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Composition for the Immunity Stimulatory Activity Comprising Calystegia dahuricus (Herb.) Choisy Water Extract
Biomed Sci Letters 2022;28:206-210
Published online September 30, 2022;  https://doi.org/10.15616/BSL.2022.28.3.206
© 2022 The Korean Society For Biomedical Laboratory Sciences.

Myeongguk Jeong*, Hyeokjin Kwon*, Seohye Jeong**, Yerin Seo**, Minguk Kim** and Go-Eun Choi†,***

Department of Biomedical Laboratory Science, College of Health Sciences, Catholic University of Pusan, Busan 46252, Korea
Correspondence to: Go-Eun Choi. Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan, Busan 46252, Korea.
Tel: +82-51-510-0563, Fax: +82-51-510-0568, e-mail: gechoi@cup.ac.kr
*Graduate student, **Undergraduate student, ***Professor.
Received September 19, 2022; Revised September 22, 2022; Accepted September 22, 2022.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
 Abstract
Calystegia dahurica (Herb.) Choisy is a natural product that has not been studied for efficacy or active ingredients. The purpose of this study is to investigate the activation effect of natural killer cells using a natural extract composition based on Calystegia dahurica (Herb.) Choisy extract (CDCE). We evaluated the activity of natural killer cells in natural products using PBMCs from healthy participants. All natural products were extracted with 50% ethanol. Based on the results of the cell viability assay, PBMCs of healthy participants were treated with extracts at various concentrations. Then, analysis was performed using flow cytometry to measure the cd107a surface expression of natural killer cells. As a result, treatment with a single extract of PBMCs increased the expression of cd107a in a concentration-dependent. Furthermore, it was confirmed that the treatment of the extract composition showed the highest expression of cd107a. In conclusion, it is expected that the extract composition containing CDCE according to this study can be used for prevention or treatment of cancer cells, tumor cells, and immune diseases.
Keywords : Calystegia dahuricus (Herb.) Choisy, Saussurea neoserrata Nakai, Nypa fruticans Wurmb, PBMC, NK cell, CD107a, Cytotoxicity
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븫 21꽭湲 紐⑤뱺 援媛뿉꽌 궗留앹쓽 二쇱슂 썝씤씠硫(Bray et al., 2018), 븫 諛쒕퀝 떆 떊泥 솢룞쓽 옣븷, 닔硫 옣븷, 궣쓽 吏 븯瑜 珥덈옒븳떎(Dehkordi et al., 2009). 떎뼇븳 쑀삎쓽 븫 移섎즺瑜 쐞빐 諛⑹궗꽑 슂踰, 솕븰 슂踰 諛 닔닠 벑쓽 移섎즺踰뺣뱾씠 궗슜맆 닔 엳떎. 븯吏留 씠윭븳 移섎즺踰뺣뱾쓽 寃쎌슦 遺옉슜쓣 珥덈옒븷 닔 엳쑝硫, 쓷엳 몢넻, 뵾濡, 깉紐, 援ы넗, 꽕궗, 蹂듯넻, 援ш컯뿼利 벑씠 諛쒖깮븷 닔 엳떎(Tan et al., 2019). 씠윭븳 遺옉슜쑝濡 씤빐 솚옄쓽 寃쎌슦 移섎즺 湲고뵾, 以묐떒쑝濡 씠뼱吏 닔 엳떎. 理쒓렐 湲곗〈 빆븫 移섎즺踰뺤쓽 븳怨꾨 洹밸났, 蹂댁셿븷 닔 엳뒗 移섎즺踰 以 븯굹濡 옄뿰궡빐꽭룷 湲곕컲 硫댁뿭 移섎즺媛 二쇰ぉ諛쏄퀬 엳쑝硫(Lorenzo-Herrero et al., 2018), 옄뿰궡빐꽭룷쓽 寃쎌슦 吏곸젒쟻쑝濡 븫 諛 醫낆뼇쓽 諛쒖깮, 利앹떇, 쟾씠 諛 옱諛쒖쓣 留됰뒗뜲 슚怨쇱쟻씠씪뒗 궗떎씠 엯利앸릺怨 엳떎(López-Soto et al., 2017). 옄뿰궡빐꽭룷뒗 븙꽦꽭룷, 蹂묒썝泥댁뿉 媛먯뿼맂 꽭룷 궗硫몄뿉 以묒슂븳 뿭븷쓣 븯뒗 꽑泥쒕㈃뿭 泥닿퀎쓽 由쇳봽援ъ씠硫(Scott-Algara and Paul, 2002), CD3(T-꽭룷 怨듬룞 닔슜泥)媛 뾾뒗 긽깭뿉꽌 몴쁽삎 留덉빱 CD56(떊寃쎌꽭룷 遺李 遺꾩옄)쓽 諛쒗쁽뿉 쓽빐 듅吏뺤뼱吏꾨떎(Becker et al., 2016). 옄뿰궡빐꽭룷 꽭룷룆꽦 怨듦꺽 利됯컖쟻씠硫 T 꽭룷 떖由 빆썝 궗쟾 媛먯옉뾾씠 븫꽭룷굹 諛붿씠윭뒪뿉 媛먯뿼맂 꽭룷뿉 븳 珥덇린 諛⑹뼱瑜 젣怨듯븳떎(Kwon et al., 2018). 옄뿰궡빐꽭룷뒗 二쇰줈 꽭룷 궗硫몄쓣 쑀룄븷 닔 엳뒗 granzyme B 諛 perforin쓣 븿쑀븳 꽭룷슜빐 怨쇰┰쓽 諛⑹텧쓣 룷븿븯뒗 湲곗쟾뿉 쓽빐 몴쟻꽭룷瑜 궗硫몄떆궎硫, 吏곸젒쟻씤 꽭룷슜빐 諛 IFN-gamma 깮궛쓣 넻븯뿬 愿묐쾾쐞븳 醫낆뼇꽭룷瑜 슚쑉쟻쑝濡 씤떇븯怨 젣嫄고븷 닔 엳떎(Viel et al., 2013).

