본 연구에서는 Ya-Ha-Rak 복합물을 대상으로 1차 열수 추출과 70% 에탄올 재추출 공정 을 단계적으로 적용하여, 추출 방법에 따른 항산화 및 항염 활성을 비교·분석하였다. 1차 열수 추출물 (YHD)은 항산화 활성을 나타냈으나 항염 활성은 제한적인 수준이었다. 반면, 70% 에탄올 재추출물 (YHE)은 항산화 활성이 향상되었으며, 2000 μg/mL 농도에서도 세포 독성이 관찰되지 않았다. 또한 YHE가 LPS 자극에 의한 iNOS의 발현을 억제함으로써 항염증 물질로서 가치가 있음을 확인하였다. 따 라서 1차 열수 추출물과 70% 에탄올 재추출은 Ya-Ha-Rak 복합물의 항산화 및 항염 효능을 효과적으 로 향상시키는 것으로 나타났다. 본 연구는 천연물 복합 추출물에서 추출 공정 최적화가 생리활성 발현 에 중요한 요소임을 제시한다.

In this study, by sequentially applying primary water extraction followed by 70% ethanol re-extraction to the Ya-Ha-Rak complex, the antioxidant and anti-inflammatory activities according to the extraction method were compared and analyzed. The first hydrothermal extract (YHD) showed antioxidant activity, but the anti-inflammatory activity was limited. On the other hand, 70% ethanol re-extract (YHE) showed improved antioxidant activity, and cytotoxicity was not observed even at a concentration of 2000 μg/mL. In addition, it was confirmed that YHE is valuable as an anti-inflammatory substance by inhibiting the expression of iNOS by LPS stimulation. Therefore, it was found that the first hydrothermal extract and 70% ethanol re-extraction effectively improved the antioxidant and anti-inflammatory efficacy of the Ya-Ha-Rak composite. This study suggests that optimization of the extraction process from natural product complex extracts is an important factor in the expression of physiological activity.

Abstract
요 약
1. Introduction
2. Materials and Methods
    2.1. Water Extraction Process
    2.2. Ethanol Extraction Process
    2.3. Samples, Reagents, and Instruments
    2.4. Measurement of DPPH RadicalScavenging Activity
    2.5. Measurement of ABTS+ RadicalCation Scavenging Activity
    2.6. Determination of Total FlavonoidContent
    2.7. Determination of Total PolyphenolContent
    2.8. Cell CultureRAW 264.7 murine
    2.9. MTT Cytotoxicity Assay
    2.10. Measurement of Nitric Oxide (NO)Inhibitory Activity
    2.11. Western Blot Analysis
    2.12. Real-Time PCR
    2.13. Statistical Analysis
3. Results and Discussion
    3.1. Total Flavonoid and PolyphenolContents
    3.2. DPPH and ABTS+ Radical ScavengingActivities
    3.3. Anti-Inflammatory Effects of YHE inRAW 264.7 Cells
4. Conclusion
Acknowledgments
References

• Su-Yeon Kim(Department of Cosmetic Science, Daegu Haany University, Gyeongsan, Gyeongbuk, 38610, South Korea)
• Young-Ah Jang(Department of Cosmetic Science, Daegu Haany University, Gyeongsan, Gyeongbuk, 38610, South Korea, Department of Cosmeceutical Science, Daegu Haany University, Gyeongsan, Gyeongbuk, 38610, South Korea, Hybrid Cosmetic Lab, Daegu Haany University, Gyeongsan, Gyeongbuk, 38610, South Korea) Corresponding author
• Ga-Eul Kim(Department of Cosmetic Engineering, Daegu Haany University, Gyeongsan, Gyeongbuk, 38610, South Korea)
• Cheol-Hee Ju(Department of Cosmeceutical Science, Daegu Haany University, Gyeongsan, Gyeongbuk, 38610, South Korea)
• Si-Hyun Park(Department of Cosmeceutical Science, Kyungsung University, Busan 48434, South Korea)
• Sareeya Reungpatthanaphong(Cosmetic Science and Natural Products, College of Health and Wellness, Dhurakij Pundit University, Bangkok, 10210, Thailand)
• Nirawan Kitprapiumpon(Cosmetic Science and Natural Products, College of Health and Wellness, Dhurakij Pundit University, Bangkok, 10210, Thailand)
• Yupaporn Sampaopan(College of Oriental Medicine, Rangsit University, Bangkok, 12000, Thailand)
• Nanthaphong Khamthong(College of Oriental Medicine, Rangsit University, Bangkok, 12000, Thailand)