Proceeding of International Conference on Medicinal Plants

ISBN : 978-62-96839-1-2 ISBN of Volume I: 978-62-96839-2-9 Proceeding of International Conference on Medicinal Plants in occasion of the 38 th Meeting of National Working Group on Indonesian Medicinal Plant 21-21 July 21 Surabaya, Indonesia Editors: Elisabeth C. Widjajakusuma Kuncoro Foe Sendy Junedi Bambang Soekardjo Retno Andayani Achmad Fudholi Lucia Hendriati Angelica Kresnamurti Organizing Committee FACULTY OF PHARMACY WIDYA MANDALA CATHOLIC UNIVERSITY in collaboration with National Working Group on Indonesian Medicinal Plants and German Academic exchange Service

DETERMINATION OF CHLOROPHYLLS AND CAROTENOIDS CONTENT IN THREE MAJOR TEAS BASED ON PEAK AREA FROM HPLC CHROMATOGRAM Wahyu Wijaya 1,2*, Heriyanto 3, Budhi Prasetyo 1,4, Leenawaty Limantara 2,3** 1 Master of Biology Program, Satya Wacana Christian University, Diponegoro 52-6, Salatiga 5711 2 Workstation of Mochtar Riady Institute for Nanotechnology, Boulevard Jend. Sudirman 1688, Lippo Karawaci, Tangerang 15811, Jakarta 3 Ma Chung Research Center for Photosynthetic Pigments, Villa Puncak Tidar N-1, Malang 65151 4 Health Science Faculty of Satya Wacana Christian University, Diponegoro 52-6, Salatiga 5711 Email : *wahyuwijaya22@gmail.com; **leenawaty.limantara@machung.ac.id Abstract : Tea is the most ancient and popular beverage world and has been traditionally known to have various merits for human health such as antibacterial, antiviral, antipyretic, diuretic, and anticarcinogenic effects. Some epidemiological studies, not only on polyphenols but also on chlorophylls, have been carried out. Various kinds of tea have been consumed as a daily beverage in Indonesia, such as green tea, oolong tea, and black tea. A simple method for the extraction of tea samples and conditions of HPLC analysis of chlorophylls and carotenoids derivatives was developed. Tea samples were extracted using acetone 1% (v/v), and this extract was injected into HPLC column. The modified HPLC procedure was developed and involved a gradient solvent system. The method consists of an elution gradient of methanol, acetone and ammonium acetate solution (1 M). After the subtraction of spectra pattern from crude extracts of tea pigment, several chlorophylls and carotenoids derivatives were found. The composition of chlorophyll and carotenoid pigments were confirmed by HPLC. Based on peak area, which was detected in λ=43 nm, pheophytin b was the dominant pigment in green tea followed by lutein, pheophytin a, β- carotene, pheophytin b-like and pheophorbide a. Lutein was the dominant pigment in black tea followed by pheophytin a, chlorophyll b, chlorophyll a, pheophorbide a. Pyropheophytin a was the dominant pigment in oolong tea, followed by pheophorbide a-like and cis-isomers form carotenoid. The pigment content based on peak area was compared in three major teas. This method is applicable to the quality control in their manufacturing processes. Keywords : HPLC analysis, peak area, green tea, oolong tea, black tea INTRODUCTION Tea is one of the most popular beverages consumed in the world. In Indonesia, tea has become one of the most popular drinks. There are three common tea products based on their processing : unfermented tea, a partially fermented tea, and a fully fermented tea [Zuo et al., 22]. These teas are popular in Indonesian markets. Besides polyphenols compound in tea, tea pigments have several health benefits, one study reported that tea pigments, a major flavonol component in black tea, have strong antioxidant potency [Leung et al., 21]. In previous studies, tea pigments were also found to be effective in preventing the occurrence and progression of precancerous liver lesions in rats [Gong et al., 2]. In addition, pheophytins a and b of the hot water extract from green tea have potent suppressive activities against chemically induced tumorigenesis in mouse skin through suppression at the tumor promotion phase [Horie and Kohata, 2]. The other chlorophyll derivatives, pyropheophytin, could be expected as one of antioxidants to prevent lipid oxidation [Cahyana et al., 1992]. Several conditions of tea manufacturing process, such as withering, rolling, drying or oxidation enzymatic, can cause degradation of chlorophylls and carotenoids. [Teranishi and Hornstein, 1995]. Chlorophylls can be degraded into their derivatives, such as 13

