[ Transaction ] Journal of the Korean Society of Clothing and Textiles Vol. 35, No. 12 (2011) p.1507~1517 http://dx.doi.org/10.5850/JKSCT.2011.35.12.1507 Analysis of Dye Extracted from Phellodendron Bark Using Liquid Chromatography Cheunsoon Ahn† Dept. of Fashion Industry, University of Incheon Received December 7, 2011; Revised December 18, 2011; Accepted December 22, 2011 Abstract Berberine, palmatine, and Phellodendron bark dye was prepared in methanol for HPLC-DAD-MS analysis of liquid dye. Silk was dyed using berberine, palmatine, and Phellodendron bark dye prepared in water. The dye was extracted from the dyed silk using the HCl/methanol/water (2:1:1 v/v/v) solvent system with a slight modification. The liquid dyes and the dye extracted from the silk samples dyed with the three dye sources were examined using the HPLC-DAD-MS analysis to simultaneously detect berberine and palmatine from the plant dye and the dyeings. Colorimetric measurement was carried out using a spectrophotometer to examine the color and the intensities of berberine, palmatine, and Phellodendron bark dyed silk samples. From the liquid dyes, berberine eluted at 5.21 min with the molecular cation m/z=336 and the UV spectrum confirming that the product was berberine. Palmatine eluted at 5.12 min with the molecular cation m/z=352 and the UV spectrum confirming that the product was palmatine. From the silk dyed with berberine and palmatine dye, berberine and palmatine species eluted at 5.35 min and 5.24 min, respectively. From the silk dyed with Phellodendron bark, berberine and palmatine were detected simultaneously at 5.35 min and 5.26 min, respectively. All three dyes had yellow hue while palmatine dyed silk showed the highest hue and chroma. Palmatine dyed silk showed the highest K/S value that indicated the strongest color intensity and the highest dye uptake. Key words: Phellodendron bark, Amur cork tree, Berberine, Palmatine, HPLC-DAD-MS I. Research Background of folk medicine in Asia from the ancient times (Drasar & Moravcova, 2004). The major chemical constituents Phellodendron, commonly called the ‘cork tree,’ is a of Phellodendron bark had been extensively studied for deciduous tree in the family of Rutaceae. The two most their application in modern pharmaceuticals owing to well known species of the cork tree are Phellodendron their medicinal effects such as anti-microbial, anti- amurense Rupr. and Phellodendron chinense Rupr. inflammatory, and antiphlogistic functions (Hu et al., These two species are commonly called amur cork tree 2010; Lee et al., 1999). Lee et al. (1999) used narrowbore and Chinese cork tree respectively (Chan et al., 2007; high performance liquid chromatography on Pellodendron Hu et al., 2010) and the plants were native to diverse bark and Coptidis Rhizoma, the biological name for regions of Asia. The bark of Phellodendron, often huang lian, to simultaneously determine berberine and called ‘huang bai,’ had been widely used as the source palmatine and the recovery rate of the two compounds from the plant material. They found out that in both †Corresponding author E-mail: [email protected] plants berberine recovery rate was about twice as that This research was supported by Basic Science Research of palmatine. Chan et al. (2007) developed a near in- Program through the National Research Foundation of frared spectroscopy (NIRS) method for the rapid di- Korea (NRF) funded by the Ministry of Education, Science fferentiation of the two species, amur cork tree and Chinese and Technology (2010-0017075). cork tree, and the content of berberine, palmatine, and – 1507 – 100 Journal of the Korean Society of Clothing and Textiles Vol. 35 No. 12, 2011 jatrorrhizine. In all the extraction methods used, berberine zing functions as pharmaceutical substance are all due was the most abundantly recovered compound fol- to its chemical structure containing the cationic nitrogen lowed by palmatine which was about three quarter of atom (Kim et al., 2003). the amount of berberine, and jatrorrhizine was present While berberine is widely known to be responsible in only a small amount (Chan et al., 2007). Hu et al. for the color of dye extracted from Phellodendron bark, (2010) examined Phellodendron amurense Rupr. and Phellodendron bark is composed of a large number of Phellodendron chinense Rupr. using high performance different compounds, many of them which belong to liquid chromatography coupled with electrospray ioni- the protoberberine group with cationic nitrogen atom in zation mass spectrometry and identified 24 different their structures. Protoberberine alkaloids isolated from alkaloids including berberine, palmatine, jatrorrhizine, Phellodendron bark in recent studies are berberine, pal- tetrahydropalmatine. Yin et al. (2009) used