Melatonin in Chinese medicinal herbs

Life Sciences 73 (2003) 19 – 26 www.elsevier.com/locate/lifescie

Melatonin in Chinese medicinal herbs Guofang Chen a, Yushu Huo b, Dun-Xian Tan c, Zhen Liang a, Weibing Zhang a, Yukui Zhang a,* a

Dalian Institute of Chemistry and Physics, Chinese Academy of Sciences, 161 Zhong Shan Road, Dalian 116011, China b Department of Microbiology, The University of Texas, Health Science Center at San Antonio, San Antonio, TX, USA c Department of Cellular and Structural Biology, The University of Texas, Health Science Center at San Antonio, San Antonio, TX, USA Received 1 August 2002; accepted 18 October 2002

Abstract Melatonin is a highly conserved molecule that not only exists in animals, but also is present in bacteria, unicellular organisms and in plants. Since melatonin is an antioxidant, in plants melatonin was speculated to protect them from intrinsic and environmental oxidative stress. More importantly, melatonin in edible plants inevitably enters animals and human through feed and food. In this study, more than 100 Chinese medicinal herbs were analyzed using the methods of solid phase extraction and HPLC-FD on-line with MS to determine whether melatonin is present in these commonly used herbs. Melatonin was detected in majority of these plants. Sixty-four of them contain melatonin in excess of 10 ng per gram dry mass. Melatonin levels in several herbs are in excess of 1000 ng/g. It is well known that normal average physiological plasma levels of melatonin are only 10– 60 pg/mL. These high level-melatonin containing plants are traditionally used to treat diseases which presumably involve free radical damage. The current study provides new information concerning one potentially effective constituent present in a large number of medicinal herbs. The results suggest that these herbs should be reevaluated in reference to their nutritional and medicinal value. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Melatonin; Plant; Herbs; Antioxidant; HPLC; MS

Introduction Melatonin, a derivative of an essential amino acid, was first isolated from the bovine pineal gland and identified as N-acetyl-5-methoxy-tryptamine by Lerner and co-workers (Lerner et al., 1958). Since then, * Corresponding author. Tel.: +1-86-411-369-3427(O); fax: +1-86-411-364-5011(H). E-mail addresses: [email protected], [email protected] (Y. Zhang). 0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(03)00252-2

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G. Chen et al. / Life Sciences 73 (2003) 19–26

melatonin has been found to be present in almost all vertebrates which have been tested (Reiter, 1991). Melatonin’s synthesis and secretion from the vertebrate pineal gland exhibits a circadian rhythm with highest levels during the scotophase and baseline levels during the photoperiod. The synthesis of melatonin is regulated by the prevailing photoperiod with light exposure inhibiting the production of melatonin in vertebrates (Reiter, 1991). The circadian rhythm of melatonin is also compromised with aging and certain medical conditions including heart diseases and neurodegenerative disorders (Pang et al., 1984; Sakotnik et al., 1999; Liu et al., 1999). Several physiological functions of melatonin have been documented including reproductive regulation, immunoresponsiveness, sleep modulation and as a signal transduction of darkness (Reiter, 1991). Recently, melatonin was proved to be a potent free radical scavenger and a broad spectrum antioxidant (Tan et al., 1993, 2002). In addition to its presence in vertebrates, melatonin is also found in bacteria, unicellular organisms, fungi and plants (Manchester et al., 1995; Poeggeler et al., 1991; Hardeland, 1999; Reiter et al., 2001). It seems that melatonin may have evolved in all evolutionary life forms from simplest bacteria to humans. As a ubiquitously distributed molecule, melatonin was first identified in edible plants by Hattori et al. (1995). Since then, melatonin has been found in many plants and in the different parts of plants (Dubbels et al., 1995; Murch et al., 2000; Van Tassel et al., 2001), seeds (Manchester et al., 2000), and it has been reported to be in extremely high levels in several medical herbs including feverfew and St. John’s wort (Murch et al., 1997). In current study, using the solid phase extraction combined with HPLC/MS 108 Chinese medicinal herbs have been analyzed for the presence of melatonin and it was found, in widely varying concentrations, in all of them.

