(2020) 15:6
Jia et al. Chin Med
https://doi.org/10.1186/s13020-020-0287-0
Chinese Medicine
Open Access
RESEARCH
A comparative study of aged
and contemporary Chinese herbal materials
by using delayed luminescence technique
Yusheng Jia1,3, Mengmeng Sun1,4,9, Yuhua Shi5, Zhihui Zhu5, Eduard van Wijk6, Roeland van Wijk6,
Tinde van Andel2,3* and Mei Wang1,7,8*
Abstract
Background: Investigation of aged Chinese herbal materials will help us to understand their use and sources in
ancient time and broaden the historical perspective of Chinese material medica. To reach this aim, the basic understanding of aged herbal materials, including physical and chemical characters, is of great importance. Delayed luminescence (DL) technique was developed as a rapid, direct, systemic, objective and sample loss-free tool to characterize the properties of Chinese herbal materials. In this study, we measured DL values in aged Chinese herbal materials
that were transported from Asia to Europe during the 20th century and stored in Naturalis Biodiversity Center and the
Utrecht University museum, and compared these with modern material of the same species.
Methods: A hyperbolic function was used to extract four properties from the DL curves of Chinese herbal material
from 1900, the 1950s and recently harvested products. Statistical tools, including the Student’s t test, One-way analysis
of variance and Principal Component Analysis, were used to differentiate the DL properties of aged and contemporary collections of Glycyrrhiza spp. Curcuma aromatica Salisb., Zingiber officinale Roscoe, Alpinia officinarum Hance and
Acorus calamus L.
Results: Our results showed that DL properties were significantly different between historical and contemporary
Chinese herbal materials. Changes in DL values were species-dependent: the effects of storage time of DL properties
were specific for each species. These outcomes help us not only in the identification of historical Chinese medicine
products but also provides valuable data of the effect of storage time on herbal materials.
Conclusion: The simple, direct, rapid, and inexpensive measurements offered by DL provide a novel tool to assess
the taxonomic identity of Chinese and other herbal materials and assess the differences in chemical properties with
increasing storage time. Our results contribute to the further development of novel digital tools for the quality control
of herbal materials.
Keywords: Delayed luminescence, Chinese herbal medicine, Aged herbal materials, quality control
*Correspondence:
[email protected];
[email protected]
1
LU-European Center for Chinese Medicine and Natural Compounds,
Institute of Biology, Leiden University, Sylviusweg 72, 2333 BE Leiden, The
Netherlands
2
Institute of Biology, Leiden University, Sylviusweg 72, 2333 BE Leiden,
The Netherlands
Full list of author information is available at the end of the article
Background
Herbal medicine has been used for millennia in China
to maintain good health and for the treatment of diseases, and during the last decades it’s global popularity
is increasing [1, 2]. Recently, the World Health Organization has included Traditional Chinese Medicine
(TCM) in its medical compendium as a recognition of
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Jia et al. Chin Med
(2020) 15:6
its significant acceptance worldwide [3]. As early as the
Chinese Han Dynasty (202 BCE-220BE), the exchange of
herbal medicine between China and the outside world
has begun through the Silk Road [4]. Around the beginning of the 10th century, Xun Li wrote his work Extrinsic Materia Medica, summarizing information on more
than 120 herbs introduced into China from abroad [5].
In the same period, due to the gradual development of
maritime trade, China began exporting herbal medicines
to its surrounding countries and regions. From the Ming
Dynasty (1368–1644 AD) onwards, Chinese herbal medicines were shipped to Europe in large quantities through
maritime trade, first with Portuguese and later with the
Dutch. One of the famous and popular herbal medicines
at that time was “China root” (Smilacis Glabrae Rhizoma,
the rhizome of Smilax glabra Roxb.). It was used to treat
syphilis and as a result of increasing global movements
and trade, the product became rapidly popular worldwide
[6]. From the 17th century onwards, European scientists
and explorers collected Chinese herbs progressively for
the aim of curiosity, the study of different medical cultures and the interest in exotic medicinal plants. Several
Page 2 of 12
European museums and private persons hold collections of historical Chinese herbal medicine, some more
than 100 years old, such as the ancient Chinese medicinal material collection in the Natural History Museum in
London [7]. Several historic TCM collections are housed
by the Utrecht University Museum and Naturalis Biodiversity Center in Leiden in the Netherlands (Fig. 1).
Historic collections of Chinese herbal materials are valuable objects for the scientific study of Chinese culture,
trade and ethnopharmacology. Investigation of ancient
herbal materials will help us to understand how the use
of TCM has changed through history. Centuries ago,
the global demand in Chinese herbal materials was not
as high as in the present. In the course of time, some of
the original plant species were replaced by others. Wild
plants are now grown in high production systems [8],
while rare plants have gone extinct [9, 10]. For example,
Lignum sinensis resinatum, the resinous wood of Aquilaria sinensis (Lour.) