以極致液相層析/串聯質譜術定量朱紅密孔菌之活性成分與保健食品中微量化學物質

Determination of Bioactive Compounds in Pycnoporus sanguineus and Trace Chemicals in Nutraceuticals Using Ultra-performance Liquid Chromatography/Tandem Mass Spectrometry

莊鈞賀(Chun-Ho Chuang)

指導教授 ： 陳家揚

國立臺灣大學/公共衛生學院/食品安全與健康研究所/碩士(2022年)

https://doi.org/10.6342/NTU202204007

摘要

Part 1. Determination of Bioactive Compounds in Pycnoporus sanguineus
朱紅密孔菌 (Pycnoporus sanguineus) 為多孔密科密孔菌屬之白腐真菌，普遍生長於熱帶及亞熱帶地區中低海拔之森林，如：澳洲和台灣等，生長季節為春夏之間。白腐真菌可以分泌蟲漆酶 (laccase) 分解植物中的木質素及纖維素，因而常應用於造紙工業、染料廢水脫色處理及環境汙染物降解等。朱紅密孔菌在藥理上有抗發炎、抗腫瘤、抗氧化及降低血脂等效果顯著，在醫藥保健領域具有相當的潛力。在過往研究中尚無朱紅密孔菌之定量分析，本研究以其在生物分類上相似之品種–靈芝 (Ganoderma lucidum) 作為參考，其含有豐富三萜類成分與植物固醇，定量台灣地區野生之朱紅密孔菌中活性成分，包含靈芝酸A (ganoderic acid A)、赤芝酸B (lucidenic acid B)、檞皮素 (Quercetin) 及麥角固醇 (ergostetol)，樣本數量共計14種菌株。
前處理方法為均質自然風乾之菌株子實體後，取50 mg粉末至1.5 mL eppendorf中，加入0.5 mL甲醇以超音波震盪萃取50分鐘。萃取完成後至離心機以4000 rpm (2867 g-force) 離心3分鐘，取上清液通過0.2 μm nylon過濾膜，次等量甲醇稀釋後上機分析，最終樣本體積為0.4 mL。分析麥角固醇則是取 50 μL上清液以甲醇稀釋100倍後上機分析。
樣本以Waters I-Class PLUS極致液相層析 (ultra-performance liquid chromatography, UPLC) 搭配Waters Xevo TQ-XS串聯式質譜儀，於多重反應偵測模式 (multiple reaction monitoring; MRM) 進行定量分析。靈芝酸A、赤芝酸B及檞皮素使用Unispray游離源，以Waters CORTECS (30 × 2.1 mm, 1.6 μm) 層析液相管柱分離，管柱溫度為45℃，有機移動相為甲醇，水性移動相為0.1%醋酸水溶液 (pH 3.26)，梯度流析流速0.5 mL/min，總層析時間為5分鐘。麥角固醇使用大氣壓力化學游離法(atmospheric pressure chemical ionization; APCI) 游離，以Waters CORTECS (30 × 3.0 mm, 1.6 μm) 層析液相管柱分離，管柱溫度為45℃，有機移動相為甲醇，水性移動相使用Milli-Q純水(pH 7.0)，梯度流析流速1.0 mL/min，總層析時間為4.5分鐘。定量分析之標準品檢量線線性範圍在 2-500 ng/mL，判定係數R2皆大於0.99以上，儀器偵測極限0.04-1.14 pg，儀器定量極限0.13-3.84 pg。樣本偵測極限60.8-61.9 pg/g，樣本定量極限202-204 pg/g。
定量結果顯示朱紅密孔菌中含有麥角固醇，濃度因不同菌株介於450至2500 μg/g，與靈芝與香菇相比高出一至二倍，靈芝酸A及赤芝酸B則是低於偵測極限。靈芝酸A在靈芝樣本中檢出濃度介於4.32至841 μg/g。本研究初步定量朱紅密孔菌中之活性成分，未來能針對其他活性物質進行研究，以得到更完整之成分分析結果。又其生長環境簡單，可於野外採集而得，且人工栽培已在開發階段，未來能使產率與品質大幅提升，可做為保健食品或是中藥材使用。
Part 2. Determination of Trace Chemicals in Nutraceuticals