pheophytin, pheophorbide and pyropheophytin because of heating process and acid condition. However, carotenoids can be converted to cis-isomers especially by heating. Besides on tea flavor and taste, pigments content and composition have relationship with several health outcomes [Suzuki and Shioi, 1998]. The purpose of this study was to determine chlorophylls and carotenoids and their derivatives in three major teas based on peak area from HPLC chromatogram. The pigments were compared among three tea products and fresh tea leaves as a comparison control. Wide applications of this study are expected, for example, for safety and quality control of manufacturing teas. MATERIALS AND METHODS Materials. Fresh leaves of tea (Camellia sinensis var assamica) were obtained from Wonosari s tea plantation, an unfermented tea (Kepala Djenggot, Indonesia), a fully fermented tea (Sari Wangi, Indonesia) and a partially fermented tea (Shui Xian, Fujian, China) were purchased from a local market. Pigment Extraction. Pigments were extracted from fresh tea leaves (.5 g) by grinding with 2 ml acetone 1%. The residue was extracted again with 2 ml acetone 1%. In the case of extraction pigments from teas, teas were extracted from tea products (.5 g) by grinding with 75 ml acetone 1%. The solvent was evaporated (Heidolph Laborata 41 digital) and purged with argon gas until the solvent became completely dry. The dry pigments were re-dissolved in 1 ml acetone 1% and then filtered with.45 µm syringe filter (Milipore, MA). After filtration, crude extract of pigments was evaporated again. Before analysis, the dry pigments were diluted in 5 ml acetone 1%, then 2 µl pigment solution was directly injected for HPLC analyses. UV-Vis Spectrophoto-meter Analysis. The dry crude-pigment extracts were re-dissolved in 125 ml acetone 1%. The absorption spectra was obtained at λ=3-8 nm with UV 17, UV-Visible Spectophotometer (Shimadzu, Kyoto). Spectra patterns of crude-pigment extracts were performed using Plots32. Different spectra was performed in Spina version 3 software (created by Y. Katsumoto, Hiroshima University) HPLC Analysis. HPLC was carried out using Shimadzu chromatographic system (Shimadzu, Kyoto, Japan) equipped with a column temperature controller, using a 25 4,6 mm i.d., Shim-Pack VP-ODS C 18 column (Shimadzu, Kyoto). Analysis of chlorophylls and carotenoids was performed according to the method of Hegazi et al., and pigments were eluted at a flow rate of 1. ml per min at 3 C with a programmed binary gradient elution system according to the method. The method used consists of an elution gradient of methanol, acetone and ammonium acetate solution (1 M). Pigments were detected spectrophotometrically with a SPD-M2A photodiode array detector, measuring from 2 to 8 nm and monitoring four channels at representative wavelengths of 4, 43, 45, and 661 nm. The wavelengths used to indicate the presence of pigments: those were 4 nm for pheophorbide a, 43 nm and 661 nm for chlorophylls, and 45 nm for chlorophyll b and carotenoids. RESULT AND DISCUSSION Spectra Patterns of Crude-Pigment Extract of Tea. The spectra patterns of fresh tea leaves and three major teas were normalized at Q y band of chlorophylls (OD=1), before they were compared among each other. 14

