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: ykzhang1@online.ln.cn, ykzhang@dicp.ac.cn (Y. Zhang).

0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved.
doi:10.1016/S0024-3205(03)00252-2
20 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% methanol-
water solution was used to elute the interference impurities. Finally, the retained melatonin was eluted at
 G. Chen et al. / Life Sciences 73 (2003) 19–26 21

Table 1
Melatonin recovery rates from the SPE
Samples Melatonin (Ag) Melatonin measured (Ag) Recovery rate %
1 5 5.14 103
2 10 9.48 95
3 15 13.52 90
4 20 19.41 97
Average 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 (HPLC-
FD) 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.
22 G. Chen et al. / Life Sciences 73 (2003) 19–26

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.
 G. Chen et al. / Life Sciences 73 (2003) 19–26 23

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 3,771 Periostracum cicadae Kushen 190 Sophora flavescens Ait.
Diding 2,368 Viola philipica Cav. Dansnen 187 Salvia miltiorrhiza Bge
Gouteng 2,460 Uncaria rhynchophylla Qinjiu 180 Gentiana macrophylla Pall.
Shiya tea-leaf 2,120 Babreum coscluea Huangqin 178 Scutellaria amoena C.H. Wright
Sangye 1,510 Morus alba L (Leaf) Jinqiancao 169 Desmodium styracifolium Merr
Huangbo 1,235 Phellodendron amurense Ru pr. Sanqi 169 Panax notoginsneg Burk
Sangbaipi 1,110 Mori Albae (Cortex) Yimucao 169 Leonurus japonicus Houtt.
Yinyanghuo 1,105 Epimedium brevicornum Maxim Juhua 160 Dendranthema morifolium
Huanglian 1,008 Coptis chinensis Franch Zicao 158 Arnebia euchroma
Dahuang 1,078 Rheum palmatum L., Gegen 150 Pueraria lobata Willd
Yuanzhi 850 Polygala tenuifolia Willd. Dazao 146 Ziziphus jujuba Mill.
Shanyurou 821 Coruns officinalis Sieb. Yejiaoteng 143 Caulis Polygonam
multiflorum Thunb
Longdacao 780 Gentiana scabra Bge. Shuanhua 140 Lonicera japonica Thunb
Luxiancao 750 Pirola decorata H. Erzhu 120 Curcuma aeruginosa Roxb.
Danghui 698 Angelica sinensis Oliv. Gancao 112 Glycyrrhiza uralensis Fisch
Sangjisheng 648 Taxillus chinensis DC Dilong 97 Pheretima aspergillum
Fuling 585 Poria cocos Schw. Wolf. Shengdi 97 Rehmannia glutinosa
Gouqi 530 Lycium barbarum L. Wuweizi 86 Schisondra chinensis
Bihu 523 Gekko japonicus (Dumeril et biron) Qinghao 84 Artemisis annua L.
Luhui 516 Aloe vela L Banlangen 79 Isatis indigotica Fort
Chuanxinlian 511 Andrographis paniculats Burm. Fangfeng 60 Saposhmikovia divaricata
Duzhong 497 Eucommia ulmoides Oliv Zhuye 55 Not available
Laifuzi 485 Raphanus sativus L. Shidagonglao 52 Mahonia bealei (Fort.)
Dingxiang 446 Syzygium aromaticum L. Lianqiao 45 Forsythia suspensa (Thunb.)
Fupenzi 387 Rubus chingii Hu Huangjing 45 Polygonatum sibiricum Delar
Xuanshen 342 Scrophularia ningpoensis Hemsl. Damzjuye 38 Lophartherum gracile Brongn
Huoxiang 302 Agastaches rugosa Xiakucao 34 Prunella vulgaris L.
Banzhilian 257 Lobelia chinesis Lour. Baijing 32 Herba Patriniae scabiosaefoliae
Suanzhaoren 256 Ziziphus jujuba Mill. Duhuo 31 Angelica biserrata
Wugong 248 Scolopendra subspinipes mutilang. Rouchongrong 28 Cistanche desericola Y
Jiangcan 227 Bombyx batryticatus Chenpi 25 Citrus reticulata Blanco
Maidong 198 Ophiopogon japonicus Zhizi 12 Galdenia jasminoides Ellis
24 G. Chen et al. / Life Sciences 73 (2003) 19–26

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
 G. Chen et al. / Life Sciences 73 (2003) 19–26 25

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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