Materials and Methods Chemicals Melatonin and other reagents were purchased from Sigma (St. Louis, MO, USA). All medicinal herbs (dried powder) derived from the flowers, seeds, leaves, roots, and stems of each herb were obtained from Dalian Medical Material Company (Dalian, China). The quality of these medicinal herbs was controlled with a national standard. The water used was purified using a Milli-Q system (Millipore, Bedford, MA, USA). Melatonin extraction Two grams of herbal powder were suspended in 20 mL methanol and ultrasonicated for 30 min at room temperature. After centrifugation at 4000 rpm for 10 min, the supernatant was filtered by a filter (0.2 Am 25 mm, Millipore). The supernatant was then dried under the nitrogen gas. The residues were redissolved in 2 mL of 5% methanol-water solution for further isolation of melatonin using the solid phase extraction (SPE). The C18 SPE cartridge (5 mL, C18, 200 mg, Dalian Institute of Chemistry and Physics, Chinese Academy of Sciences) was first activated with 10 mL methanol followed by 10 mL of pure water. Two mL prepared samples were added to the column. After drying 10 mL of 5% methanolwater solution was used to elute the interference impurities. Finally, the retained melatonin was eluted at


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Table 1 Melatonin recovery rates from the SPE Samples

Melatonin (Ag)

Melatonin measured (Ag)

Recovery rate %

1 2 3 4 Average

5 10 15 20

5.14 9.48 13.52 19.41

103 95 90 97 96.0 F 4.6%

a low flow-rate using 5 mL of 80% methanol-water solution; the samples were then subjected for HPLC/ MS analysis. Melatonin detection Melatonin extracted from the herbs was identified using the HPLC-fluorescence detector (HPLCFD) on-line with a mass spectrometer (MS). The HPLC system consisted of a reversed-phase 5 Am Hypersil ODS (4.6 250 mm) column connected with an Elite200II HPLC (Dalian, China), equipped with a JASCO FP-1520 intelligent fluorescence detector (Jasco, Tokyo, Japan). The mobile phase was constituted by methanol: 50 mM Na2HPO4/H3PO4 buffer (40:60, v/v) pH 4.5. The mobile phase was delivered using a P200II high-pressure constant flow-rate pump and a 7125 injector (Rheodyne, Cotati, CA, USA) at a flow rate of 1.0 mL/min. Twenty AL extracted samples were injected for the analyses. The excitation wavelength was selected at 280 nm and the emission wavelength was selected as 348 nm for melatonin detection using fluorescence. The MS instruments were the Finnigan LCQk DUO apparatus (Finnigan MAT, San Jose, CA, USA) with the LCQk DUO workstation (Core Data System Software, version 1.2). The analysis conditions for MS were sheath-gas (80 unit), auxiliary-gas (15 unit), heated capillary temperature (200 jC),vaporizer temperature (450 jC) and collision energy (28%).

Fig. 1. Relationship of the melatonin concentrations and the HPLC peak areas. Y = 12.2209 + 3.1486X. r = 0.99934.

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Fig. 2. HPLC spectrum of an extract of Shiya tea-leaf (Babreum coscluea) (a Chinese medicinal plant). The melatonin peak is indicated by an arrow with the retention time of 28 min.

Quality control for the melatonin extraction and detection To measure the recovery rate of SPE melatonin standard with known concentrations (from 5 AM to 20 AM) were subjected to the same SPE column analysis as were the herbal samples. Both the melatonin standard solutions and their respective elutes were measured by HPLC-FD and the values were compared to obtain the recovery rate. To quantify herbal melatonin levels, the melatonin standard curve was performed using the HPLC-FD within the range of 50-1000 nM. Melatonin concentrations were calculated based the HPLC peak areas.

Fig. 3. MS spectrum of the extract of Shiya tea-leaf (Babreum coscluea). The profile of this spectrum matches that of the melatonin standard.


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Statistical analysis All samples are extracted 3 times and the data are expressed as the mean values of 3 independent measurements.

