常用烏頭屬藥材及相關製劑

之藥理與毒理活性評估

研究生 賴美州

中國醫藥大學 藥學院 中國藥學研究所

 摘要

烏頭屬植物為毛茛科多年生草本植物，具有毒性，需經炮製，以降低毒性、增進療效，使得藥物在臨床使用上更加安全。烏頭屬植物中的川烏、草烏、天雄及附子為常用之中藥。在中國及日本許多傳統中藥方劑中，所使用Aconitum屬植物，多廣泛在鎮痛、抗發炎、降血糖和神經傳導方面的應用。因此，在本研究中，針對常用之炮製後的川烏、草烏、天雄及附子等中藥，進行藥理、毒理、成分分析及複方應用之探討。

 藥理部分包括：鎮痛、抗發炎、抗氧化及降血糖等作用之評估。毒理部分包括：急性毒性試驗、慢性毒性評估及組織切片檢查。並將上述所得結果，利用DIO(diet-induced
obesity)所誘導的第2型糖尿病的大鼠，應用在含有烏頭屬的中藥方劑-八味地黃丸、六味地黃丸、肉桂及天雄，單獨或合併的給藥方式，進行評估。

首先，本研究以醋酸扭體法、甩尾試驗及福馬林疼痛試驗法，探討川烏、草烏、天雄及附子粗抽物之鎮痛作用。利用λ-角叉菜膠誘導小鼠足蹠腫脹，來探討川烏、草烏的抗發炎作用。藉由測定小鼠肝臟組織之超氧歧化酶、麩胱甘肽還原酶及麩胱甘肽過氧化酶的活性；還有小鼠發炎足蹠組織中丙二醛的含量；及血清中腫瘤壞死因子含量變化，來探討川烏、草烏之粗萃物是否經由提升抗氧化酵素活性及抑制發炎性介質之含量進而達到抗發炎作用。抗氧化方面主要目的則在探討川烏、草烏、天雄及附子之體外抗氧化功能，包括DPPH染色法、清除DPPH清除自由基能力、Trolox當量、總抗氧化能力、還原力等方法，並進行總多酚類、類黃酮類和黃酮醇類成分之含量測定，以分析其抗氧化作用與總酚類、類黃酮類和黃酮醇類含量之間的關係。在降血糖的表現上，分別利用由streptozotocin（STZ）及diet-induced obesity (DIO)所誘導的第1及第2型糖尿病的大鼠，分別以口服方式給藥，觀察是否具有調降血糖的作用。

急性毒性試驗及慢性毒性試驗，則測試待測物可能產生之毒性影響，且提供更長期試驗劑量設定之依據。藉由尿液微量白蛋白 (urine albumin)及24小時尿微量白蛋白排泄率(urinary albumin excretion rate, UAER)數值的改變，同時配合離體腎臟的組織病理切片，評估大鼠腎細胞腫脹及傷害的程度。此外，運用免疫組織化學染色法(immunohistochemistry staining)觀察待測藥物對正常大鼠腎臟中的轉化生長因子(trans forming growth factor-β1,TGF-β1)表現量的影響。

結果發現：於鎮痛試驗中，常用的毛茛科烏頭屬植物(川烏、草烏、天雄及附子)均可減少由醋酸所引起疼痛之扭體次數。川烏及草烏能減少福馬林引起之前後期舔足反應時間；天雄及附子能減少福馬林引起之後期舔足反應時間。於抗發炎試驗中，川烏、草烏、天雄及附子 60mg/kg粗萃物可明顯抑制λ-carrageenan誘導的急性足蹠腫脹。

於肝組織抗氧化酵素活性測定中，川烏及草烏粗萃物對肝臟中之超氧岐化酶（superoxide dismutase,SOD）、麩胱甘肽還原酶（glutathoine
reductase,GR）、麩胱甘肽過氧化酶(glutathione
peroxidease,GPx)活性均具增強作用，並會減少小鼠發炎足蹠組織中丙二醛（malondialdehyde,

