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1 微生物学通报 Microbiology China Jan. 20, 207, 44(): DOI: /j.microbiol.china.602 生物实验室高效液相色谱法测定不同产地及品种灵芝三萜类成分的含量贾红岩,2 王亚涛,2 张芝华,3 冯娜刘艳芳周帅张忠张劲松 ( ) ( ) ( ) 唐庆九 * 摘要 : 目的建立同时测定灵芝子实体中灵芝酸 C 2 灵芝酸 B 灵芝酸 A 灵芝酮三醇灵芝酸 DM 灵芝酸 T 灵芝醇 B 灵芝酸 S 灵芝酸 G 灵芝酸 F 灵芝酸 D 灵芝稀酸 B 2 种三萜成分的高效液相色谱法方法选用 Aglient ZORBAX SB-Aq (4.6 mm 250 mm,5 μm) 色谱柱, 以乙腈 - 醋酸 (0.0%) 水溶液为流动相进行梯度洗脱, 流速.0 ml/min, 分析波长 252 nm, 柱温 30 C, 对不同产地及品种的灵芝样品进行测定分析结果同一产地不同品种间三萜类成分含量具有显著差异, 不同产地的灵芝样品三萜成分含量也有显著差异, 所建方法精密度高, 稳定性和重复性好结论所建方法适用于 2 种三萜成分的同时测定关键词 : 灵芝, 高效液相色谱法, 三萜类化合物 Determination of triterpenoids in Ganodema lingzhi from different areas and species by HPLC JIA Hong-Yan,2 WANG Ya-Tao,2 ZHANG Zhi-Hua,3 FENG Na LIU Yan-Fang ZHOU Shuai ZHANG Zhong ZHANG Jing-Song TANG Qing-Jiu * (. National Engineering Research Center of Edible Fungi, Key Laboratory of Edible Fungi Resources and Utilization(South), Ministry of Agriculture, China, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai 20403, China) (2. College of Food Science, Shanghai Ocean University, Shanghai 20306, China) (3. Shanghai Institute of Technology, Shanghai 2048, China) Abstract: [Objective] We established an HPLC (High Performance Liquid Chromatography) method to determine Ganoderic acid C 2, Ganoderic acid B, Ganoderic acid A, Ganodermanontriol, Ganoderic acid DM, Ganoderic acid T, Ganoderiol B, Ganoderic acid S, Ganoderic acid G, Ganoderic acid F, Ganoderic acid D, Ganoderenic acid B in Ganodema lingzhi. [Methods] Aglient ZORBAX SB-Aq (4.6 mm 250 mm, 5 μm) was adopted as the chromatographic column, with Foundation item: Key Technologies Research & Development Program of China (No. 203BAD6B08-02) *Corresponding author: Tel: ; tangqingjiu@saas.sh.cn Received: January 29, 206; Accepted: April 4, 206; Published online ( May 04, 206 基金项目 (No. 203BAD6B08-02) * 通讯作者 :Tel tangqingjiu@saas.sh.cn 收稿日期 : ; 接受日期 : ; 优先数字出版日期 (

2 : 239 acetonitrile-0.0% acetic acid solution using gradient elution at 30 o C with a flow rate of ml/min, and the UV detection wavelength was set at 252 nm. Composition and content of different varieties of triterpenoids from different areas were determined by the above designed method. [Results] Triterpenoids in different varieties of Ganodema lingzhi in one area and samples from different areas were significantly varied in their compositions and contents; the method was sensitive, reliable and reproducible. [Conclusion] This method can be used to determine triterpenoids. Keywords: Ganodema lingzhi, HPLC, Triterpenoids (Ganodema lingzhi) Ganodema lingzhi Sheng H. Wu, Y. Cao & Y.C. Dai [-2] [3] [4-5] [6] [7-8] A B 982 [9] [0-] [2-3] [4-20] HPLC HPLC [20] C 2 HPLC [4] [2] C 2 G A [6] 9 NY/SJ NY/T NY/SJ NY/T A B HPLC 2 材料与方法. 材料 (FJ) 9 (ZJ) (AH) (JL-) (JL-2) 2 (JL-3).2 主要试剂 C 2 B A DM T B S G F D B Sigma Dikma.3 主要仪器 Waters 600 Waters 77 plus Waters 2996 Empower Waters KQ-600B ELGA PURELAB Ultra ( ) 545D

3 240 微生物学通报 Microbiol. China 207, Vol.44, No. Eppendorf MS05DU Metter Toledo.4 实验方法.4. 高效液相色谱条件 : Agilent ZORBAX SB-Aq (4.6 mm 250 mm 5 μm) - (0.0%).0 ml/min 90 nm 400 nm 3D 30 C 20 μl 252 nm (A)- (0.0%)(B) 0 25 min A 28% 30% min A 30% 39% min A 39% 60% min A 60% 00% min A 00% 00 0 min A 00% 28% 0 0 min A 28%.4.2 标准品溶液的制备 :() 2 C 2 B A GT-2 DM T B S G F D B g/l (2) 25 ml C 2 (00 mg/l) B (00 mg/l) A (00 mg/l) GT-2 (05 mg/l) DM (00 mg/l) T (95 mg/l) B (00 mg/l) S (05 mg/l) G (2 mg/l) F (30 mg/l) D (06 mg/l) B (00 mg/l) 样品溶液的制备 : 0.5 g 0 ml 0 ml ( 600 W 40 khz) h 2.0 ml r/min 2 min 0.22 μm HPLC 2 结果与分析 2. 三萜类标准样品的定性分析.4.2(2) ( ) 2 C min G min B min B min A min D min F min min DM min S min T min B min 2.2 方法学验证 2.2. 线性关系考察 :.4.2(2) 6 2. HPLC X Y 精密度实验 :.4.2(2) 6 RSD 重复性实验 : RSD h.4. RSD 2 h

4 : 24 图三萜类标准品混合组分与样品的 HPLC 图谱 Figure Chromatogram of mixture of triterpenoids standards and tested sample A B. C 2 2 G 3 B 4 B 5 A 6 D 7 F 8 9 DM 0 S T 2 B. Note: A: Standard; B: Sample. : Ganoderic acid C 2 ; 2: Ganoderic acid G; 3: Ganoderenic acid B; 4: Ganoderic acid B; 5: Ganoderic acid A; 6: Ganoderic acid D; 7: Ganoderic acid F; 8: Ganodermanontriol; 9: Ganoderic acid DM; 0: Ganoderic acid S; : Ganoderic acid T; 2: Ganoderiol B. 表不同三萜类成分的方法学数据 Table Data of methodology of different triterpenoids Standard samples Regressive equations Correlative coefficient Linear bounds (mg/l) C 2 Ganoderic acid C 2 Y=.36e+004X 7.82e G Ganoderic acid G Y=.58e+004X.26e B Ganoderenic acid B Y=3.02e+004X.7e B Ganoderic acid B Y=.9e+004X.37e A Ganoderic acid A Y=.43e+004X 9.73e D Ganoderic acid D Y=.8e+004X 5.2e F Ganoderic acid F Y=.54e+004X 4.2e Ganodermanontriol Y=2.6e+004X 2.04e DM Ganoderic acid DM Y=.63e+004X 2.54e S Ganoderic acid S Y=.88e+004X 4.23e T Ganoderic acid T Y=2.7e+004X 2.25e B Ganoderiol B Y=.49e+004X.33e