隨著世界各國醫療資源的進步及人口老化加速，「保健食品」備受重視，市面上相關產品種類繁多，民眾對其需求量也逐年增加。在台灣，「保健食品」除了《健康食品管理法》認證之「健康食品」外，亦涵蓋許多機能性食品及營養補充劑，而此類產品在製程中可能因為機器設備汙染，或是原物料受環境汙染等因素，造成最終產品中有天然毒素、殺蟲劑、農藥、加工附劑或環境汙染物之殘留，經人體攝入後造成健康危害。本研究將針對台灣市售之「保健食品」進行化學參雜物定量分析，並探討其健康風險。
本實驗室在先前研究已針對保健食品前處理方式優化，以QuEChER搭配Oasis PRiME HLB萃取匣作為樣本前本處方式，並藉由超高效液相層析飛行時間串聯式質譜儀 (UHPLC-QTOF/MS) 將市售53種保健食品樣本進行篩查，與6個資料庫包含Agilent建立之藥物與毒物資料庫 (Forsenic and Toxicology)、黴菌毒素資料庫 (Mycotoxins)、農藥資料庫 (Pesticides)、畜產用藥資料庫 (Veterinary)、水體環境新興汙染物資料庫 (Water) 及本研究團隊自建高度灌注物質資料庫 (Concerned Chemicals)共計3376種化學物比對，後續以ToxPi根據檢出頻率、訊號強度、人體或實驗動物代謝半衰期、毒性或危害性(LD50、oral reference dose )等排序優先定量之化學物。共計12種化學物包含己二酸二辛酯 (Di(2-ethylhexyl)adipate; DEHA), 鄰苯二甲酸二（2-乙基己基）酯 (Bis(2-ethylhexyl)phathalate; DEHP)、咖啡因 (caffeine)、洛伐他汀 (lovastatin)、毒藜鹼 (anabasine)、地西泮 (diazepam)、阿糖腺苷 (vidarabine)、水楊酸 (salicylic acid)、龍膽酸 (gentisic acid)、棒麴黴素 (patulin)、益必寧 (empenthrin) 及3,4,5,6四氫苯酐 (3,4,5,6 tetrahydrophathlic anhydride) 於本研究定量分析。
前處理方法使用QuEChERS (quick, easy, cheap, effective, rugged and safe)，固體樣本均質後取1 g 與5 mL 水混和，而液體樣本取5 mL使用。以10 mL 乙腈作為溶劑，劇烈搖晃後再加入Extran 7萃取粉劑。離心後取5 mL上清液通過Oasis PRiME HLB萃取匣淨化，最後濃縮至1 mL 過濾後上機分析。
本研究方法使用Waters I-Class PLUS極致液相層析 (ultra-performance liquid chromatography, UPLC) 搭配Waters Xevo TQ-XS串聯式質譜儀，以多重反應偵測模式 (multiple reaction monitoring; MRM) 進行定量分析。游離源使用Unispray，以Lunar Omega C18 (30 × 2.1 mm, 1.6 μm) 層析液相管柱分離，管柱溫度為40℃，正電模式下有機移動相為甲醇，水性移動相使用5 mM醋酸胺水溶液 (pH 6.45)，梯度流析流速0.5 mL/min，總層析時間為8分鐘。負電模式有機移動相為含0.04% 醋酸之甲醇 (pH 3.50)，水性移動相使用0.04%醋酸水溶液 (pH 3.45)，總層析時間為7分鐘。定量分析之標準品回歸線線性範圍在 2-500 ng/mL，判定係數R2皆大於0.99以上，儀器偵測極限0.01-8.71 pg，儀器定量極限0.02-29.1 pg；樣本偵測極限0.02-4.46 ng/g，樣本定量極限0.07-2.67 ng/g。
定量結果顯示，少量殺蟲劑如：3,4,5,6四氫苯酐殘留於樣本中，濃度介於50.9至861 ng/g，低於殘留標準，視為低健康風險。大豆精華之DEHP濃度為1625 ng/g，以60公斤成人每日攝取量計算，超過我國規定之每日耐受量 (tolerable daily intake, TDI)參考值：0.05 mg/kg BW/day。地西泮於少量樣本如：龜鹿飲與芝麻錠中殘留，濃度介於10.5至32.1 ng/g，遠低於有效劑量亦視為低健康風險。內生性物質如咖啡因或洛伐他汀等，在產品中皆由天然來源所貢獻。綜觀以上，市售「保健食品」雖有微量化學物殘留，其濃度皆低於限量標準，表示健康風險危害低。而塑化劑在現今被大量使用，於此結果中也有一項超標情形，未來針對塑化劑之殘留與汙染需多加留意。