MDA）的含量。且川烏及草烏粗萃物亦會減少發炎後腫瘤壞死因子（Tumor Necrosis Factor-α,TNF-α）的含量。

 綜合上述結果顯示：常用的毛茛科烏頭屬植物(川烏、草烏、天雄及附子)具有鎮痛、抗發炎作用。其鎮痛效果與周邊抑制花生四烯酸代謝，減少前列腺素合成有關；而抗發炎作用機轉可能藉由提升肝臟中抗氧化酵素，而利於清除自由基；此外，亦藉由抑制發炎足蹠組織中MAD含量，來減少脂質過氧化；並抑制TNF-α的活性，進而減少前列腺素的生成，以達到抗發炎的作用。在清除DPPH自由基能力、Trolox當量總抗氧化能力、還原力等實驗評估上，以草烏均具有最好的抗氧化能力；川烏次之，而天雄、附子則較無抗氧化之活性。總酚類、類黃酮類和黃酮醇類含量之測定結果，亦顯示總酚類、類黃酮類和黃酮醇類含量以草烏最高，而川烏、天雄和附子等繼之。在降血糖表現上，毛茛科烏頭屬植物(川烏、草烏、天雄及附子)皆可降低血糖，在STZ（streptozotocin）及DIO(diet-induced obesity)所誘導的第1及第2型糖尿病的大鼠，分別以附子及天雄效果最佳。

在急性毒性試驗上，經炮製後的烏頭屬植物(川烏、草烏、天雄及附子)，其毒性均可降低。在慢性毒性試驗，烏頭屬(川烏、草烏、天雄及附子)藥材於中劑量(600 mg/kg/day)或是高劑量(3000 mg/kg/day)連續使用3個月後，大鼠即會出現腎損傷；尤其是以高劑量草烏(3000 mg/kg/day)處理的大鼠腎損傷的情況最為嚴重且其腎球體間質細胞明顯的擴大，同時腎臟中轉化生長因子(TGF-β₁)有過度表現的現象，而天雄引至腎臟傷害的能力最輕微。

在成分分析方面以aconitine、mesaconitine及hypaconitine為指標成分，以草烏的生物鹼含量最高，其次為川烏、天雄及附子。

將上述所得的結果，將毒性較低藥效較佳的天雄，應用在含有烏頭屬成分常用方劑-八味地黃丸、六味地黃丸、天雄及肉桂對DIO所誘導的第2型糖尿病的大鼠進行比較。六味地黃丸及八味地黃丸對第2型糖尿病大鼠的血糖、血脂及腎功能上有很好的改善效果。但單獨或合併使用天雄或肉桂，對第2型糖尿病的大鼠腎功能反而有害。

綜合本研究結果，烏頭屬植物在鎮痛、抗炎、降血糖之作用，具有一定之效果，未來應可開發作為臨床上輔助治療的藥物使用。

Evaluation of the Toxicity
and the Pharmacology of Aconitum Herbs

Mei-Chou Lai

Institute
of Chinese Pharmaceutical Sciences, College of Pharmacy

China Medical
University

Abstract

 The
Aconitum is a perennial plant that belongs to the Ranunculaceae family. It is a
poisonous plant. It should be prepared to reduce it toxicity for curative
effects and clinical safety. In China
and Japan,
for example, preparations of Aconitum
carmichaeli (Aconitum roots) are employed for their analgestic,
anti-inflammatory, hypoglycemia and neurological properties. Therefore, this
research aims to evaluate the pharmacology, the toxicity, and the ingredient
analysis of Aconitum, which is commonly used in Chinese herbal medicine—herbs
like Chuan Wu, Cao Wu,
Tian Syong and Fu Zih.

 The
pharmacology includes the analgesia, the anti-inflammation, the anti-oxidation
and the hypoglycemia. The toxicology part includes the acute toxicity test, the
chronic toxicity and biopsy smear. Type 2 DM rats which are applied by the
diet-induced obesity are tested with the traditional Chinese medicine—Baiwei
Duhuang pill, Liuwei Dihuang pill, cinnamon and the Tian Syong, all of which
belong to the monkshood. They can be applied to the rats respectively or in
combination.

 First,
with the acetic acid induced writhing, tail flick assay and formalin pain
tests, I study the analgesic effect of the extract of herbs—like Chuan Wu, Cao Wu, Tian Syong and Fu Zi—in
rats. With the λ-carrageenan-induced paw edema mechanism, the ant-inflammation
effect in rats of Chuan Wu, Cao and; similarly, SOD, GR and GPx in MDA levels
in mice livers can be analyzed. Through the analysis of the content change of
the tumor necrosis in the blood serum, I will study whether or not the
effectiveness of anti-inflammation function of the wide aconite thick gathering
varies comsumerate with the use of oxidation resistance enzyme and
inflammational medium. The study of the antioxidant activities is to analyze
vitro extracts of the Chuan Wu, Cao
Wu, Tian Syong and Fu Zih through the means of DPPH staining,
free radical scavenging capacity, Trolix equivalent antioxidant capacity (TEAC)
assay, total and antioxidant capacity and reducing power methods. In terms of
hypoglycemic activities, Type 1 and Type 2 rats, when tested with oral
medication and induced with streptozotocin (STZ) and diet-induced obesity
(DIO), the function of the lowering blood sugar can be thus observed.

