Background : Recently, there has been an increase in the use of herbal drugs for healthcare. The reference standards of herbal medicine is used to quality control for herbal medicines. It have been produced and re-evaluated by Ministry of Food and Drug Safety, starting from 2002. In this study, we re-tested the quality of a variety of Herbal Reference Standards (HRS). We analyze the HRSs with identification test by High Performance Thin Layer Chromatography (HPTLC). All test is based on experimental method of the Korean Pharmacopoeia, the Korea Herbal Pharmacopoeia. Methods and Results : To check the stability of reference standards of herbal medicine, we annually performe real-time monitoring under storage condition (4℃). The aim of this study is to perform a stability testing for 200 national reference standards to ensure the reliability of standards. For example, there are reference standards of Osterici Radix, Polygoni Multiflori Radix, Lithospermi Radix etc. It was achieved by comparing the colors and Rf values of the bands in TLC with those of the marker compounds using High Performance Thin Layer Chromatography (HPTLC). Tests for each item are based on experimental methods of the official compendium or the previous study on establishment of HRSs. Through re-test system, we confirm stability and reliability of HRSs condition. Also, based on the results, these HRSs will be continuously supplied as Herbal Reference Standards of MFDS. Conclusion : As the result, the HPTLC results showed the same TLC patterns as compared with previous reports. Based on the all results that we will update the each certificate of analysis. We want to contribute for quality control of herbal medicines.

Background : The reference standards of herbal medicine is used to quality control for herbal medicines. It is important that National Reference Standards maintain high stability of herbal medicines during all periods from the manufacture to the storage. In order to control quality of herbal medicines, the control reference standards (CRSs) have been produced by Ministry of Food and Drug Safety, starting from 2002. The designated CRSs are essential tests and assays to be carried out in accordance with the official compendium such as the Korean Pharmacopoeia and the Korean Herbal Pharmacopoeia. Methods and Results : The aim of this study is to perform a stability testing for 80 national reference standards to ensure the reliability of standards. For example, there are reference standards of Sweroside, Liquiritin, Wogonin etc. First of all, We randomly select three samples of CRSs and weight them and mobile phase is made suitable for the experimental conditions. We analyzed the purity value of each CRSs in calculating absolute peak area percent, using High Performance Liquid Chromatography (HPLC) based on the method in the official compendium. And the result showed that 80 evaluted CRSs were revealed more than 95% purity value. The stability of the CRSs under storage condition (4℃) were tested annually for 3 randomly selected samples. Conclusion : There are no significant difference among observed contents. The results of stability assessment represent suitable for quantitative analysis: less than 5% uncertainty of measurement. Even though this statistical analysis identified that stability was maintained for most of the products, some of them still need to be continuously monitored.

Background : Alpinia Officinarum Rhizome (高良薑) is recorded as a roots of Alpinia officinarum Hance (Zingiberaceae) in Korea Pharmacopoeia and there is no established confirmation test and method for quantitative determination using marker compound. Using the six marker compounds, pinocembrin, (5S)-5-hydroxy-7- (4-hydroxy-3-methoxyphenyl) -1-phenyl-3-heptanone, pinocembrin, galangin, kaemferide, galangin 3-methyl ether, A simultaneous analysis method was developed to utilize the quality control. Methods and Results : In this study, YMC-pack column (ODS C18, 250 × 4.6 ㎜, 5 ㎛) from YMC was used and the temperature was set at 30℃. A soultion of 0.1% formic acid was using water (mobile phase A) and acetonitrile (mobile B), respectively, and the mobile phase B was set to 15 - 35% for 0 - 15 minutes and 35 - 40% for 15 - 60 minutes. And 270 ㎚ was set as the detection wavelength. The order of the components is pinocembrin (1, Rt: 23.2 min), (5S)-5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-phenyl-3-heptanone (2, Rt: 37.8 min), pinocembrin (3, Rt: 44.9 min), galangin (4, Rt: 46.7 min), kaemferide (5, Rt: 48.6 min), galangin 3-methyl ether (6, Rt: 52.8 min) and Based on the retention time, ultraviolet spectrum and MS value, it was confirmed by comparison with standard compounds. Conclusion : In this study, we developed the simultaneous analysis method by six marker components of the Alpina officinarum Hance extract to develop a quality test method to scientifically identify Alpinia officinarum Hance.

Background : Kalopanacis Cortex (海桐皮) is listed in「The Korea Pharmacopoeia (KP)」as the original plant of Kalopanax septemlobus (Thunb.) Koidz. However, the dried cortex of Erythrina eariegata L (刺桐) is an adulterant, one of the most indiscriminately used herbal medicines because of its similar morphologic. Due to the morphological similarities of the dried cortex of this plant to those of K. septemlobus which is used as a substitute herbal for E. eariegata, distinguish these two species is extremely difficult. Meanwhile, K. septemlobus is a polymorphic species, its morphological characteristics showed great diversity due to the different geographical and environmental factors. For this reason, it is conducted to develop molecular markers to distinguishing these K. septemlobus with E. eariegata by using conventional polymerase chain reaction (PCR). Methods and Results : In this study, In order to clearly identify origin of K. septemlobus, E. eariegata was analyzed from four barcode regions of chloroplast DNA (psbA-trnH, rbcL, matK, atpH-atpF) and nuclear ribosomal DNA (ITS2) to evaluate the ability of discrimination for each barcode region. The present study aimed to analyze the percent of variable sites were provided the highest ITS2 (2.3%) followed by rbcL (8.2%), in oder to develop a species-specific primer that can distinguish K. septemlobus form E. eariegata. Conclusion : The INDEL markers were developed based on the divergence of each sequence, and it is possible now to identify the two species of K. septemlobus with just a single performance of PCR. This will not only prevent misused of the plant, but also to maintain the quality of the herbal medicine as well as to verify and guarantee safety for public health.