Results The quality-control studies showed that the melatonin recovery rates of SPE under the current conditions ranged from the 90 to 103% (Table 1). The average recovery rate was 96.0 F 4.6%.

Table 2 Melatonin levels in 64 commonly used Chinese medicinal herbs (listed according to their concentrations of melatonin) Name

ng/g

Scientific name

Name

ng/g

Scientific name

Chantui Diding Gouteng Shiya tea-leaf Sangye Huangbo Sangbaipi Yinyanghuo Huanglian Dahuang Yuanzhi Shanyurou

3,771 2,368 2,460 2,120 1,510 1,235 1,110 1,105 1,008 1,078 850 821

Periostracum cicadae Viola philipica Cav. Uncaria rhynchophylla Babreum coscluea Morus alba L (Leaf) Phellodendron amurense Ru pr. Mori Albae (Cortex) Epimedium brevicornum Maxim Coptis chinensis Franch Rheum palmatum L., Polygala tenuifolia Willd. Coruns officinalis Sieb.

Kushen Dansnen Qinjiu Huangqin Jinqiancao Sanqi Yimucao Juhua Zicao Gegen Dazao Yejiaoteng

190 187 180 178 169 169 169 160 158 150 146 143

Longdacao Luxiancao Danghui Sangjisheng Fuling Gouqi Bihu Luhui Chuanxinlian Duzhong Laifuzi Dingxiang Fupenzi Xuanshen Huoxiang Banzhilian Suanzhaoren Wugong Jiangcan Maidong

780 750 698 648 585 530 523 516 511 497 485 446 387 342 302 257 256 248 227 198

Gentiana scabra Bge. Pirola decorata H. Angelica sinensis Oliv. Taxillus chinensis DC Poria cocos Schw. Wolf. Lycium barbarum L. Gekko japonicus (Dumeril et biron) Aloe vela L Andrographis paniculats Burm. Eucommia ulmoides Oliv Raphanus sativus L. Syzygium aromaticum L. Rubus chingii Hu Scrophularia ningpoensis Hemsl. Agastaches rugosa Lobelia chinesis Lour. Ziziphus jujuba Mill. Scolopendra subspinipes mutilang. Bombyx batryticatus Ophiopogon japonicus

Shuanhua Erzhu Gancao Dilong Shengdi Wuweizi Qinghao Banlangen Fangfeng Zhuye Shidagonglao Lianqiao Huangjing Damzjuye Xiakucao Baijing Duhuo Rouchongrong Chenpi Zhizi

140 120 112 97 97 86 84 79 60 55 52 45 45 38 34 32 31 28 25 12

Sophora flavescens Ait. Salvia miltiorrhiza Bge Gentiana macrophylla Pall. Scutellaria amoena C.H. Wright Desmodium styracifolium Merr Panax notoginsneg Burk Leonurus japonicus Houtt. Dendranthema morifolium Arnebia euchroma Pueraria lobata Willd Ziziphus jujuba Mill. Caulis Polygonam multiflorum Thunb Lonicera japonica Thunb Curcuma aeruginosa Roxb. Glycyrrhiza uralensis Fisch Pheretima aspergillum Rehmannia glutinosa Schisondra chinensis Artemisis annua L. Isatis indigotica Fort Saposhmikovia divaricata Not available Mahonia bealei (Fort.) Forsythia suspensa (Thunb.) Polygonatum sibiricum Delar Lophartherum gracile Brongn Prunella vulgaris L. Herba Patriniae scabiosaefoliae Angelica biserrata Cistanche desericola Y Citrus reticulata Blanco Galdenia jasminoides Ellis