궛怨⑥랬(Saussurea neoserrata Nakai)뒗 援솕怨쇱뿉 냽븯뒗 뿬윭빐궡씠 떇臾쇱씠떎. 슚뒫쑝濡쒕뒗 빆뿼利, UVB濡 씤븳 떊寃쎌꽭룷 넀긽 삁諛 벑씠 엳쑝硫, 쑀슚꽦遺꾩쑝濡쒕뒗 럹궛쓽 씪醫낆씤 protocatechuic acid (3,4-dihydroxy benzoic acid) 벑씠 蹂닿퀬릺뿀떎(Lee et al., 2021a; Lee et al., 2021b). 빐二쎌닚(Nypa fruticans Wurmb) 빞옄怨쇱뿉 냽븯硫 룞븘떆븘쓽 媛踰뚭낵 뒿吏뿉꽌 옄씪뒗 빞옄닔쓽 씪醫낆씠떎. 슚뒫쑝濡 빆怨좏삁떦, 빆넻媛곸닔슜꽦, 빆떦눊蹂 諛 빆궛솕 슚怨쇰 룷븿븯뒗 떎뼇븳 깮臾쇳븰쟻 솢꽦씠 엳뒗 寃껋쑝濡 蹂닿퀬맂 諛 엳쑝硫, 뤃由ы럹 諛 뵆씪蹂대끂씠뱶 媛숈 떎뼇븳 솕빀臾쇱쓣 룷븿븯怨 엳떎(Yusoff et al., 2015). 꽑硫붽퐙(Calystegia dahuricus (Herb.) Choisy) 硫붽퐙怨쇱뿉 냽븯뒗 뿬윭빐궡씠 떇臾쇱씠硫, 슚뒫 諛 쑀슚꽦遺 벑 뿰援щ맂 諛붽 뾾떎. 蹂 뿰援ъ뿉꽌뒗 泥대궡 硫댁뿭젰쓣 씪젙 닔以 씠긽쑝濡 쑀吏떆궗 닔 엳떎硫, 洹 옄泥대줈 깉濡쒖슫 쓽誘몄쓽 룷愿꾩쟻 삁諛⑸쾿씠씪怨 븷 닔 엳떎. 뵲씪꽌 옄뿰궡빐꽭룷 湲곕뒫쓽 솢꽦쓣 洹쇰낯쟻쑝濡 쉶蹂듯븷 닔 엳뒗 泥쒖뿰 떇臾쇱옄썝 異붿텧臾쇱쓣 諛쒓뎬븯뿬 씤泥 궡 硫댁뿭젰쓣 씪젙 닔以 씠긽쑝濡 뼢긽떆耳 떎뼇븳 媛먯뿼꽦 吏덊솚, 븫 移섎즺 諛 븫쑝濡쒕꽣 삁諛⑺븯뒗 諛⑸쾿쓣 紐⑥깋븯怨좎옄 븳떎.

