On screening various oriental herbs which have long been used as medicines to treat some cancer-like diseases we have found in our laboratory that the root extracts of $\mbox{\underline{Scutellaria}}$ $\mbox{\underline{baicalensis}}$ have strong inhibitory effect on mouse leukemia L1210 cells. The present investigation was planed to isolate and identify the cytotoxic principle. Furthermore, The substance and its analogues were synthesized to search a pharmacophoric moiety and obtain enough amount for the quantitative animal tests. The work for clarifying the cytotoxic mechanism was also carried out.
To isolate the cytotoxic principle the ether extract of the roots was chromatographed on silica gel and the fractions obtained were tested on the L1210 cells. By repeated chromatography and crystallization the cytotoxic substance was isolated, and its structure was identified as skullcapflavon II (5,2'-dihydroxy-6,7,8,6'-tetramethoxyflavone) based on the chemical and spectral data obtained.
The majority of other flavones isolated from $\mbox{\underline{S.}}$ $\mbox{\underline{baicalensis}}$ showed no significant inhibition on L1210 growth. From the modification of A-ring of baicalein, 5,8-dihydroxy-6,7-dimethoxyflavone (III) was found as a moderate cytotoxic flavone ($ED_50=7.5μg/ml$), but any obivious role of A-ring for the activity was not observed except that the fully oxidized A-ring was essential for the cytotoxic flavones.
To observe the relation between the activity and the substitutions in B-ring of a flavone, skullcapflavon II and its analogues were synthesized by Baker-Venkataraman rearrangement, partial debenzylation and methylation. By this method skullcapflavon II was obtained from 2,6-dibenzyloxybenzoyl chloride and 2,6-dihydroxy-3,4,5-trimethoxyacetophenone with 33% yield and other analogues with comparable yields. Among the synthesized flavones, 5,2',6'-trihydroxy-6,7,8-trimethoxyflavone (XVII) and skullcapflavon II (I) showed the strong cytotoxicities on L1210 cells ($ED_50=4.5 and 2.3 μg/ml$, respectively). From the analysis of the structures with corresponding activities it was supposed that the coplanarity of A-C-ring and B-ring might play a certain role for the cytotoxicity.
To elucidate the cytotoxic mechanism, the effects of skullcapflavon II on the lactate formation, macromolecule syntheses and active transport of L1210 cells were observed. Skullcapflavon II showed no inhibition on the glycolysis of the cell and some inhibitions on the incorporations of $^3H-thymidine$, $^3H-uridine$ and $^3H-leucine$ into DNA, RNA and protein respectively, but not specifically. The activity of membrane bounded ATPase was inhibited significantly by skullcapflavon II. By combining the moderate coplanarity of skullcapflavon II molecule and the inhibition on ATPase activity by this compound, a possible mechanism of the inhibition of growth by skullcapflavon II was postulated.
The significant antitumor activity on arcoma 180 in both ascite and solid forms (T/C = 166% and Inhibition rate = 71% at 40 mg/kg, respectively) and the moderate activity on L1210 (T/C = 122%) suggest a possibility of using skullcapflavon II in the treatment of neoplasms.
새로운 항암제를 찾기위한 목적으로 동양에서 오랫동안 암이나 암과 유사한 질병의 치료에 이용되어 왔던 천연물중의 하나인 황금(Scutellaria baicalensis)을 선정하여 암세포 성장 억제물질을 분리하고 동정하였으며 반합성및 전합성의 방법으로 구조와 작용간의 관계를 관찰하고 가능한 억제기전과 실험동물에 대해 항암효과가 있음을 밝혔다. 황금의 작용물질을 분리하기 위하여 여러 용매 및 시리카겔 컬럼을 이용하여 분획하였고 각분획을 암세포 성장 억제시험을 하여 작용을 추적하는 방법으로 작용원인 물질을 단리하고 화학적, 물리적 방법으로 구조에 대한 분석을 하여 그 결과를 참고문헌과 비교하여 Skullcapflavon II (5, 2'-Dihyd-roxy-6, 7, 8, 6'-tetramethoxyflavone) 임을 밝혔다. 이물질은 $1.5 \mu g/ml$ 의 농도에서 50\%의 L1210 세포를 억제하였다. Skullcapflavon II의 A-링의 작용에 대한 역할을 보기위해 황금으로부터 분리한 Baicalein을 Persulfate 산화, 부분 메칠화 반응 등을 이용하여 구조를 변형하여 시험하여 또하나의 비교적 강한 억제 작용을 가진 후라본인 5, 8-Dihydroxy-6, 7-dimethoxyflavone을 얻었으며 $(ED_{50} = 7.5 \mu g/ml)$, 모두 산화된 A-링이 작용에 중요한 역할을 할것으로 추측하였다. B-링의 역할을 보기위해 Skullcapflavon II와 그유도체들을 Baker-Venkataraman 전위, 부분 탈벤질화, 부분 메칠화 반응 등을 이용하여 합성하고 그 작용을 각각 시험한 결과 5, 2', 6'-Trihydroxy-6, 7, 8-trimethoxyflavone 도 역시 강한 작용 $(ED_{50} = 4.5 \mu g/ml)$이 있음을 알았다. 작용에는 어떤 하나의 작용기보다는 A-C-링과 B-링간의 평면적인 배열위치가 중요할 것으로 추측되었다. 작용기전을 규명하기위해 L1210의 해당작용, 핵산및 단백질 합성, 세포막의 ATPase 활성등에 미치는 영향을 조사하였는데 주로 세포막의 ATPase의 활성을 억제하여 영양물질의 능동적 이동을 저해하는 것으로 추측되었다. Skullcapflavon II를 L1210을 이식한 생쥐에 투여하였을 때에는 약간의 수명연장 효과 (T/C = 122\%)를 보였지만 Sarcoma 180을 이식한 생쥐에 대해서는 복강암에서 현저한 수명연장효과 (T/C = 166\%), 고형암에서 강한 암세포 억제작용 (억제도 = 71\%)를 보임으로써 암치료제로서의 개발 가능성을 제시하였다.
