재배황기의 Astragalosides 함량의변이

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1 韓藥作誌 (Korean J. Medicinal Crop Sci.) 20(5) : (2012) 재배황기의 Astragalosides 함량의변이 김승현 1 전윤미 1 임주진 김성협 정일민 김은혜 건국대학교생명환경과학대학응용생물과학과 Variation of Astragalosides Contents in Cultivated Astragalus membranaceus Seung Hyun Kim 1, Yoon Mi Jun 1, Ju Jin Lim, Sung Hyop Kim, Ill Min Chung and Eun Hye Kim Department of Applied Bioscience, College of Life and Environmental Science, Konkuk University, Seoul , Korea. ABSTRACT : This study was conducted to determine the contents of astragalosides in Astragalus membranaceus. The total astragaloside content (1701 μg g -1 ) of 2 year-cultivated astragalus was the highest among the variously cultivated astragalus plants. Upon an increase in cultivation time, the average value of the total astragaloside content decreased (from 1650 μg g -1 to 645 μg g -1 ). Especially, the content of astragaloside decreased rapidly. Comparison between the top and subterranean parts of harvested astragalus plants showed that as the cultivation time increased, the astragaloside content of the top part decreased. On the other hand, the subterranean part demonstrated the opposite pattern. The astragaloside content of the top root increased compared to that in the lateral root (from 1.9 μg g -1 to 33.4 μg g -1 ). Further, the content of astragaloside increased in the top root as the cultivation time increased. Key Words : Astragalus membranaceus, Astragaloside, Variation INTRODUCTION Astragalus (Astragalus membranaceus) is considered to be one of the largest genera, containing about 2,500-3,000 species (annual, perennials, and shrubs) distributed throughout the northern temperate zones (Podlech, 1986; Kim et al., 2008). The greatest number of Astragalus species is found in the arid, continental regions of western North America (400 species) and central Asia (2,000-2,500 species). An additional 150 species are present in temperate South Africa (Liston and Wheeler, 1994). Especially, the roots of A. membranaceus, called Huangqi, which is a herb native to northern China and the elevated regions of the Chinese provinces, Yunnan and Sichuan, are commonly used in traditional Chinese medicine (TCM) and are considered an alternative medicine in the west a satonicon par with ginseng. This drug has been used in the treatment of night sweats, deficiency of qi (e.g., fatigue, weakness, and loss of appetite) and diarrhea (Foster and Yue, 1992; Shi et al., 2011; Song et al., 2008). A. membranaceus is often harvested in autumn and dried use in decorations, powder, and tinctures. It also possesses hepatoprotective, antioxidative, antiviral, antihypertensive, and immuno-stimulant properties (Rios and Waterman, 1997; Gui et al., 2006). A. membranaceus roots have been reported to contain triterpene saponins, phenolic compounds, and polysaccharides (Hirotami et al., 1994; Subarnas et al., 1991). A. membranaceus is an important ingredient in many traditional Chinese formulas. It is often combined with Angeloca polymorpha var. sinensis or treatment of cold sensitivity, poor circulation, and low energy as well as with Atractylodes macrocephala and Ledebouriella seseloides for treatment of allergies, frequent colds, diabetes, kidney problems, prolapsed organs, anemia, and slow-healing skin eruptions. It also improves recovery and longevity in cancer patients undergoing chemotherapy or radiation treatment. Astragalosides, a type of triterpenoid saponin, are substances with phytochemical and pharmacological properties. Triterpenoid saponins are major active ingredients Corresponding author: (Phone) ( ) mandu17@konkuk.ac.kr Received 2012 September 25 / 1st Revised 2012 September 28 / Accepted 2012 October 9 1 Kim SH and Jun YM contributed equally to this paper. 372