蹂 뿰援ъ뿉꽌 궗슜븳 PBMC뒗 떊泥댁쟻쑝濡 듅젙븳 吏덊솚쓣 媛吏怨 엳吏 븡쑝硫, 뿰援ъ쓽 紐⑹쟻怨 닔뻾릺뒗 寃궗 諛 떎뿕뿉 愿븳 궡슜쓣 뱽怨 옄諛쒖쟻쑝濡 뿰援ъ뿉 李몄뿬븷 寃껋쓣 룞쓽꽌뿉 꽌紐낇븳 20꽭 씠긽쓽 嫄닿컯븳 꽦씤 以 50紐낆쓣 긽쑝濡 빆쓳怨좎젣(EDTA) 뒠釉뚮 궗슜븯뿬 留먯큹삁븸쓣 梨꾪삁븯떎. 蹂 뿰援щ뒗 遺궛媛넧由븰援 湲곌깮紐낆쑄由ъ쐞썝쉶쓽 떖쓽瑜 嫄곗퀜 닔뻾릺뿀떎(CUPIRB-2020-022). Histopaque-1077 (Sigma-Aldrich, St. Louis, USA) 슜븸쓣 씠슜븳 諛룄援щ같 썝떖遺꾨━뿉 쓽빐 빆쓳怨좎젣(EDTA) 뒠釉뚯쓽 삁븸쑝濡쒕꽣 PBMC瑜 遺꾨━븯떎. 遺꾨━맂 꽭룷瑜 李④슫 PBS뿉꽌 2쉶 꽭泥숉븯怨 10% fetal bovine serum (Gibco, Paisley, UK), 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco, Paisley, UK)씠 蹂댁땐맂 RPMI-1640 (Gibco, Paisley, UK) 諛곗뿉 諛곗뼇븯떎. 紐⑤뱺 怨쇱젙 梨꾪삁 썑 4떆媛 씠궡뿉 닔뻾릺뿀떎. 留뚯꽦 怨⑥닔꽦 諛깊삁蹂 븫꽭룷씤 K-562 (Korean Cell Line Bank, Seoul, Korea) 꽭룷뒗 10% fetal bovine serum (Gibco, Paisley, UK), 100 U/mL penicillin, 100 μg /mL streptomycin (Gibco, Paisley, UK)씠 蹂댁땐맂 RPMI-1640 (Gibco, Paisley, UK) 諛곗瑜 궗슜븯뿬 37℃, 5% CO2 議곌굔뿉꽌 諛곗뼇븯떎.

궛怨⑥랬(Saussurea neoserrata Nakai), 빐二쎌닚(Nypa fruticans Wurmb), 꽑硫붽퐙(Calystegia dahuricus (Herb.) Choisy) 嫄댁“삎깭쓽 쟾珥덈 떎 씤꽣궡뀛꼸(Hanam, Korea)뿉꽌 援ъ엯븯떎. 뿉깂삱 異붿텧臾쇱쓽 젣議곕뒗 (Adebiyi et al., 2017)쓽 諛⑸쾿쓣 蹂삎븯뿬 궗슜븯떎. 떆猷뚮 遺꾩뇙븳 뮘 50% 뿉깂삱쓣 떆猷 1 : 뿉깂삱 10쓽 鍮꾩쑉濡 샎빀븯뿬 80℃뿉꽌 3떆媛 以묓깢쓣 3쉶 諛섎났 異붿텧븯떎. 