Evaluation of the Bioactivity of Polygonium tinctorium Leaf: Potential Clinical Uses

쪽잎의 생리활성 평가

Abstract

The leave of Polygonum tinctorium (LPT) have been used for centuries as a traditional medicine and as a food ingredient and natural dye. The aim of the current study was to develop high-value added products using LPT. Hot water extract (HWE) and ethanol extract (EE) of LPT were prepared, respectively, and their bioactivity was evaluated. The extraction ratio for the HWE was 27.6%, which was two-fold higher than that of the EE. The contents of total polyphenol in the HWE and total sugar in the EE were 51.2 mg/g and 297.8 mg/g, respectively. The total flavonoid and reducing sugar contents were similar in the extracts, irrespective of the extraction solvent. The HWE did not show antimicrobial activity in a disc-diffusion assay, but the EE showed strong growth inhibition against gram-positive bacteria. The EE exhibited stronger DPPH and ABTS radical scavenging activities and reducing power than those of the HWE. The HWE was particularly effective as a scavenger of nitrite ($RC_{50}$ of $6.0{\mu}g/ml$). In an antithrombosis activity assay, the EE showed significant anticoagulation activity as determined by an extended blood coagulation time (thrombin time, prothrombin time, and activated partial thromboplastin time), in addition to platelet aggregation activity. The HWE also showed platelet aggregation inhibitory activity. This report provides the first evidence of antithrombosis activities of LPT. Our results suggest that LPT has potential as a new antioxidant and antithrombosis agent.

본 연구에서는 천연 염재, 약용 및 식용으로 사용되고 있는 쪽잎의 고부가가치화를 위해, 쪽잎의 열수 추출물 및 ethanol 추출물을 조제하고, 이의 항산화, 항균, 항당뇨, 항응고, 혈소판 응집저해 활성을 평가하였다. 쪽잎의 열수 추출효율은 27.6%로 ethanol 추출효율보다 약 2배 높았으며, 열수 추출물의 TP 함량은 51.2 mg/g으로 ethanol 추출물보다 높았으며, 특이하게 총당 함량은 ethanol 추출물(297.8 mg/g)에서 열수 추출물보다 1.2배 높게 나타났다, 반면 TF 및 환원당 함량은 열수 및 ethanol 추출물에서 유사하게 나타났다(p<0.05). 항균 활성 평가 결과, ethanol 추출물은 L. monocytogenes, S. epidermidis, S. aureus 및 B. subtilis의 그람 양성균에 대해서만 강력한 항균력을 나타내었으며, 열수 추출물은 항균 활성이 나타나지 않았다. 또한 ethanol 추출물이 열수 추출물보다 강력한 38.8%의 DPPH 소거능, 84.5%의 ABTS 소거능과 환원력($OD_{700}=0.357$)을 나타내었으나, nitrite 소거능은 열수 추출물이 효과적임을 확인하였다. 한편 쪽잎 ethanol 추출물은 5 mg/ml 농도에서 무첨가구에 비해 TT는 1.86배, PT는 1.79배, aPTT는 1.65배 연장시켜 매우 강력한 항응고 활성을 나타내었으나, 빠르게 혈소판 응집을 촉진하는 것을 확인하였다. 반면 열수 추출물은 강력한 혈소판 응집저해를 나타내었다. 현재까지 쪽잎의 항혈전 활성은 보고된 바 없으며, 이러한 쪽잎의 강력한 항산화 및 항혈전 활성은 쪽잎을 이용한 항혈전 소재 개발이 가능함을 제시하고 있다.

Fig. 1. Component analysis of the (1) hot water and (2) ethanol extract of jjok leaf (leaf of Polygonium tinctorium). Different superscripts within a column differ significantly (p<0.05).

Fig. 2. Anti-oxidation activities of the (1) hot water and (2) ethanol extract of jjok leaf (leaf of Polygonium tinctorium). The concentrations used for DPPH and ABTS scavenging activity assay, and nitrite scavenging activity assay were 500 μg/ml and 200 μg/ml, respectively. Different superscripts within a column differ significantly (p<0.05).

Fig. 3. Effect of the extracts of jjok leaf (leaf of Polygonium tinctorium) on the activities of (A) α-amylase, (B) β-amylase, and (C) α-glucosidase. (1) acarbose (0.065 mg/ml), (2) acarbose (0.5 mg/ml), (3) hot water extract (0.5 mg/ml), (4) ethanol extract (0.5 mg/ml).

Fig. 4. Effect of the hot water and ethanol extract of jjok leaf (leaf of Polygonium tinctorium) on blood coagulation. Anti-coagulation activity is calculated on the clotting time of sample divided by the clotting time of solvent control in blood coagulation assay. The thrombin time (TT), prothrombin time (PT) and activated partial thromboplastin time (aPTT) of solvent control (dimethylsulfoximide) were 36.7 sec, 20.4 sec and 103.95 sec, respectively. Data are means ± SD of triplicate determinations.

Fig. 5. Diagram of impedance changes during platelet aggregation after addition of aspirin and the extracts of jjok leaf (leaf of Polygonium tinctorium) in whole blood aggregometer. (A) DMSO, (B) aspirin (0.25 mg/ml), (C) aspirin (0.125 mg/ml), (D) aspirin (0.065 mg/ml), (E) the hot water extract of jjok leaf, and (F) the ethanol extract of jjok leaf. Aggregation was induced by addition of 2.5 μl of collagen (1 mg/ml) into cuvette containing 50 μl of washed PRP and measured the impedance change for 12 min. The sample concentrations used were 0.25 mg/ml, respectively.

Table 1. Anti-microbial activities of the hot water and ethanol extract of jjok leaf (leaf of Polygonium tinctorium) against pathogenic and food spoilage microorganism

Table 2. Calculated RC₅₀s of the hot water and ethanol extract of jjok leaf (leaf of Polygonium tinctorium)

Table 3. Effect of the hot water and ethanol extract of jjok leaf (leaf of Polygonium tinctorium) on human platelet aggregation