Background : In the KHP (the Korea Herbal Pharmacopoeia), the Cuscutae Semen (菟絲子) is defined as the seed of the Cuscuta chinensis Lamark (family: Convolvulaceae). Using authentic raw herbal materials is fundamental to herbal medicine quality and Cuscutae Semen is widely distributed in many asian countries. Due to having tiny bodies of seeds, it is extremely difficult to differentiate them from adulterants and closely related species by morphologic characteristics, leading to serious safety problems. For this reason, there was conducted to develop molecular markers to distinguishing these Cuscuta chinensis with Cuscuta japonica and Cuscuta pentagona by using conventional polymerase chain reaction (PCR). Method and Results : In this study we developed a clearly and efficient method to identify Cuscutae Semen on the market. These samples (C. chinensis, C. japonica and C. pentagona) were analyzed from two barcode regions of chloroplast DNA (rbcL, psbA-trnH) and nuclear ribosomal DNA (ITS2). Based on genetic distance, the precent of variable sites were provided the highest psbA-trnH value (38.7%), followed by ITS2 (23.4%), rbcL (9.9%), in order to develop a specific primer that can distinguish C. chinensis, C. japonica and C. pentagona. Conclusion : From the above results, DNA barcoding was proved to be a successful tool for authentication the three species of Cuscutae semen. The adoption of DNA barcoding as an authentication tool by food safety agencies can safeguard the interests of both consumers and traders.

Background : Curcumae Longae Rhizoma (薑黃) is listed in「The Korea Pharmacopoeia (K P)」as the original plant of Curcuma longa L (Zingiberaceae). Meanwhile, Zeodariae Rhizoma (莪朮) is listed in 「The Korea Pharmacopoeia (KP)」as the original plant of C. phaeocaulis, C. aromatica and C. Kwangsiensi (Zingiberaceae). Due to the morphological similarities of the dried roots of this plant to those of C. phaeocaulis, C. aromatica and C. Kwangsiensis which is used as a substitute herbal for C.longa, distinguish these four species is extremely difficult. Methods and Results : A total of 90 collected samples were used in this study, In order to clearly distinguish of Curcumae Longae Rhizoma and Zeodariae Rhizoma were analysis based on sequence of the chloroplast DNA (trnK, rbcL, trnL-F, atpB-rbcL) and nuclear ribosomal DNA (ITS2). The present study aimed to analyze the percent of variable sites were provided the highest trnK (2.3%), in oder to develop a species-specific primer that can distinguish C.longa form C. phaeocaulis, C. aromatica and C. Kwangsiensis. In addition, the complete chloroplast genome of C. longa were sequenced by a 454 sequencing platform, and the structure of the obtained chloroplast genome was also analyzed. the result used that INDEL (insertion/deletion) marker for distinguish C.longa form C. phaeocaulis, C. aromatica and C. Kwangsiensis. Conclusion : The INDEL markers were developed based on the divergence of each sequence, and it is possible now to identify the four species of Curcumae Longae Rhizoma with just a single performance of PCR. This will not only prevent misused of the plant, but also to maintain the quality of the herbal medicine as well as to verify and guarantee safety for public health.

Background : Chrysanthemi Indici Flos (甘菊) is listed in 「The Korea Herbal Pharmacopoeia (KHP)」as the original plant of Chrysanthemum indicum L. C. indicum was one of the most representative medicinal plants in Asteraceae, Dried flowers of this plant have been valid chemical composition such as flavonoids, phenylpropanoids, terpenoids, and polysaccharides, possessing broad spectrum antibacterial, antiviral, antihypertensive and anti-oxidation functions. Meanwhile, C. indicum was a polymorphic species, its morphological characteristics showed great diversity due to the different geographical and environmental factors. For this reason, there was conducted to develop molecular markers to distinguishing these C. indicum with C. morifolium, C. zawadskii var. latilobum and Aster spathulifolius by using conventional polymerase chain reaction (PCR). Methods and Results : In this study, In order to clearly identify origin of Chrysanthemi Indici Flos, these samples (C. indicum, C. morifolium, C. zawadskii var. latilobum and A. spathulifolius) were analyzed from five barcoding regions of chloroplast DNA (rbcL, matK, rpoB, atpF-atpH) and nuclear ribosomal DNA (ITS2) to evaluate the ability of discrimination for each barcoding region. Based on genetic distance, the percent of variable sites were provided the highest ITS2 value (56.9%), followed by atpF-atpH (48.18%), matK (27.2%), psbK (8.2%), and rbcL (2.9%). Comparative analysis based on the complete genome sequence of the petL-petG region INDEL (insertion/deletion) that the gene annotations were registered to the GenBank (accession number: JN-867592.1, NC-020092.1, MF-034027.1, NF-279514.1). Conclusion : From the above results, we may suggest that the petL-petG region INDEL analysis were conducted for molecular authentication of four plants (C. indicum, C. morifolium, C. zawadskii var. latilobum and A. spathulifolius). The findings of results indicated that petL-petG region might be established INDEL analysis systems and hence were proved to be an effective tools for molecular evaluation and comparison of “Chrysanthemi Indici Flos” with other plants.