2 김승현 전윤미 임주진 김성협 정일민 김은혜 (Wang et al., 2002; Bedir et al., 2000; Cui et al., 2003; Kwon and Park, 2012) and are characterized by a four or five ring configuration consisting of 30 carbons with several oxygens attached. They are assembled from a C 5 isoprene unit through the cytosolic mevalonate pathway to make a C 30 compound, and they are steroidal in nature. Triterpenes are separated into some 20 groups according to their particular structures. Although all terpenoid compounds have bioactivity in mammals, triterpenes are the most responsible for the adaptogenic ability of plants. Most triterpenoid compounds in adaptogenic plants are present as saponin glycosides, which consist of various sugar molecules attached to a triterpene unit. These sugars can be easily cleaved off in the gut by bacteria, allowing aglycone (triterpene) to be absorbed. This allows their insert into cell membranes (Attele et al., 1999), which modifies the composition and influences membrane fluidity (Lee et al., 2003), potentially affecting cell signaling involving ligands and cofactors (Lindsey et al., 2003). Saponin glycosides reduce the surface tension of water by foaming and break down lipids. Usually, triterpene saponins are designated by the suffix side, as in ginsenoside or astragaloside, and are named for the plant genus in which they first discovered. In addition, astragalosides Ⅰ and Ⅱ are chemically very unstable and easily converted into astragaloside Ⅳ through loss of acetoxyl groups. Therefore, they are often commercially unavailable even though they are important bioactive saponins. As mentioned above, herbal medicines possess rely on biologically functional substances. For this reason, more research is needed to determine whether or not Astragalus is safe and has few side effects. Therefore, this study focused on the concentrations of astragalosides and in Astragalus plants raised for different time periods and harvested at different dates. Further, we investigated the relationship between astragaloside content and environmental conditions. MATERIALS and METHODS 1. Plant materials Twenty-seven astragalus (Astragalus membranaceus) plants were obtained from the Department of Herbal Medicine Resource, Kangwon National University (Dogye Campus, Hwangjori 3, Dogye-up, Samcheok, Kangwon-Do, Korea) and Jeongseon Agricultural Technology and Extension Center (Jeongseongun, Kangwon-Do, Korea) in They were divided based on sample types as shown in Table 1. Astragalus samples were divided into three types based on harvesting time. Nine samples were harvested on June 5, nine samples were harvested on July 8, and nine samples were harvested on September 21. Astragalus samples from each harvesting time were further subdivided based on their cultivation time: 2 year-cultivated astragalus, 3 year-cultivated astragalus, and 5 year-cultivated astragalus. Lastly, these astragalus samples were even further classified based on plant part: top part, subterranean parttop root and lateral root. 2. Analysis of astragaloside by ELSD 1) Sample preparation The extraction of astragalosides Ⅰ, Ⅱ, Ⅲ, and Ⅳ was performed as follows. 3 g of finely ground astragalus powder was extracted with 10 ml of 100% MeOH for 2 h using an ultrasonic generator. The extracted samples were filtered through a 0.2 μm nylon membrane syringe filter (TITAN, USA) and analyzed by Evaporative Light Scattering Detection (ELSD). HPLC grade (J. T. Baker, USA) solvents were used in this procedure. 2) Analysis of astragalosides The instrumentation for ELSD analysis was applied by the following method. The ELSD system consisted of an Alltech 2000 ES ELSD, a TSP (Thermo Separation Products, USA) AS 1000 auto injector, and an ACME 9000 pump (Young- Lin, Korea). The analysis operation differed in accordance with the astragaloside type (two types of analysis were performed): astragaloside Ⅰ and astragaloside Ⅱ, Ⅲ, and Ⅳ. Astragaloside Ⅰ analysis was performed as follows. Nitrogen was used as the ELSD nebulizer gas (2.5 ml min -1 ), and the tube temperature was set to The separation of astragaloside was performed using a Kromasil KR100-5C 18 (Dimensions 250 mm 4.6mm ). The mobile phase was follows: acetonitrile : water = 45 : 55, isocratic. The analysis time was 20 min and the flow rate 1.0 ml min -1. Astragaloside Ⅱ, Ⅲ, and Ⅳ analysis was as follows. Nitrogen was used as the ELSD nebulizer gas (2.6 ml min -1 ), and the tube temperature was set to The separation of astragalosides Ⅱ, Ⅲ, and Ⅳ was performed using an Kromasil KR100-5C 18 (Dimensions 250 mm 373

