본 연구에서는 11종 약용식물의 기능성 성분 및 생리활성을 비교 분석하고 그 중 함량이 높고 활성이 좋은 어성초와 야관문를 이용하여 분획 추출 하였다. 어성초와 야관문의 분획물 중 기능성 성분 함량 및 활성이 가장 좋은 ethyl acetate 분획물을 이용하여 페놀산과 플라보노이드 성분을 HPLC와 UPLC/MS/MS로 정성 및 정량 분석하였다. 확인된 성분을 이용하여 항산화, 항염증, 항암 활성을 비교 측정하였다.
약용식물의 메탄올 추출물의 총 폴리페놀 함량은 야관문(102.7 mg GAE/g), 오가피(98.0 ㎎ GAE/g), 어성초(66.1 mg GAE/g) 순으로 높았으며, 총 플라보노이드 함량은 야관문(62.8 mg QE/g), 어성초(23.6 mg QE/g), 오가피(16.6 mg QE/g) 순으로 높았다. DPPH와 ABTS 라디칼 소거활성은 오가피(87.9%, 90.9%), 어성초(83.1%, 87.9%), 야관문(79.3%, 85.8%) 순으로 높았다. FRAP 활성은 야관문(169.5 mg TE/g), 오가피(165.5 mg TE/g), 해동피(62.4 mg TE/g) 순으로 높았다. ACE 저해활성은 구기자(19.2%), 해동피(16.8%), 야관문(13.5%) 순으로 높았다. α-Glucosidase 저해활성은 야관문(93.7%), 산수유(50.9%), 천궁(28.9%) 순으로 높았다. 분석 항목간의 상관관계 분석결과 총 폴리페놀과 총 플라보노이드(R=0.754), DPPH 라디칼 소거활성(R=0.846), ABTS 라디칼 소거활성(R=0.736), FRAP 활성(R=0.927)은 상관관계가 있었다. 총 플라보노이드 함량도 DPPH 라디칼 소거활성(R=0.707), ABTS 라디칼 소거활성(R=0.551), FRAP 활성(R=0.742), α-glucosidase 저해활성(R=0.649)과 상관관계가 있었다. DPPH 라디칼 소거활성은 ABTS 라디칼 소거활성(R=0.852)와, FRAP 활성(R=0.813), α-glucosidase 저해활성(R=0.365)과 ABTS 소거활성은 FRAP 활성(R=0.748)과 α-glucosidase 저해활성(R=0.432)과 상관관계가 있으며, FRAP 활성은 α-glucosidase 저해활성(R=0.484)과 상관관계가 있었다.
페놀성 화합물 함량과 항산화 활성이 높았던 어성초와 야관문 메탄올 추출물을 용매 극성 차이를 이용한 계통분획법으로 hexane, chloroform, ethyl acetate, butanol, water 층으로 분획하여 각 분획물의 기능성 성분 함량 및 항산화 활성, ACE 저해활성, α-glucosidase 저해활성 등 다양한 생리활성을 측정하였다. 총 폴리페놀 함량은 어성초(200.5 mg GAE/g), 야관문(142.9 mg GAE/g) 모두 ethyl acetate 분획물에서 가장 높았고, 총 플라보노이드 함량도 ethyl acetate 분획물에서 어성초(157.1 mg QE/g), 야관문(130.3 mg QE/g) 모두 가장 높았다. DPPH 라디칼 소거활성은 ethyl acetate 분획물에서 어성초(90.8%), 야관문(91.2%) 모두 소거활성이 가장 높았다. ABTS 라디칼 소거활성은 어성초는 ethyl acetate 분획물에서(86.1%) 가장 높았고, 야관문은 chloroform 분획물에서(95.6%) 으로 가장 높았다. FRAP 활성은 ethyl acetate 분획물에서 어성초(360.1 mg TE/g), 야관문(239.2 mg TE/g) 모두 가장 높았다. ACE 저해활성은 chloroform 분획물이 어성초(13.2%), 야관문(35.2%) 모두 가장 높았다. α-Glucosidase 저해활성은 어성초는 ethyl acetate 분획물이(56.3%), 야관문은 water 분획물이(93.6%) 가장 높았다.