3 2 A B C D Absorbance 1 1 2 4 5 6 7 8 Wavelength (nm) Figure 1. Absorption spectra of acetone extracts from tea products, normalized at Q y band of chlorophylls. (A) fresh tea leaves; (B) green tea; (C) black tea; (D) oolong tea. The first circle (1) showed the absorption region of carotenoids, and the second circle (2) showed the Q y absorption of chlorophylls. As a result, the absorption region of carotenoids (λ=4-5 nm) was not detected in oolong tea. The highest absorption of this region in fresh tea leaves was detected because it has not been treated like the other teas. However, this absorption in green tea and black tea still appeared. Furthermore, a small hypsochromic shift in Q y band of chlorophylls (λ max =662 nm) occurred in three major teas. In green tea and oolong tea, Q y band shifted from 662 nm to 665 nm. However, in black tea, a shift occurred from 662 nm to 664 nm. This phenomenon indicated that the formation of chlorophylls degradation products. Different Spectra of Crude Pigment Extract. The different spectra were obtained by subtracting each spectra pattern of tea products with fresh tea leaves as reference spectra. Figure 2 shows the appearance of new positive absorption bands which indicated several chlorophylls derivatives emerging due to tea manufacturing. In green tea, new bands emerged with λ max 41 nm, 56 nm, 534 nm, 63 nm, and 672 nm. Similar to green tea, new bands of oolong tea had λ max absorption 41 nm, 51 nm, 537 nm, 63 nm, and 672 nm. These absorptions were similar to λ max absorption of pheophorbide a (49 nm, 55 nm, 539 nm, 68 nm, 665 nm), pheophytin a (48 nm, 55 nm, 535 nm, 68 nm, 665 nm), pyropheophytin a (411 nm, 55 nm, 534 nm, 69 nm, 667 nm) [Suzuki and Shioi, 1998; Hegazi et al., 1998; Jeffrey et al., 1997]. However, in black tea, the other bands emerged with λ max 397 nm and 478 nm. These bands indicated another chlorophyll derivatives, such as epimer of chlorophylls [Kohata et al., 1998]. Further identification of several chlorophyll and carotenoid derivatives was confirmed by HPLC chromatogram. Pigment Identification. Each peak represents type of pigment in fresh tea leaves and three tea products. Each peak was identified by sequence of pigment polarity, comparison with peak retention times and absorption spectrum from the photodiode array data. In this study, 45 peaks, 33 chlorophylls and 12 carotenoids, were detected [Suzuki and Shioi, 1998; Hegazi et al., 1998; Jeffrey et al., 1997]. 15

1.5 1 41 A..5 56 534 63 672 -.5-1 1.5 Absorbance 1.5 397 49 57 534 478 63 672 B. -.5-1 1.5 41 1 C..5 51 537 63 672 -.5-1 4 5 6 7 8 Wavelength (nm) Figure 2. The different spectra of three major teas. (A)green tea; (B)black tea; (C)oolong tea. Table 1. Identification and Peak Area of Chlorophylls and Carotenoids Chlorophyll No. t R (min) Components Tea Leaves Green Black Oolong 1 3.72 Chlorophyllide b-like 14184 - - - 2 5.36 Chlorophyllide a-like 5733 1368 - - 3 6.99 Pheophorbide b-like 19521 71547 6889-4 7.56 Pheophorbide a-like - 454 1226 1399 5 8.17 Pheophorbide b-like - 93254-86922 6 1.56 Pheophorbide a 2822 1263214 6218-7 12.84 Pheophorbide a-like - 191844 9212-8 15.5 Pheophorbide a-like - 1723 - - 9 16.12 Pheophorbide a-like - 4827 11978 577488 1 18.68 Pheophorbide a-like - 4496-3448 11 19.72 - - - 481 12 27.26 Chlorophyll b-like - - 21694-13 29.99 Chlorophyll b-like - - 18641-14 3.4 Chlorophyll b-like - 2316 - - 15 33.27 Chlorophyll b 114574 48295 823141-16 34.27 Chlorophyll b 18977 246454 15472-17 35.2 - - + - 16