3 황기의 astragaloside 의함량변이 Table 1. List of astragalus used in this study. Sample No. Area Harvest day Cultivation year Plant structure location Resource classification 1 Taebaeg 6/5 2 top part improved variety 2 Taebaeg 6/5 2 subterranean part - main root improved variety 3 Taebaeg 6/5 2 subterranean part - lateral root improved variety 4 Taebaeg 6/5 3 top part improved variety 5 Taebaeg 6/5 3 subterranean part - main root improved variety 6 Taebaeg 6/5 3 subterranean part - lateral root improved variety 7 Taebaeg 6/5 5 top part improved variety 8 Taebaeg 6/5 5 subterranean part - main root improved variety 9 Taebaeg 6/5 5 subterranean part - lateral root improved variety 10 Taebaeg 7/8 2 top part improved variety 11 Taebaeg 7/8 2 subterranean part - main root improved variety 12 Taebaeg 7/8 2 subterranean part - lateral root improved variety 13 Taebaeg 7/8 3 top part improved variety 14 Taebaeg 7/8 3 subterranean part - main root improved variety 15 Taebaeg 7/8 3 subterranean part - lateral root improved variety 16 Taebaeg 7/8 5 top part improved variety 17 Taebaeg 7/8 5 subterranean part - main root improved variety 18 Taebaeg 7/8 5 subterranean part - lateral root improved variety 19 Taebaeg 9/21 2 top part improved variety 20 Taebaeg 9/21 2 subterranean part - main root improved variety 21 Taebaeg 9/21 2 subterranean part - lateral root improved variety 22 Taebaeg 9/21 3 top part improved variety 23 Taebaeg 9/21 3 subterranean part - main root improved variety 24 Taebaeg 9/21 3 subterranean part - lateral root improved variety 25 Taebaeg 9/21 5 top part improved variety 26 Taebaeg 9/21 5 subterranean part - main root improved variety 27 Taebaeg 9/21 5 subterranean part - lateral root improved variety 4.6 mm ). The mobile phase was as follows : acetonitrile : water = 40 : 60, isocratic. The analysis time was 20 min and the flow rate 1.0 ml min -1. The standard astragaloside materials were used to establish a calibration curve. Standards for astragalosides Ⅰ, Ⅱ, Ⅲ, and Ⅳ were prepared in 100% MeOH at concentrations of 12.6, 63.29, , and μg ml -1, respectively, and high linearity was obtained. The interpretation of astragalosides Ⅰ, Ⅱ, Ⅲ, and Ⅳ was supported by the retention times. 3. Statistical analysis Statistical analyses were conducted by the general linear model procedure (GLM) of the statistical analysis program (SAS, 2000). The experimental design was a completely randomized design with three replications. The least significant different (LSD) test was based on a 0.05 probability level. RESULTS and DISCUSSION 1. Distribution of astragaloside contents in astragalus Study of the total astragaloside content of astragalus (Fig. 1) found that the 2 year-cultivated 7/8 harvesting astragalus plants had the highest total astragaloside content (2573 μg g -1 ), whereas 5 year-cultivated 9/21 harvesting astragalus (632 μg g -1 ) had the lowest. More exactly, astragaloside Ⅰ (538 μg g -1 ) was detected at the highest concentration in astragalus compared to other astragalosides. On the other hand, astragalosides Ⅱ (176 μg g -1 ), Ⅲ (158 μg g -1 ), and Ⅳ (199 μg g -1 ) were detected. The gap between astragaloside Ⅰ (538 μg g -1 ) and astragalosides Ⅱ, Ⅲ, and Ⅳ (533 μg g -1 ) was 5 μg g -1. The sum totals of astragalosides Ⅱ, Ⅲ, and Ⅳ (49.8 μg %) were almost equal compared to that of astragaloside Ⅰ (50.2 μg %). In conclusion, 2 year-cultivated 7/8 harvesting astragalus has commercial value. 374

4 김승현 전윤미 임주진 김성협 정일민 김은혜 Fig. 1. The astragaloside contents of astragalus according to the different cultivated-year and harvesting-time. Fig. 2. Total astragalosides contents according to the cultivated-year. 2. Distribution of astragaloside contents in astragalus according to cultivation year Total astrogaloside content according to cultivation year ranged from 645 μg g -1 to 1650 μg g -1 in the astragalus (Fig. 2). Two year-cultivated astragalus had the highest astragaloside content (1701 μg g -1 ) among the astragalus plants cultivated for different time periods. In contrast, 3 year-cultivated astragalus had the lowest concentration (645 μg g -1 ) among astragalus. The average value of total astragaloside contents was 1071 μg g -1. As the time of cultivation increased, the average total astragaloside content decreased (from 1650 μg g -1 to 645 μg g -1 ). Especially, the content of astragaloside rapidly decreased. The proportion of astragaloside among the total astragalosides (astragaloside Ⅱ, Ⅲ, and Ⅳ) decreased from about 66.4% percent in 2 year-cultivated astragalus to about 24.4% percent in 5 year-cultivated astragalus. The gap in astragaloside content between 2 year-cultivated and 5 year-cultivated astragalus was 42.1%. To conclude, 375