활성이 좋은 ethyl acetate 분획물을 이용하여 페놀산과 플라보노이드 성분을 정성 및 정량 분석하였다. 페놀산 성분은 어성초와 야관문 각각 7종이 확인되었는데, protocatechuic acid이 어성초(1.121 mg/g), 야관문(0.790 mg/g) 모두 가장 함량이 높았고, 플라보노이드 성분은 어성초 12종, 야관문 10종이 확인 되었는데, 어성초는 quercitrin (13.960 mg/g), 야관문은 vitexin (12.329 mg/g) 이 가장 함량이 높았다. 어성초와 야관문에서 확인된 페놀산 및 플라보노이드 성분의 DPPH 라디칼 소거활성을 측정한 결과 어성초는 페놀산 중 caffeic acid, syringic acid 플라보노이드 중 quercetin, myricetin, kaempferol, luteolin, orientin, isoquercitrin, hyperoside, quercitrin이 80% 이상의 높은 활성을 나타내어 주된 항산화 성분으로 추정되고, 야관문은 페놀산 중 caffeic acid, syringic acid, 플라보노이드 중 quercetin, myricetin, luteolin, orientin, isoquercitrin, hyperoside, quercitrin이 80% 이상의 높은 활성을 나타내어 주된 항산화 성분으로 추정된다. 어성초와 야관문에서 확인된 페놀산 및 플라보노이드 성분의 항염증 활성 측정 결과 apigenin, quercetin이 세포 생존율의 저해 없이 항염증 활성을 나타내었다. 확인된 페놀산 및 플라보노이드 성분의 항암 활성 연구를 위해 세포생존율 측정을 수행하였다. 수행 결과 4-hydroxybenzoic acid은 HCT116 세포의 세포 생존율을 농도 의존적으로 감소시켰으며, 세포 생존율도 가장 낮았다.
본 연구는 첨단 장비를 이용하여 어성초와 야관문의 페놀성 화합물을 정량분석 하였다. 특히, 어성초에서 myricetin과 apigenin 성분과 야관문에서 protocatechuic acid, hyperoside, myricetin, apigenin, luteolin 성분을 확인하였다. 연구논문 검색결과 이들 성분에 관한 분리 동정 및 확인 정량에 관한 논문은 미흡하여, 향 후 어성초와 야관문 연구에 활용 가치가 있다고 판단된다. 종합적으로 본 연구의 결과는 약용식물의 기능성 성분 및 다양한 생리활성 연구에 도움이 될 것이며, 또한 천연물을 이용한 다양하고 광범위한 분야에 활용 가능할 수 있는 기초자료를 제공했다고 사료된다.

This study compared and analyzed the functional components and various physiological activity of 11 medicinal herbs, then fractionation using only Houttuynia cordata and Lespedeza cuneata which are high in contents. Among those fractions, using ethyl acetate layer which has the best functional components and activity, quantified and qualified compounds of Phenolic acid and Flavonoid helped by HPLC and UPLC/MS/MS. With above mentioned verified compounds, Anti-oxidant, Anti-inflammatory and Anti-cancer activities in fraction extracts were measured.
The total polyphenol contents of methanol extract from 11 medicinal herbs were higher in the order of Lespedeza cuneata (102.7 ㎎ GAE/g), Eleutherococcus senticosus (98.0 ㎎ GAE/g) and Houttuynia cordata (66.1 ㎎ GAE/g). Total flavonoid contents of methanol extract from 11 medicinal herbs were higher in the order of Lespedeza cuneata (62.8 ㎎ QUE/g), Houttuynia cordata (23.6 ㎎ QUE/g) and Eleutherococcus senticosus (16.6 ㎎ QUE/g). DPPH and ABTS radical scavenging activities of methanol extract from 11 medicinal herbs were higher in the order of Eleutherococcus senticosus (87.9%, 90.9%), Houttuynia cordata (83.1%, 87.9%) and Lespedeza cuneata (79.3, 85.8%). The results of FRAP assay of methanol extract from 11 medicinal herbs were higher in the order of Lespedeza cuneata (169.5 ㎎ TE/g), Eleutherococcus senticosus (165.5 ㎎ TE/g) and Kalopanax pictus (62.4 ㎎ TE/g). The results of ACE inhibition assay of methanol extract from 11 medicinal herbs were higher in the order of Lycium chinense (19.2%), Kalopanax pictus (16.8%) and Lespedeza cuneata (13.5%). The results of α-glucosidase inhibition assay were of methanol extract from 11 medicinal herbs higher in the order of Lespedeza cuneata (93.7%), Cornus officinalis (50.9%), Cnidium officinale (28.9%). As a results of correlation analysis between analysis items, it is proven that total polyphenol and Flavonoid (R=0.754), DPPH radical scavenging activity (R=0.846), ABTS radical scavenging activity (R=0.736), FRAP activity (R=0.927) have its mutual correlation. Total Flavonoid content was also highly correlated with DPPH radical scavenging activity (R=0.707), ABTS radical scavenging activity (R=0.551), FRAP activity (R=0.742), and α-glucosidase inhibition assay (R=0.649). DPPH scanvenging activity was also correlated with ABTS scanvenging activity (R=0.852)와, FRAP (R=0.813), α-glucosidase inhibition assay (R=0.365) while ABTS scavenging activity was correlated with FRAP activity (R=0.748) and α-glucosidase inhibition assay (R=0.432), also FRAP activity was correlated with α-glucosidase inhibition assay (R=0.484).