 The
acute toxicity test and the chronic toxicity experiment can test the possible
effectiveness of the object tested and offer the basis of the long-term test
dosage hypothesis. The extent of kidney cell damage and swelling will assessed
by the levels of urine microalbumin and 24-hour urinary albumin excretion rate
(UAER), together with kidney biopsy smear. In addition, the influence of
medicine tested on the protein levels of transforming growth factor-β1 (TGF-β1) are determined by
immunohistochemistry staining.

The results have shown that the
commonly used Aconitum herbs—Chuan Wu, Cao Wu, Tian Syong and Fu Zih—significantly inhibit acetic
acid-induced writhing response. Chuan
Wu and Cao Wu can
significantly inhibit the formalin-induced licking time during the early phase
and late phase. Likewise, Tian Syong and Fu Zih significantly inhibit the formalin-induced
licking time in the late phase. In the anti-inflammation experiments, the
60mg/kg thick gatherings of Chuan Wu, Cao
Wu, Tian Syong, and Fu Zih can also significantly inhibit the
λ-carrageenan-induced paw edema.

In the liver
anti-oxidation enzyme activeness determination, Chuan Wu and Cao Wu can significantly increase
superoxide dismutase (SOD), glutathione reductase (GR) and glutathione
peroxidease (GPx) levels in livers, and decrease the malondialdehyde MDA level
in paw. And Chuan Wu and Cao Wu
also reduc tumor necrosis factor (TNF-α) content.

The results have
shown that the commonly used Aconitum herbs (Chuan Wu, Cao Wu, Tian Syong and Fu Zih)
have the analgesia and the anti-inflammation effects. Their analgestic effects
and peripheral suppressive arachidonic acid metabolism are related to the
reduction of the prostaglandin synthesis. By means of promoting the
anti-oxidation enzyme in livers, mechanism of anti-inflammation may increase
significantly antioxidant activities and can be conducive to the elimination of
free radicals. In addition, the mechanism can reduce the MDA level in paw
content and subsequently the lipid peroxidaton. TNF-α activities are
accordingly suppressed and the prostaglandin production reduced; however, the
anti-inflammation can be achieved. In terms of the scavenging of free radicals
and Trolox equivalent capacity (TEAC), Cao Wu has the best anti-oxidation effects in
experiment; Chuan Wu comes in second. However, Tian Syong and Fu Zih have the
least anti-oxidation effects. In terms of total polyphenols, flavonoids and
flavonols tests, Cao Wu
ranges the highest in containing the three elements tested, followed by Chuan
Wu, Tian Syong and Fu Zih. In terms of reducing blood sugar, the commonly used
Aconitum herbs (Chuan Wu, Cao Wu,
Tian Syong and Fu Zih) are all very effective. Type 1 and Type 2 rats with
STZ-induced diabetic and DIO-induced diabetic Fu Zih and Tian Syong are most
effective in this respect.

In acute toxicity
tests, the toxicity of the Aconitum herbs (Chuan Wu, Cao Wu, Tian Syong and Fu Zih) is
reduced after preparation. In chronic toxicity tests, rats who have received
repeated oral treatment at medium dose (600 mg/kg/day) and high dose (3000
mg/kg/day) for three months is tested with serious renal damage. Those rats
having received the highest dose (3000 mg/kg/day) suffer from the most renal
damage and the most enhanced degree of mesangial expansion and higher expression of TGF-ß_1.
In contrast, Tian Syong causes the most minor effect on
renal damage.

Aconitine,
mesaconitine, and hypaconitine are the three key indicators in the component
analysis. The highest concentration of alkaloids in Aconitum herbs is found in Cao Wu, followed by Chuan Wu,
Tian Syong and Fu Zih.

Results obtained from the analysis have
shown that Tian Syong with low toxicity but better drug efficacy can be applied
into commonly prepared medicines, such as Bawei Dihuang pills, Liuwei Dihuang
pills, Tian Syong pills and cinnamon pills. We apply these pills into the
testing of Type 2 rats with diabetes. We find that both Bawei Dihuang pills and
Liuwei Dihuang pills are effective in reducing blood sugar, blood fats and
boosting kidney functions. However, Tian Syong pills and cinnamon pills, when
applied in the same rats respectively or jointly, produce harmful results to
the rats’ kidney functions.

In summary, aconitum herbs show significant
antinociceptive, anti-inflammatory and hypocemia effects. They have great
potentials for further development of auxiliary drugs in clinical uses.