關鍵字

朱紅密孔菌 ； 活性成分 ； 極致液相層析/串聯質譜術 ； 定量 ； 保健食品 ； 微量化學物質

並列摘要

Part 1. Determination of Bioactive Compounds in Pycnoporus sanguineus
Pycnoporus sanguineus is a kind of white rot fungus, and distributes in subtropical and tropical regions, e.g., Australia and Taiwan. It features in producing laccase, a phenoloxidase, which degrades lignin and cellulose and is so widely used in the paper industry or textile industry. Furthermore, researchers have demonstrated that Pycnoporus sanguineus is capable of various bioactivities, including anti-inflammation, anti-tumor, or anti-oxidation. Because of the similarity in taxonomy, Ganoderma lucidum was taken as a reference for bioactive compounds, with abundant content of triterpene and pytosterol. This study developed a method for quantification of four chemicals, including ganoderic acid A, lucidenic acid B, quercetin and ergosterol.
For sample preparation, natural dried fruiting bodies and mycelia were homogenized into powder. 50 mg powder was mixed with 0.5 mL methanol in the 1.5 mL eppendorf. After sonication for 50 minutes, samples were centrifugation at 4000 rpm (2867 g-force) for 3 minutes, and the supernatant was collected and passed through a 0.2 μm nylon filter. For analysis of ganoderic acid A, lucidenic acid B and quercetin, the extract was diluted by equivalent volume of methanol, with final volume of 0.4 mL; for analysis of ergosterol, 50 μL of the extract was diluted 100 times of methanol.
The method was developed using ultra-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Ganoderic acid A, lucidenic acid B and quercetin were analyzed by Unispray ionization. Waters CORTECS C18 column (30 × 2.1 mm, 1.6 μm) was used for separation with 0.1% acetic acid(aq) (pH 3.35) and methanol as mobile phase. The flow rate was 0.5 mL/min, and the column temperature was set at 45℃. Ergosterol was ionized by atmospheric pressure chemical ionization (APCI) and Waters CORTECS C18 column (30 × 3.0 mm, 1.6 μm) was used for separation with Milli-Q water and methanol as mobile phase. The flow rate was 1.0 mL/min and the column temperature was set at 45℃. The linear ranges of all chemicals were from 2 ng/mL to 500 ng/mL, with a coefficient of determination higher than 0.99. The limits of instrumental detection (IDL) were between 0.04 pg and 1.14 pg and the limits of instrumental quantification (IQL) were between 0.13 pg and 3.84 pg. The limits of detection (LOD) were between 60.8 pg/g and 61.9 pg/g, and the limits of quantification (LOQ) were between 202 pg/g and 204 pg/g.
Concentration of ergosterol in Pycnoporus sanguineus were between 450 to 2500 μg/g, which were one to two times higer than those in Ganoderma lucidum and mushrooms in this study. Ganoderic acid A and lucidenic acid B were not detectable in Pycnoporus sanguineus. Concentrations of Ganoderic acid A in Ganoderma lucidum were between 4.32 to 841 μg/g. Pycnoporus sanguineus have specific pytosterol compounds, which can be potential in traditional Chinese medicine or food supplements. In addition, it can grow in an austere environment, and artificial cultivation has also been developed, which will promote the production and quality of ergosterol.
Part 2. Determination of Trace Chemicals in Nutraceuticals