By the grid fractionation analysis using solvent polarity difference, those were fractionated of the metanol extract from Houttuynia cordata and Lespedeza cuneata which have the highest content and the highest activity into hexane, chloroform, ethyl acetate, butanol, and the water layer, then analyzed its Physiological activity in variety such as content of functional components and anti-oxidant activity, ACE inhibitory activity, α-glucosidase inhibitory activity.
In the ethyl acetate fraction, total polyphenol content was highest in Houttuynia cordata (200.5 mg GAE/g) and Lespedeza cuneata (142.9 mg GAE/g). In the ethyl acetate extract, total flavonoid content was highest in Houttuynia cordata (157.1 mg QUE/g) and Lespedeza cuneata (130.3 mg QUE/g). DPPH radical scavenging activities were highest in ethyl acetate fraction in Houttuynia cordata (90.8%) and Lespedeza cuneata (91.2%). ABTS radical scavenging activities were highest in ethyl acetate fraction in Houttuynia cordata (86.1%) and Lespedeza cuneata (95.6%) was highest in chloroform fraction. FRAP activities were highest in ethyl acetate fraction in Houttuynia cordata (360.1 mg TE/g) and Lespedeza cuneata (239.2 mg TE/g). The ACE inhibitory activities was the highest in chloroform fraction in Houttuynia cordata (13.2%) and Lespedeza cuneata (35.2%). α-Glucosidase inhibitory activities was Houttuynia cordata (56.3%) the highest in ethyl acetate extract and Lespedeza cuneata (93.6%) was highest in water fraction.
Quantitative and quantitative analysis of compounds of phenolic acid and flavonoid was performed using the ethyl acetate fraction which has the best activity. Pheonlic acid compounds was confirmed in 7 types of Houttuynia cordata ethyl acetate fraction and in 7 types of Lespedeza cuneata ethyl acetate fraction. Protocatechuic acids was the highest in both Houttuynia cordata (1.121 mg/g) and Lespedeza cuneata (0.790 mg/g). Flavonoid compounds was found in 12 types of Houttuynia cordata ethyl acetate fraction and in 10 types of Lespedeza cuneata ethyl acetate fraction, while Houttuynia cordata ethyl acetate fraction contains the highest quercitirin (13.960 mg/g) and Lespedeza cuneata ethyl acetate fraction contains the highest vitexin (12.329 mg/g).
As a result of the antioxidant activity analysis helped by the DPPH radical scavenging of the phenol acid and the flavonoid compounds found in Houttuynia cordata and Lespedeza cuneata ethyl acetate fraction, caffeic acids, syringic acids have shown high activity over 80% in case of the pheonol acid compounds while quercitrin, hyperoside, orientin, isoquercitrin, luteolin, kaempferol, quercetin, myricetin in case of the individual Flavonoid compounds.
As a result of anti-inflammatory activity measurement of the phenol acid and the flavonoid compounds found in Houttuynia cordata and Lespedeza cuneata ethyl acetate fraction, apgenin and quercitrin have shown its anti-inflammatory activity without the obstruction of cell viability.
Cell viability assay was performed to study the anti-cancer activity of the above idenfitied Phenolic acid and flavonoid compounds in Houttuynia cordata and Lespedeza cuneata ethyl acetate fraction. As a result of the assay, 4-hydroxybenzoic acids reduced the cell viability of HCT116 cells in a concentration dependent matter, with the lowest cell viability.
This study quantitatively analyzed the phenolic acid compounds of Houttuynia cordata and Lespedeza cuneata using advanced equipment. In particular, there is insufficient domestic and abroad study of the identification of myricetin and apigenin in Houttuynia cordata and the identificacion of protocatechuic acid, hyperoside, myricetin, apigenin and luteolin in Lespedeza cuneata. Accordingly, it is expected that this analysis has its value in the study of Houttuynia cordata and Lespedeza cuneata in the future.
The results of this study will be helpful for the study of functional components and various biological activities of medicinal herbs, and also provide basic data that can be used in various and wide fields using natural products.