18 35.52 Chlorophyll a-like - - - 5827 19 36.2 Chlorophyll a-like 21655 + - 249 2 38.39 Chlorophyll a 666399-716393 - 21 39.16 Chlorophyll a 3994 47157 41529 3461 22 41.7 Pheophytin b-like - 85575 122935-23 42.83 Pheophytin b-like - 691728 86553-24 46.99 Pheophytin b - 379983 386845-25 48.2 Pheophytin a-like - 368 24335 49881 26 49.47 Pheophytin b - 749857 - - 27 5.16 Pheophytin a-like - 273355 187412 49754 28 56.85 Pheophytin a 342242 299216 1187271-29 57.33 Pheophytin b - - - 292147 3 6.25 Pheophytin a - 817114 176656-31 61.85 - - - 194184 32 67.43 - - - 92394 33 71.81 Pyropheophytin a - 178943 93772 1672599 Carotenoid No. C1 C2 C3 C4 C5 C6 C7 C8 C9 C1 C11 C12 t R (min) Components Tea Leaves Green Black Oolong 11.7 6 Neoxanthin 234287-139986 - 16.9 7 56847 - - - 17.1 Violaxanthi 6 n 3121 69918 - - 18.1 3 17561 4496 - - 18.3 8 599 - - - 18.9 8 655 - - - 19.8 1 Lutein 2416325 3487751 1688499-21.3 3 cis form - 12436 3942-23.3 7 cis form 38363 279166 69145-24. 4 cis form 139213 25671 11197-33.6 2 cis form - 122199-418313 62. 2 ß-carotene 149744 1859366 39926 - Blank, not identified Pigment Content Based on Peak Area. To compare the composition and content of pigments from tea leaves before and after processing, pigment analyses were carried out using fresh tea leaves (Camellia sinensis var. assamica) which were common and popular material for tea products in Indonesia. An HPLC separation profile of the acetone extract from the fresh tea leaves and three tea products is shown in Figure 1. Chlorophylls, carotenoids and their other derivatives were found in the acetone extract of fresh tea leaves and three tea products (Table 1). Pigment contents based on peak area which were detected in λ=43 nm, pheophytin b was the dominant pigment in 17

Indonesia s green tea, and followed by lutein, pheophytin a, β-carotene, pheophytin b- like, pheophorbide a and so on. The emergence of several chlorophyll and carotenoid derivatives which were not detected in fresh leaves, appeared in green tea and the other teas. Pheophytin a, pheophytin b and pheophorbide a which were the dominant pigments in green tea, had the largest peak area compared with black tea and oolong tea (Figure 4.). 3 2 1 6 C7 A. 15 2 C12 25 3 6 C7 24 28 B. 2 C12 Intensity (mau) 1 3 2 6 C7 15 2 28 C. 1 24 3 D. 33 2 9 C11 1 25 5 75 Retention time (min) Figure 3. HPLC chromatograms of acetone extracts. (A) fresh tea leaves; (B) green tea; (C) black tea; (D) oolong tea. Detection was at 43 nm. Peak number shown the dominant pigments. For peak number, see table 1. The other derivatives of chlorophylls such as epimer of chlorophyll a. chlorophyll b, pyropheophytin a were also seen in green tea and the other teas. Formation of derivatives of chlorophylls a and b is considered to be the heating effect that happened during the tea processing, contributed to the conversion of chlorophyll a to pheophytin a and chlorophyll b to pheophytin b [Carper, 1989; Higashi-Okai et al., 1998; Canjura et al., 1991; Schwartz et al., 1981]. This might be caused by multiple drying in Indonesia s green tea processing at the range of temperature of about 7-3 C, at which chlorophyll a and b might be easily changed to form epimers by heat [Kohata et al., 1991]. Figure 4 shows that chlorophyll a was not present, but chlorophyll b was still detected in green tea (with peak area = 48295), this indicated that chlorophyll a was more unstable than chlorophyll b. On the other hand, the peak area of pheophytin b was larger than pheophytin a, this also indicated that chlorophyll b and its derivatives were more stable than chlorophyll a. Chlorophyll a, when it was converted to pheophytin a might be more easily and quickly degraded into its derivatives, especially pyropheophytin a [Schwartz and Von Elbe, 1983]. In addition, chlorophyll a was degraded earlier than chlorophyll b into its derivatives such as, chlorophyll a-like, chlorophyll a epimer and especially pheophorbide a-like which was found in many quantities in acetone extract of green tea. 18