5 242 微生物学通报 Microbiol. China 207, Vol.44, No 加样回收率实验 : HPLC 样品含量测定.4.3 HPLC A FJ ZJ AH JL- A 表 2 三萜类成分加样回收率实验结果 Table 2 Determination on recoveries of triterpenoids Triterpenoids Precision (%) Repeatability (%) Stability (%) Recovery (%) RSD (%) C 2 Ganoderic acid C G Ganoderic acid G B Ganoderenic acid B B Ganoderic acid B A Ganoderic acid A D Ganoderic acid D F Ganoderic acid F Ganodermanontriol DM Ganoderic acid DM S Ganoderic acid S T Ganoderic acid T B Ganoderiol B 表 3 不同产地灵芝子实体中三萜类成分含量测定 Table 3 Determination of triterpenoids in G. lingzhi from different areas (μg/g) Sample FJ ZJ AH JL- JL-2 JL-3 C 2 Ganoderic acid C ± ± ± ±2.92 G Ganoderic acid G ± ± ± ±2.97 B Ganoderenic acid B 80.78± ± ± ±0.44 B Ganoderic acid B ± ± ± ±0.66 A Ganoderic acid A 76.97± ± ± ± ±0.96 _ D Ganoderic acid D 926.9± ± ± ±0.57 F Ganoderic acid F 59.44± ± ± ± ± ±0.59 Ganodermanontriol ± ± ± ± ± ±0.08 DM Ganoderic acid DM 64.94± ± ± ± ± ±.09 S Ganoderic acid S 68.79± ± ± ± ± ±0.7 T Ganoderic acid T 5.98± ± ±. 5.47±.02.56± ±0.39 B Ganoderiol B 36.3± ± ± ± ± ±0.8 Total amount ± ± ± ± ± ±0.09. Note: All results are means of three determinations; : Not detected.

6 : 243 图 2 不同产地灵芝子实体的 HPLC 图谱 Figure 2 Chromatogram of the G. lingzhi from different areas 23.69% 39.06% 33.78% 28.25% F DM S T B JL-2 A 3 讨论 D F HPLC [22] 3 C 2 B A GT-2 DM T B S G F D B 参考文献 [] Dai YC, Cao Y, Zhou LW, et al. Notes on the nomenclature of the most widely cultivated Ganoderma species in China[J]. Mycosystema, 203, 32(6): (in Chinese),,,. [J]., 203, 32(6): [2] Cao Y, Wu SH, Dai YC. Species clarification of the prize medicinal Ganoderma mushroom Lingzhi [J]. Fungal Diversity, 202, 56(): [3] Shao LP, Shen RX, Zhang SX, et al. Fungus Taxonomy[M]. Beijing: Forestry, 983 (in Chinese),,,. [M]. :, 983 [4] Zhang XY, Yang CQ. The chemical composition and pharmacological action of Ganoderma lucidum[j]. Foreign Medical Sciences (Herbal Drug), 2006, 2(4): (in Chinese),. [J]. ( ), 2006, 2(4): [5] Feng DJ. The chemical composition, efficacy and pharmacological action of Ganoderma lucidum[j]. Special Economic Animal and Plant, 2006, 9(8): (in Chinese). [J]., 2006, 9(8): [6] Gan WW, Chang JD. Study on extraction of polysaccharides from Ganoderma lucidum using response surface methodology[j]. Jiangsu Agricultural Sciences, 2009(): (in Chinese),. [J]., 2009(): [7] Lin ZB. Modern Research of Ganoderma Lucidum[M]. 3rd Edition. Beijing: Medical Publishing House of Peking University, 2007: (in Chinese)