異붿텧臾쇱쓣 媛먯븬냽異뺢린濡 媛먯븬냽異뺥븳 썑 룞寃곌굔議곌린(Ilshin Lab, Yangju, Korea)瑜 궗슜븯뿬 -80℃뿉꽌 룞寃곌굔議 썑 遺꾨쭚삎깭쓽 떆猷뚮 뼸뿀떎. 媛 異붿텧臾쇰퀎 냽룄뿉 뵲瑜 PBMC쓽 깮議댁쑉쓣 痢≪젙븯湲 쐞빐 Cell Counting Kit-8 (Dojindo, Kumamoto, Japan)쓣 씠슜븯뿬 깮議댁쑉쓣 遺꾩꽍븯떎. 꽭룷瑜 96 well plate뿉 well떦 2×105媛쒓 릺룄濡 遺꾩<븳 썑 37℃, 5% CO2 議곌굔뿉꽌 24떆媛 諛곗뼇븯떎. 24떆媛 諛곗뼇 썑 냽룄蹂(0, 0.02, 0.04, 0.2, 0.4, 0.8, 1, 2 mg/mL) 異붿텧臾쇱쓣 泥④븯뿬 24떆媛 룞븞 諛곗뼇븳 썑 CCK-8 슜븸쓣 泥④븯떎. 30遺 諛곗뼇湲곗뿉꽌 諛섏쓳 썑 Varioskan LUX microplate reader (Thermo Scientific, MA, USA)瑜 궗슜븯뿬 450 nm 뙆옣뿉꽌 씉愿묐룄瑜 痢≪젙븯떎. 궛怨⑥랬(Saussurea neoserrata Nakai) 뿉깂삱 異붿텧臾쇱 냽룄 0.02~0.8 mg/mL 궗씠쓽 냽룄뿉꽌 95% 씠긽 깮議댄븯쑝硫, 1 mg/mL뿉꽌 92±0.81%, 2 mg/mL뿉꽌 90.66±2.35% 깮議댄븯떎(Fig. 1. (A)). 빐二쎌닚(Nypa fruticans Wurmb) 뿉깂삱 異붿텧臾쇱 냽룄 0.02~1 mg/mL 궗씠뿉꽌 95% 씠긽 깮議댄븯쑝硫 2 mg/mL 냽룄뿉꽌 93±0.81% 깮議댄븯떎(Fig. 1. (B)). 꽑硫붽퐙(Calystegia dahuricus (Herb.) Choisy) 뿉깂삱 異붿텧臾쇱 0.02~0.4 mg/mL 궗씠쓽 냽룄뿉꽌 95% 씠긽 깮議댄븯쑝硫 0.8 mg/mL뿉꽌 94±2.94%, 1 mg/mL뿉꽌 93.66±0.94%, 2 mg/mL뿉꽌 92.66±1.24% 깮議댄븯떎(Fig. 1. (C)). 異붿텧臾쇱뿉 븳 룊媛瑜 쐞빐꽌뒗 3媛쒖쓽 臾쇱쭏뿉 빐 깮議댁쑉씠 95% 씠긽씤 냽룄(0.04, 0.2, 0.4 mg/mL)瑜 궗슜븯떎. 異붿텧 샎빀臾쇱쓽 寃쎌슦 뿰援щ맂 諛붽 뾾뼱 엫쓽쓽 諛고빀 鍮꾩쑉(1:1, 1:1:1)쓣 궗슜븯쑝硫, 씠썑 紐⑤뱺 떎뿕뿉 궗슜븯떎.