Nutraceuticals have garnered more and more attention from people in recent years because of promotions of medical and increasing of aging. There are various types of nutraceuticals and the market potential is expanding. However, some chemical contaminants or adulterants may exist in commercial products from materials or procedures, including natural toxins, pesticides or environmental pollutants, and health risks are concerned. This study determined chemical contaminants in nutraceuticals and discussed the potential risks.
Previous studies from our team had optimized the sample preparation of nutraceuticals, using QuEChERS with Oasis PRiME HLB cartridge, and operated suspect screening of chemicals in nutraceuticals by ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry. The mass spectra of peaks in samples were compared with six libraries, including five libraries (Forsenic and Toxicology, Mycotoxins, Pesticides, Water, Veterinary) provided by Agilent and one library (Concerned Chemicals) developed from our lab. The results were prioritized by detectable rates, signal intensities, metabolic half-life and LD50, and 12 chemicals were decided for quantification, including Di(2-ethylhexyl)adipate (DEHA), Bis(2-ethylhexyl)phthalate, (DEHP), caffeine, lovastatin, anabasine, diazepam, vidarabine, salicylic acid, gentisic acid, patulin, empenthrin, and 3,4,5,6 tetrahydrophthalic anhydride.
For sample preparation, 1 g solid samples and 5 mL Milli-Q water were mixed, and 5 mL of liquid sample were directly used. 10 mL of acetonitrile were added to the centrifuge tube and vortexed, and then a pack of Extran 7 was added to the tube. After centrifugation, 5 mL of supernatant from each sample was collected for cleaning up through Oasis PRiME HLB. Extracts were concentrated to 1 mL and passed through a 0.2 μm nylon filter before the analysis.
The method was developed using ultra performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and ionized by Unispray ionization. Lunar Omega C18 (30 × 2.1 mm, 1.6 μm) was used for separation with mobile phase consisted of (A) 5 mM ammonium acetate(aq) (pH 6.45) and (B) methanol for positive mode and (A) 0.04% acetic acid(aq) (pH 3.45) and (B) methanol with 0.04% acetic acid (pH 3.50) for negative mode. The flow rate was 0.5 mL/min, and the column temperature was 40℃. The linear ranges of all chemicals were from 2 ng/mL to 500 ng/mL, with a coefficient of determination higher than 0.99. The IDLs were from 0.01 pg to 8.71 pg and IQLs were from 0.02 pg to 29.1 pg. The LODs were from 0.02 to 4.46 ng/g, and LOQs were from 0.07 to 2.67 ng/g.
The results revealed that low concentrations of 3,4,5,6 tetrahydrophthalic anhydride were detected in samples, with concentrations from 50.9 to 861 ng/g. Diazepam was detected in some samples such as Turtle Deer Drink and Sesame Seed Extract, with concentrations from 10.5 to 32.1 ng/g which were lower than dosages and was considered as low healthy risk. The concentration of DEHP in Soy Extract was 1.62 μg/g. For a 60 kg human with daily intake of Soy Extract, DEHP was higher than TDI from the TFDA. Endogenous chemicals such as caffeine or lovastatin were from natural sources in samples. To sum up, most nutraceuticals had low residues, which were considered as low healthy risk. While plasticizer have been heavily used, it is important to focus on its contamination and healthy risk.

並列關鍵字

Pycnoporus sanguineus ； bioactive compounds ； ultra-performance liquid chromatography/mass spectrometry ； quantification ； nutraceuticals ； trace chemicals

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