High Performance Liquid Chromatographic Determination of Pheophorbide-a and Its Related Chlorophyll Derivatives in Tea Leaves.

Abstract

A simple method for the extraction of tea samples and conditions of HPLC analysis of pheophorbide-a (PB-a) and its related chlorophyll derivatives was developed. Tea samples were extracted with 85% acetone (v/v) with this solution injected directly into the HPLC column. The modified HPLC procedure developed included a gradient solvent system in which solvent A (95% ethanol (v/v) containing 0.005 M sodium chloride) and solvent B (80% ethanol (v/v) containing 0.005 M sodium chloride) were the mobile phase. PB-a, its derivatives and their C-10 epimers could be clearly separated and determined within 35 min. This analytical method could be routinely used to determine low levels of PB-a content (<10 mg/100g) and its related individual chlorophyll derivatives in green teas. Hence, it is applicable to the safety and quality control of green teas.Keywords: pheophorbide-a, chlorophyll derivatives, HPLC analysis, tea leaves Pheophorbide-a (PB-a) is one of the photosensitive derivatives formed after chlorophyll decomposition and is known to cause dermatitis to human and animal skins (Hashimoto & Tsutsumi, 1963; Tamura et al, 1979). In 1981, the standards of the contents of PB-a and total PB-a (PB-a+pB-a produced by chlorophyllase activities after incubation (Kan et al, 1982)) in processed chlorella were established by the notification of the chief of a environment sanitation bureau in the Ministry of Welfare (Kanshoku No. 99).Green tea is thought to contain low levels of PB-a, because the enzyme activity in green tea leaves is inactivated by steaming immediately after plucking; however, very recently we first found the important fact that chlorophyllase activitles still remain in green teas (Kohata et al, 1997). The measurement of PB-a content in green tea was first carried out about 30 years ago by paper chromatography (Saijo, 1970). There has been no recent research on PB-a levels in green tea. We therefore should reinvestigate the accuracy of the PB-a content in green tea. PB-a is not water-soluble; therefore, it is not extracted in tea infusion at all, suggesting that green tea is absolutely safe for drinking. However, the demantd for green tea as an edible tea has recently been increasing. We therefore should also reevaluate the safety of green tea on the basis of PB-a content.The conversion rate of chlorophyll to pheophytin is a very useful indicator of the degree of discoloration of green tea, hence green tea quality. That is because the first step in discoloration during manufacturing or storage is due to the conversion of chlorophyll to pheophytin (Tanaka & Hara, 1972). This conversion rate has been obtained according to the colorimetric method (Tanaka & Hara, 1971) Hashimoto's procedure (Hashimoto, 1985) applied to the deterrnination of PB-a in chlorella is widely used. Whereas this method is a modification of a conventional colorimetric method (Kan et al, 1982) and has much better accuracy than the colorimetric method, there is still room for improving the otherwise tedious sample preparation method for HPLC an...

Comparison of the Chemical Components of Powdered Green Tea Sold in the US

“…We used a HPLC system (LC10ADvp, Shimadzu, Kyoto, Japan) for the analysis. First, we determined the contents of chlorophyll a (Chl a), chlorophyll b (Chl b), pheophytin a (Phy a), and pheophytin b (Phy b) according to the method presented by Kohata et al (1998) using the system, as these pigments easily deteriorate (Haraguchi 2002). The conversion percentage of chlorophyll to pheophytin was calculated according to Kohata et al (1999) using the analytical results.…”

Antihipercolesterolemia Effects and Acute Toxicity Test of Pheophytin on Green Tea

“…Green tea is chosen because it is believed to reduce weight. The compounds contained in green tea are caffeine, polyphenols, essential oils, cellulose, crude fiber, protein, theobromine, tannins, amino acids, manganese, potassium, phosphorus, vitamins and chlorophyll [6,7,8]. Chlorophyll is the dominant pigment in green tea [7], but chlorophyll is easily degraded into its derivatives due to the effects of heating and storage [9].…”

“…The compounds contained in green tea are caffeine, polyphenols, essential oils, cellulose, crude fiber, protein, theobromine, tannins, amino acids, manganese, potassium, phosphorus, vitamins and chlorophyll [6,7,8]. Chlorophyll is the dominant pigment in green tea [7], but chlorophyll is easily degraded into its derivatives due to the effects of heating and storage [9]. Some examples of chlorophyll degradation products that are generally formed due to these effects are pheophytin and pheeophorbide [7,9,10].…”

“…Chlorophyll is the dominant pigment in green tea [7], but chlorophyll is easily degraded into its derivatives due to the effects of heating and storage [9]. Some examples of chlorophyll degradation products that are generally formed due to these effects are pheophytin and pheeophorbide [7,9,10]. The content of pheophorbide in green tea according to [7] is less than that of pheophytin.…”

“…Some examples of chlorophyll degradation products that are generally formed due to these effects are pheophytin and pheeophorbide [7,9,10]. The content of pheophorbide in green tea according to [7] is less than that of pheophytin. This fact occurs because the activity of the chlorophyllase enzyme in the tea is deactivated due to the steaming process after picking, while the formation of pheophytin is caused by the storage process [7].…”