7 244 微生物学通报 Microbiol. China 207, Vol.44, No.. [M]. 3. :, 2007: [8] Chen RY, Yu DQ. Advances in studies on chemical constituents on Ganoderma triterpene[j]. Acta Pharmaceutica Sinica, 990, 25(2): (in Chinese),. [J]., 990, 25(2): [9] Kubota T, Asaka Y, Miura I, et al. Structures of ganoderic acid A and B, two new lanostane type bitter triterpenes from Ganoderma lucidum (Fr.) Karst[J]. Helvetica Chimica Acta, 982, 65(2): 6-69 [0] Huang YJ, Xiao GL. The progress of pharmacology on ganoderma triterpene[j]. Cuiding Journal of Traditional Chinese Medicine and Pharmacy, 2008, 4(9): 87-88,97 (in Chinese),. [J]., 2008, 4(9): 87-88,97 [] Xu JW, Zhao W, Zhong JJ. Biotechnological production and application of ganoderic acids[j]. Applied Microbiology and Biotechnology, 200, 87(2): [2] Fu LZ, Wu XQ, Li MY, et al. Crude polysaccharide and triterpene content of ganoderma fruit bodies at different stages of development[j]. Acta Edulis Fungi, 2008, 5(3): (in Chinese),,,. [J]., 2008, 5(3): [3] Fu LZ, Wu XQ, Li MY, et al. Analysis and evaluation of polysaccharides and triterpenes contents of fruitbody of Ganoderma lucidum strains[j]. Edible Fungi of China, 2009, 28(4): (in Chinese),,,. [J]., 2009, 28(4): [4] Dong HL, Xia GP, Zhao NX, et al. Quantitative analysis of ganoderic acid C 2, G, and A in sporophore and spore powder of Ganoderma by HPLC[J]. Drugs & Clinic, 203, 28(): 4-43 (in Chinese),,,. HPLC C 2 G A[J]., 203, 28(): 4-43 [5] Xing ZT, Yu QH, Zhang JS, et al. Comparative study on triterpenes in different Ganoderma species[j]. Journal of Chinese Medicinal Materials, 2004, 27(8): (in Chinese),,,. [J]., 2004, 27(8): [6] Li BM, Gu HF, Li Y, et al. Determination of nine triterpenoid acids from Ganoderma lucidum of different producting areas by HPLC[J]. China Journal of Chinese Materia Medica, 202, 37(23): (in Chinese),,,. HPLC 9 [J]., 202, 37(23): [7] Ma L, Wu F, Chen RY. Analysis of triterpene constituents from Ganoderma lucidum[j]. Acta Pharmaceutica Sinica, 2003, 38(): (in Chinese),,. [J]., 2003, 38(): [8] Wang XM, Yang M, Guan SH, et al. Quantitative determination of six major triterpenoids in Ganoderma lucidum and related species by high performance liquid chromatography[j]. Journal of Pharmaceutical and Biomedical Analysis, 2006, 4(3): [9] Li BM, Liu C, Wang HQ, et al. Study on determination method of total triterpen acid content of Ganoderma lucidum[j]. China Journal of Chinese Materia Medica, 2007, 32(2): (in Chinese),,,. [J]., 2007, 32(2): [20] Wang XJ, Lin HB, Xu J, et al. Determination of concentrations of triterpene constituents and polysaccharides in Ganoderma by HPLC and UV-VIS[J]. Contemporary Medicine, 2009, 5(3): (in Chinese),,,. HPLC UV-VIS [J]., 2009, 5(3): [2] Yao SJ. Study on technology of ultrasonic extraction of triterpenoids from Ganoderma lucidum and its fingerprint method[d]. Guangzhou: Master s Thesis of South China University of Technology, 200: (in Chinese). [D]. :, 200: [22] Zhang SL. Studies on active ingredients and fingerprint of Ganoderma[D]. Hefei: Master s Thesis of Anhui University of Chinese Medicine, 203: 3-43 (in Chinese). [D]. :, 203: 3-43

g ml 10% ph 色谱条件 kinetex C μm 100 A 4. 6 mm 150 mm 25 5 μl A B 10 mmol /L ml /min

g ml 10% ph 色谱条件 kinetex C μm 100 A 4. 6 mm 150 mm 25 5 μl A B 10 mmol /L ml /min 25 4 2013 8 监测技术 5 1 2 1 ( 1. 江苏省疾病预防控制中心, 江苏南京 210009; 2. 南京医科大学第二附属医院, 江苏南京 210029) 5 Phenomenex kinetex C 18 2. 6 μm 4. 6 mm 150 mm 10 mmol /L ph 2. 5 + 60 40 DAD 280 nm 5 20. 0 mg /L ~ 100 mg /L 0.