Fig. 1. Cytotoxicity of the three extracts in culture with PBMC. Cytotoxicity of PBMC was determined in the culture of PBMC treated with increasing doses of the three extracts for 24 h, followed by the analysis of relative metabolic activity by CCK-8 assays. (A) Cytotoxicity of the Saussurea neoserrata Nakai, (B) Cytotoxicity of the Nypa fruticans Wurmb, (C) Cytotoxicity of the Calystegia dahuricus (Herb.) Choisy. The summarized data (A-C) is shown as mean ± SD from three independent experiments.

옄뿰궡빐꽭룷 꽭룷룆꽦 깉怨쇰┰쓽 痢≪젙쓣 쐞빐 꽭룷 몴硫 CD107a쓽 諛쒗쁽쓣 Flow cytometry瑜 궗슜븯쑝硫(Lee et al., 2021c)瑜 李멸퀬븯뿬 蹂삎맂 諛⑸쾿쓣 궗슜븯떎. 닔吏묐맂 PBMC瑜 U-bottom plate (Thermo Scientific, MA, USA)뿉 1×106媛쒖뵫 遺꾩<븯怨 냽룄蹂(0, 0.04, 0.2, 0.4 mg/mL) 뿉깂삱 異붿텧臾쇱쓣 泥섎━븳 썑 37℃, 5% CO2 솚寃쎌뿉꽌 20떆媛 諛곗뼇븯떎. 怨듬룞 諛곗뼇 썑 몴쟻꽭룷瑜 1 : 1쓽 Effector : Target 鍮꾩쑉뿉 留욎떠 遺꾩< 썑 2떆媛 諛곗뼇븯뿬 옄洹뱁븯떎. 옄洹 썑 꽭룷瑜 V-bottom plate (Thermo Scientific, MA, USA)뿉 삷湲곌퀬 쑀꽭룷 遺꾩꽍쓣 쐞빐 뿼깋븯떎. 뿼깋쓣 쐞빐 CD3-PerCP (Becton Dickinson, MA, USA), CD56-PE (Becton Dickinson, MA, USA), and CD107a-FITC (Becton Dickinson, MA, USA) 빆泥대 궗슜븯뿬 鍮 李⑤떒 썑 4℃뿉꽌 1떆媛 뿼깋븯떎. 紐⑤뱺 뜲씠꽣뒗 BD Accuri C6 plus (Becton Dickinson, MA, USA) 쑀꽭룷 遺꾩꽍湲곗뿉꽌 닔吏묐릺뿀怨 FlowJo version 10.8.1 (Becton Dickinson, OR, USA) 냼봽듃썾뼱瑜 궗슜븯뿬 遺꾩꽍븯떎. 궛怨⑥랬(Saussurea neoserrata Nakai), 빐二쎌닚(Nypa fruticans Wurmb), 꽑硫붽퐙(Calystegia dahuricus (Herb.) Choisy) 媛 뿉깂삱 異붿텧臾쇱쓽 떒씪 泥섎━떆 옄뿰궡빐꽭룷쓽 CD107a 諛쒗쁽 젙룄媛 냽룄 쓽議댁쟻쑝濡 利앷븯떎. 듅엳 슚怨쇨 媛옣 긽썡븳 꽑硫붽퐙 異붿텧臾쇨낵 옄뿰궡빐꽭룷쓽 솢꽦쓣 利앹쭊떆궎뒗 寃껋쑝濡 솗씤맂 몢 媛吏 異붿텧臾(궛怨⑥랬, 빐二쎌닚)怨 븿猿 1 : 1 : 1濡 議고빀븯쓣 븣 옄뿰궡빐꽭룷 꽭룷 몴硫 CD107a 諛쒗쁽 젙룄媛 넻怨꾩쟻쑝濡 쑀쓽븯寃 利앷븯떎(Fig. 2). 꽑硫붽퐙 異붿텧臾쇱쓣 쑀슚꽦遺꾩쑝濡 븿쑀븯뒗 옄뿰궡빐꽭룷쓽 솢꽦 利앹쭊슜 議곗꽦臾쇱 硫댁뿭 솢꽦怨 愿젴븳 Granzyme B 諛 Perforin쓽 諛쒗쁽쓣 利앷떆궡쑝濡쒖뜥 옄뿰궡빐꽭룷쓽 솢꽦쓣 利앹쭊떆궗 닔 엳떎.

Fig. 2. The three extracts enhances the natural cytotoxicity of NK cells. PBMCs exposed at varying concentrations for 20 h and then mixed with K562. After 2 h incubation at 37℃, cytotoxic degranulation of NK cells was measured by cell surface expression of CD107a on CD3-CD56+ NK cells. (A) The expression of CD107a of the Saussurea neoserrata Nakai, (B) The expression of CD107a of the Nypa fruticans Wurmb, (C) The expression of CD107a of the Calystegia dahuricus (Herb.) Choisy. (D) The combination treatment (1:1 and 1:1:1, respectively) with the Calystegia dahuricus (Herb.) Choisy. The right column shows Representative flow cytometry profiles. The left column shows the percentage of CD107a+ NK cells in four independent experiments. Data are expressed as means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 by one-way ANOVA with the Dunnett's test.

슂빟븯硫, 슦由ъ쓽 寃곌낵뒗 씠쟾뿉 씤떇릺吏 븡븯뜕 꽑硫붽퐙 異붿텧臾쇱씠 옄뿰궡빐꽭룷 슚怨쇱뿉 븳 湲곕뒫쓣 珥됱쭊븷 닔 엳뒗 옞옱젰쓣 媛吏怨 엳쓬쓣 蹂댁뿬以떎. 씠윭븳 留λ씫뿉꽌 異뷀썑 뿰援щ 넻빐 옄뿰궡빐꽭룷뿉 븳 꽑硫붽퐙 옉슜 硫붿빱땲利섏뿉 븳 깉濡쒖슫 넻李곕젰쓣 젣怨듯븷 븘슂媛 엳떎. 뵲씪꽌 슦由ъ쓽 뿰援щ뒗 씤泥 궡 궗 옉슜쑝濡 씤빐 꽑硫붽퐙 異붿텧臾쇱쓣 빆븫젣濡 怨좊젮븯뒗 뜲 二쇱쓽媛 븘슂븯吏留(Kwon et al., 2018), 삁諛 諛 移섎즺愿젏뿉꽌 솕븰 삁諛 諛 솕븰 슂踰 옞옱젰쓣 솢슜븯뒗 슚怨쇱쟻씤 쟾왂 媛쒕컻뿉 湲곗뿬븷 닔 엳떎.

ACKNOWLEDGEMENT

This study was supported by the Undergraduate Research Program (URP) support project, Korea Foundation for the Advancement of Science & Creativity (KOFAC) and a grant of the National Research Foundation of Korea (NRF) by the Ministry of Science, (No. 2019R1C1C1004820).

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

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