs, the hens’ diet should be supplemented with flax seed, linseed, or the corresponding oils to achieve enrichment of a-linolenic acid (ALA), which is a precursor of docosahexaenoic acid (DHA). Similarly, active compounds in herbal plants or their extracts could be introduced into conventional foods, including eggs, to provide health benefits once consumed.
Conclusion
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Functional foods can improve the general condition of the body, decrease the risk of certain diseases, and even cure certain illnesses. In this context, AGN is a candidate to produce hen’s eggs containing biofunctional components, such as decursin and decursinol angelate. The present study showed that the main bioactive metabolites were stably transferred into eggs after supplementation of feed with AGN root and byproducts (stem and leaf). These results indicated that byproducts and root of AGN could be utilized for production of value-added eggs, and for improving the health of hens in the poultry industry as well.
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Acknowledgements
This work was also supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Agri-Bio Industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (316005-5).
Conflict of Interest
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.
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9. Accepted Article
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18. Accepted Article
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