천연으로부터 보체 저해활성 물질을 선발할 목적으로 용혈성 보체측정법(hemolytic complement assay, $CH_{50}$)을 이용한 미량 탐색법을 확립하였다. 보체결합 반응은 microplate에서 수행하였으며, 표준 용혈(50% hemolysis)을 설정하기 위한 최적조건은 classical pathway (CP)의 경우 양 적혈구 농도를 $5.0{ imes}10^8;cells/ml$로 하였을때 hemolysin 희석농도 $1/75{sim}1/100$ 및 보체 희석농도 $1/80{sim}1/120$이었으며, alternative pathway (AP)의 경우는 토끼 적혈구 농도 $4.0{ imes}10^8;cells/ml$에서 보체 희석농도 l/5, EGTA 4mM 및 $Mg^{2+};4{sim}8mM$ 이었다. 비수용성 화합물의 검정에는 dimethyl sulfoxide를 1% 수준이 되도록 시료의 농도를 조절하여 첨가하였다. 본 실험 조건에서 3종의 phenylpropanoid는 모두 농도에 비례하여 항보체 활성을 나타내었는데, 그중 rosmarinic acid의 항보체 활성은 CP의 경우 $0.063{sim}0.5mM$에서 $5.4{pm}3.6%{sim}95.8{pm}0.2%$, AP의 경우 $0.063{sim}1;mM$에서 $35.1{pm}0.9%{sim}95.6{pm}1.1%$으로 측정되었다.
To screen inhibitors on complement system from natural resources, micro-screening method was established by using hemolytic complement assay. Complement fixation reaction was carried out in the microplate system. For standard hemolysis (50% hemolysis) of the classical pathway (CP), hemolysin and complement serum were diluted to $1/75{sim}1/100;and;1/80{sim}1/120$, respectively, when sheep erythrocytes were $5.0{ imes}10^8;cells/ml$. In case of the alternative pathway (AP), complement serum was diluted to 1/5 and EGTA and $Mg^{2+}$ were added 4 mM, $4{sim}8;mM$, respectively, when rabbit erythrocytes were $4.0{ imes}10^8;cells/ml$. Dimethyl sulfoxide was used for the assay of non-aquous soluble compounds or extracts and its final concentration was not more than 1%. Three phenylpropanoids showed anticomplementary activities in proportion to the concentration for both pathways and rosmarinic acid exihibited the highest inhibitory activities: $5.4{pm}3.6%(0.063;mM){sim}95.8{pm}0.2%(0.5;mM);and;35.1{pm}0.9%(0.063;mM){sim}95.6{pm}1.1%(1;mM)$ on the CP and the AP, respectively.