5 황기의 astragaloside의 함량 변이 Fig. 3. The astragalosides contents according to the their harvesting-time in the astragalus. Fig. 4. The comparison of astragalosides contents between top part and subterranean part according to the different cultivatedyear and different harvesting-time in the astragalus. 376

6 김승현 전윤미 임주진 김성협 정일민 김은혜 Fig. 5. The comparison of astragalosides contents between top part and subteeranean part according to the different cultivated-year in the astragalus. A) showed the each of astragalosides contents according to the different-year; B) showed the tendency of the total astragalosides according to the different-year. astragalus cultivated for 2 years is appropriate for cultivation. 3. Distribution of astragaloside content in astragalus according to harvesting time Total astragalosides ranged from 769 μg g -1 to 1127 μg g -1. 7/8 harvesting astragalus had the highest content of total astragalosides (1127 μg g -1 ) among astragalus harvested at different times. In contrast, 9/24 harvesting astragalus had the lowest concentration of astragalosides (769 μg g -1 ). This experiment followed the changes in astragaloside content. Astragaloside content grew by 12.1% from 6/5 to 7/8 harvesting astragalus, then decreased 24.4% fall from 7/8 to 9/21 harvesting astragalus (Fig. 3). Astragalosides Ⅱ, Ⅲ, and Ⅳ demonstrated the opposite pattern. The contents of astragalosides Ⅱ, Ⅲ, and Ⅳ decreased by 3.18, 8.51, and 0.4%, respectively, from 6/5 to 7/8 harvesting astragalus, then decreased by 9.9, 5.9, 8.6%, respectively, from 7/8 to 9/21 harvesting astragalus. It may be guessed the tendency of good harvest time from the shown data. The harvest season of astragalus, July is the appropriated time of harvest. 4. Comparison of astragaloside contents between the top and subterranean parts of astragalus Comparison of total astragaloside contents between the top and subterranean parts of 2 year- (Fig. 4-A), 3 year- 377

7 황기의 astragaloside 의함량변이 Fig. 6. The comparison astragalosides contents of the top root and lateral root according to the different cultivated-year in the astragalus. A) showed the total astragalosides contents; B) showed the each contents of astragalosides. (Fig. 4-B), and 5 year- (Fig. 4-C) cultivated astragalus plants was performed as follows. The total astragaloside contents in 2 year-cultivated astragalus ranged from 544 μg g -1 to 2870 μg g -1, and this value reflected the phenolic content in the top part minus that in the subterranean part. 7/8 harvesting astragalus cultivated for 2 years had the highest gap in total astragaloside content (2870 μg g -1 ) between the top part and subterranean part. In contrast, 9/21 harvesting astragalus plants cultivated for 2 years had the lowest gap in total astragaloside content (544 μg g -1 ) between the top part and subterranean part. The total astragaloside contents in 3 year-cultivated astragalus ranged from 63.5 μg g -1 to 557 μg g -1. 6/5 harvesting astragalus cultivated for 3 years had the highest gap in total astragaloside content (557 μg g -1 ) between the top part and subterranean part. In contrast, 7/8 harvesting astragalus cultivated for 3 years had the highest gap in total astragaloside content (63.5 μg g -1 ) between the top part and subterranean part. The total astragaloside contents in the 5 year-cultivated astragalus plants ranged from 26.5 μg g -1 to 55 μg g -1. 6/5 harvesting astragalus plants cultivated for 5 years had the highest gap in total astragaloside content (55 μg g -1 ) between the top part and subterranean part. In contrast, 7/8 harvesting astragalus cultivated for 3 years had the lowest gap in total astragaloside content (26.5 μg g -1 ) between the top part and subterranean part. 5. Comparison of astragaloside contents between the top and subterranean parts of astragalus plants according to time of cultivation Comparison between the total astragaloside contents of the top and subterranean parts of 2 year-, 3 year-, and 5 378