high perfor- matine, jatrorrhizine, magnoflorine, phellodendrine, mance liquid chromatography coupled with a diode etc. and among these compounds more attention has + array detection method to investigate the simultaneous been paid to berberine and palmatine (C21H24NO4 , detection of 11 active pharmaceutical compounds inclu- molecular weight: 352.40) (Lee et al., 1999; Lee et al., ding berberine from the well-known Chinese traditional 2007; Li et al., 2007; Suto et al., 1997) (Fig. 1). The iso- medicine. lated pure powders (or crystals) of berberine and pal- Another important characteristic of Phellodendron matine are yellow in color (Lee et al., 2007). Another bark is that it has a deep yellow color which can be protoberberine alkaloid of Phellodendron bark which extracted to dye fibers. Having its root in Asia, Phello- received interest next to palmatine in the pharmaceu- + dendron bark has been one of the major sources of tical area is jatrorrhizine (C20H20NO4 , molecular weight: yellow dye used in historic Asia for fibers and paper 338.38). While jatrorrhizine is known to have the same materials not only for its color but also for its anti- medicinal effect as berberine or palmatine, the review bacteria and anti-insect effects (Leona & Lombardi, of previous literatures in the pharmaceuticals indicated 2007; Nakamura et al., 2009). With the growing interest that it has been seldomly used as the standard com- in natural dyes and dyeings, Phellodendron bark has pound in the medicinal studies. This is probably due to been studied widely in terms of its dyeing properties on the fact that the standard compound of jatrorrhizine is different fiber types. not sold by the reliable global chemical suppliers. While Major coloring compound of Phellodendron bark is several studies reported the success in the simultaneous + known as berberine (C20H18NO4 , molecular weight: isolation of berberine and jatrorrhizine from the similar 336.36), which belongs to the protoberberine group plant (Hu et al., 2010) their identification based on the within the isoquinoline alkaloids (Wikipedia, 2011) mass spectral data is difficult since the molecular weight (Fig. 1). Berberine is one of the few cationic dyes difference between the two compounds are extremely among the natural plant dyes. Its high affinity to protein small, 336.36 for berberine and 338.38 for jatrorrhizine. fibers as dyeing substance and its anti-microbial, deodori- And Rueffer et al. (1983) reported that when (S)-reticu- line and (S)-protosinomenine were fed to Berberis stolo- nifera plant, berberine was first synthesized and then it transformed into jatrorrhizine in vivo, suggesting the transformation of berberine into jatrorrhizine during the analytical procedure. Jatrorrhizine is said to be brown red in color (Shaanxi Hongkong Biological Techno- logy Co., Ltd., 1999-2010). It is reported that berberine is the most abundant of all the protoberberine alkaloids in the bark of Phello- Fig. 1. Structures of berberine and palmatine (m.w. is dendron. According to Lee et al. (1999) berberine and molecular weight). palmatine contents in Phellodendron bark were 0.529% – 1508 – Analysis of Dye Extracted from Phellodendron Bark Using Liquid Chromatography 101 and 0.299% respectively when the bark was extracted search was to examine the dye extracted from Phello- using 2% hydrochloric acid with cobalt thiocyanate dendron bark to simultaneously detect berberine and and dichloroethane. Li et al. (2007) examined the effect palmatine using the HPLC-DAD-MS instrument. And of water and drought condition on the production of to verify the method of extracting dye from the dyed berberine and palmatine in 100g of amur cork tree see- silk samples so that both berberine and palmatine can dlings. The seedlings were extracted using 60% ethanol be detected simultaneously from the silk samples dyed and analyzed using HPLC. The results indicated that with Phellodendron bark. This research also examined berberine content was by far higher than palmatine or the color difference between the Phellodendron bark any other protoberberines regardless of the growth con- dyed silk and the silk dyed with reference standards to dition. Simultaneous detection of berberine and pal- investigate the effect of berberine and palmatine on the matine in Phellodendron bark and other similar bark yellow color of the Phellodendron bark dyed silk. This species had been studied extensively in pharmaceuticals research utilized the high performance liquid chroma- using the HPLC instrument (Lee et al., 1999; Suto et tography (HPLC) instrument tripled with a diode array al., 1997; Yin et al., 2009). detector (DAD) and a mass selective detector (MSD) While berberine has been generally regarded as the for examining