In Sari Wangi, one of the popular black tea in Indonesia, lutein was the dominant pigment in black tea, followed by pheophytin a, chlorophyll b, chlorophyll a, and pheophorbide a (Table 1). Chlorophyll a remained in black tea with a peak area larger than green tea and oolong tea, while chlorophyll b appeared in black tea and green tea but it was not present in oolong tea. In addition to the evidence mentioned above, the peak area between the peak of chlorophyll a and chlorophyll b in black tea was almost the same, the peak area of chlorophyll a was 716393 and chlorophyll b was 823141. For this comparison, the ratio of both percentage peaks area in fresh tea leaves was about 5:1, this indicated that chlorophyll a was more unstable than chlorophyll b (Figure 4). In manufacturing black tea, the main process is enzymatic oxidation rather than drying process, so they are very rich in flavor. [Teranishi and Hornstein, 1995]. This was another evidence of chlorophyll a, chlorophyll b and some carotenoids such as lutein and β-carotene which were still present in black tea. Figure 4. Comparison of the peak area of chlorophylls and their derivatives contained in acetone extracts from tea leaves and three tea products. Shui Xian oolong tea is made by a combination of enzymatic oxidation and multiple heating. First, enzymatic oxidative process was activated with gradual evaporation of water in leaves after withering in the sun at 3-4 C for 3-9 minutes, then following pan-firing, these leaves were kneaded and dried twice at 95-1 C for about 12 minutes and at 6-89 C for 2-4 hours. Because of this process, pyropheophytin a and cis type carotenoids had the largest area among three major teas tested in this study (Table 1, Figure 4.). In addition, the other chlorophyll derivatives such as pheophytin a became lower in oolong tea due to their conversion to pyropheophytin a. It has been reported that the same degradation occurred in spinach during cooking, and that pyropheophytin a was detected in canned vegetables [Cahyana et al., 1992]. Chlorophyll a was converted to pheophytin a, and then pheophytin a was degraded into pyropheophytin a by heating [Schwartz et al., 1983]. Pheophytin a degraded by the loss of Mg whereas pyropheophytin a degraded by the loss of Mg and R 2 replaced by hydrogen in their structure (Figure 5). Oolong tea had more chlorophyll and carotenoid derivatives, indicating that this might be caused by the combination of pan-firing and enzymatic oxidation method. However, the heating process played a significant role in chlorophyll and carotenoid degradation. 19

Figure 5. Structure of chlorophylls and their derivatives [Suzuki and Shioi, 1998] A change of carotenoids due to heat was also observed. According to [Schwartz et al., 1983], some carotenoids were modified to their isomers due to heating, before they degraded into colourless compounds. Several cis-isomers form carotenoids emerged in large peak area (Table 1), and this might be caused by modification of carotenoids in fresh tea leaves especially β-carotene and lutein. In addition, neoxanthin was not detected in green tea but it was detected in black tea, while violaxanthin was not detected in black tea but it was detected in green tea. Only one carotenoid was detected in oolong tea, cisisomers form carotenoid had the highest peak area compared with others. Cis carotenoids in fresh tea leaves had two peaks, whereas in black tea and green tea the number of peak was increased to three and four peaks. cis-isomers form and other carotenoids decreased because of the combination of excessive heating and oxidation in oolong tea processing. CONCLUSION The results conclude that pheophytin a, pheophytin b and pheophorbide a in green tea had the largest peak area compared with black tea and oolong tea. Pyropheophytin a in oolong tea had the largest peak area compared with green tea and black tea. In addition, only one carotenoid was detected in oolong tea, cis-isomers form carotenoid had the highest peak area percentage compared with others. Based on peak area which was detected in λ=43 nm, pheophytin b was the dominant pigment in green tea followed by lutein, pheophytin a, β-carotene, pheophytin b-like and pheophorbide a. Lutein was the dominant pigment in black tea followed by pheophytin a, chlorophyll b, chlorophyll a, pheophorbide a. Pyropheophytin a was the dominant pigment in oolong tea, followed by pheophorbide a-like and cis form carotenoid. ACKNOWLEDMENT Wahyu Wijaya would like to thank the Ministry of National Education for the Beasiswa Unggulan program which has been held at Satya Wacana Christian University. Special gratitude to Ma Chung University for the credence and laboratory support. This work was also supported by Ma Chung Research Grant II. REFERENCES Cahyana, A. H.; Shuto, Y.; Kinoshita Y. 1992. Pyropheophytin a as an antioxidative substance from the marine alga, Arame (Eisenia bicyclis). Biosci Biotech Biochem., 56 (1), 1533-1535. Canjura, F. L., Schwartz, S. J., and Nunes, R. V. 1991. Degradation kinetics of chlorophylls and chlorophyllides. J. Food. Sci. 56 : 1639-1643. Carper, J. 1989. Seaweed or kelp, In The food pharmacy. Bantam Books, New York. p. 264-268. 11

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