8 김승현 전윤미 임주진 김성협 정일민 김은혜 year-cultivated astragalus plants was performed as follows (Fig. 5-A, -B). Total astragaloside content in 2 year-cultivated astragalus was 1378 μg g -1, and this value was the astragaloside content of the top part minus that of the subterranean part. The gap in astragaloside content between the top and subterranean parts in 2 year-cultivated astragalus was the highest in astragaloside (1416 μg g -1 ). Total astragaloside content in 3 year-cultivated astragalus was 191 μg g -1, and this value was the astragaloside content of the top part minus that of the subterranean part. The gap in astragaloside content between the top and subterranean parts in 3 year-cultivated astragalus was the highest in astragaloside (305 μg g -1 ). Total astragaloside content in 5 year-cultivated astragalus was 41.2 μg g -1, and this value was the astragaloside content of the top part minus that of the subterranean part. The gap in astragaloside content between the top and subterranean parts in 5 year-cultivated astragalus was the highest in astragaloside (37.9 μg g -1 ). As time of cultivation increased, the astragaloside content of the top part decreased while that of the subterranean part increased. 6. Comparison of astragaloside contents between the top and lateral roots of astragalus Comparison between the total astragaloside contents of the top and subterranean roots of 2 year-, 3 year-, and 5 year-cultivated astragalus plants (Fig. 6-A, -B). Total astragaloside content in 2 year-cultivated astragalus was 1955 μg g -1, and this value was the phenolic content of the top root minus that of the lateral root. The gap in astragaloside content between the top and lateral roots in 2 year-cultivated astragalus was the highest in astragaloside (1849 μg g -1 ). Total astragaloside content in 3 year-cultivated astragalus was 514 μg g -1, and this value was the phenolic content of the top root minus that of the lateral root. The gap in astragaloside content between the top and lateral roots in 3 year-cultivated astragalus was the highest in astragaloside (420 μg g -1 ). Total astragaloside content in 5 year-cultivated astragalus was 33.4 μg g -1, and this value was the phenolic content of the top root minus that of the lateral root. The gap in astragaloside content between the top and lateral roots in 5 yearcultivated astragalus was the highest in astragaloside (25.9 μg g -1 ). ACKNOWLEDGEMENTS This work was supported by a grant from the Next- Generation BioGreen 21 Program (The National Center for GM Crops No. PJ008322), Rural Development Administration, Republic of Korea. LITERATURE CITED Attele AS, Wu JA and Yuan CS. (1999). Commentary: Ginseng pharmacology. Multiple constituents and multiple actions. Biochemical Pharmacology. 58: Bedir E, Pugh N, Calis I, Pasco DS and Khan IA. (2000). Effects of triterpene saponins from Astragalus species on in vitro cytokine release. Biological and Pharmaceutical Bulletin. 23: Cui R, He JC, Wang BF, Zhang GY and Chen SY. (2003). Suppressive effect of Astragalus membranaceus Bunge on chemical hepatocarcinogenesis in rats. Cancer Chemotherapy and Pharmacology. 51: Foster S and Yue CX. (1992). Herbal emissaries: Bringing Chinese herbs to the west. Healing Art Press. Rochester. New York, USA. p Gui SY, Wei W, Wang H, Wu L, Sun WY, Chen WB and Wu CY. (2006). Effects and mechanisms of crude astragalosides fraction on liver fibrosis in rats. Journal of Enthnopharmacology. 103: Hirotami M, Zhou Y. Lui H and Furuya T. (1994). Cycloartane triterpene glycosides from the hairyroot cultures of Astragalus membranaceus. Phytochemistry. 37: Kim DH, Park HW, Park CG, Sung JS and Seong NS. (2008). Effect of gamma irradiation on the germination and growth of Astragalus membranaceus. Korean Journal of Medicinal Crop Science. 16: Kwon HJ and Park YD. (2012). Determination of astragalin and astragaloside content in Radix Astragali using hughperformance liquid chromatography coupled with pulsed amperometric detection. Journal of Chromatography A. 1232: Lee JC, Jung HN, Kim JS, Woo WH and Jeong WY. (2003). Selective priming of Th1-mediated antigen-specific immune responses following oral administration of mixed prescriptions of traditional Korean medicines. Clinica Chimica Acta. 329: Lindsey K, Pullen ML and Topping JF. (2003). Importance of plant sterols in pattern formation and hormone signalling. Trends in Plant Science. 8: Liston A and Wheeler JA. (1994). The phylogenetic position of the genus Astragalus(Fabaceae): Evidence from the chloroplast gene rpoc 1 and C 2. Biochemical Systematics and Ecology. 22: Podlech D. (1986). Taxonomic and phytogeographical problems in Astragalus of the old world and southwest asia. Proceedings of the Royal Society of Edinbergh. 89: Rios JL and Waterman PG. (1997). A review of the 379

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