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The ratio of signal to noise was 3 and the detection limit was 50 pg using current HPLC-FD method for melatonin detection. A linear relationship between the melatonin concentrations and the HPLC peak areas was established when the selected melatonin levels were studied (Fig. 1). Using the organic solvent combined SPE extraction, melatonin was detected in Chinese medicinal herbs by HPLC-FD. The HPLC spectrum of an extract of Shiya tea-leaf (Babreum coscluea) indicated a peak with a retention time of 28 min (Fig. 2), which is the same retention time for the melatonin standard. To verify that the HPLC-peak with a retention time of 28 min was truly melatonin, the elute of this peak was analyzed on-line by means of the MS. The profile of the spectrum of the MS obtained (Fig. 3) was the same as the melatonin standard indicating that melatonin was present in the extracts of the herbs. Using the methods mentioned above, 108 commonly used Chinese medicinal herbs were screened. Melatonin was found in a majority of them. Sixty four of these herbs contained melatonin in excess of 10 ng/g dry mass (Table 2). Among these, 39 had melatonin levels in excess of 100 ng/g and 10 of them had melatonin levels in excess of 1000 ng/g.

Discussion Melatonin was initially thought to be exclusively produced in animals where melatonin was usually portrayed as a hormone. Recently, however, melatonin was identified in plants and in the different parts of plants including roots, stems, leaves, flowers and seeds (Hattori et al., 1995; Dubbels et al., 1995; Murch et al., 1997, 2000; Manchester et al., 2000; Van Tassel et al., 2001). These findings have created a new area for melatonin research. The scientific reports of melatonin in plants have increased dramatically in the last few years (Reiter and Tan, 2002). It is speculated that one primary function of melatonin in plants may be to protect plants from adverse environmental insults and the endogenous processes that metabolically generate free radicals, since melatonin is a potent free radical scavenger (Tan et al., 2000; Hardeland et al., 2001). It is inevitable that melatonin from the plants will enter the animals via the diet. When chicks were fed melatonin-rich foodstuffs, serum melatonin levels were shown to increase (Hattori et al., 1995). Also, it is known that physiological melatonin levels contributed significantly to total antioxidant capacity of human serum (Benot et al., 1999), so it is likely that dietary melatonin, such as that from the medicinal plants, could be important in protecting against oxidative damage in animals (Reiter and Tan, 2002). Based on this concept, a large number of Chinese medicinal herbs were analyzed to determine whether these commonly used herbs contain melatonin. Using the technologies available including SPE exraction and HPLC-FD online with MS, melatonin was indentified in majority of these herbs. Sixty-four of 108 Chinese medicinal herbs screened contained melatonin in excess of 10 ng/g dry mass. Melatonin levels in 43 herbs were in excess of a hundred ng/g dry mass. Melatonin levels in these herbs are substatially higher than those in the serum of mammals which are usually in the pg/mL range. For several herbs (10), melatonin levels were in the Ag/g range. Interestingly, majority of the herbs containing the highest levels of melatonin are traditionally used to retard aging and to treat diseases which associate with free radicals. For example, yinyanghuo (Epimedium brevicornum Maxim) (melatonin 1105 ng/g) and sangbaipi (Mori Albae Cortex) (melatonin 1110 ng/g) were typically used to slow the aging process, and sangye (Leaf Morus alba L) (melatonin 1510 ng/g) is believed to have a beneficial effect on irradiation damage induced by UV or radiotherapy. Aging as well as UV radiation and damage from radioactivity involve free radical generation (Podhaisky


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et al., 2002; Barjs, 2002). Historically, it has been difficult to explain the alleged therapeutic effects of these Chinese herbal medicine since the already-known constituents extracted from them do not possess antioxidant properties. The findings of relatively high levels of melatonin in these herbs provide a potential pharmacological basis for their clinical use as determined over many centuries. This is the first report to screen a large number (a total of one hundred eight) of Chinese medicinal herbs using advanced methods and the analyses found melatonin levels over a very wide range of concentrations (10–3000 ng/g dry mass). HPLC analysis of melatonin in these herbs was further verified using MS. The results are consistent with the findings of Murch et al. (1997). Using HPLC to estimate melatonin levels, they also found that melatonin existed in several herbs. Our results not only confirmed their findings but also provided evidence that virtually all herbs contain detectable melatonin levels, in the case of the present study, of 108 herbs were found to contain melatonin. Identifying melatonin in these products, the current findings may provide some justification for the clinical use for some medicinal herbs, particularly those containing the highest levels of melatonin.

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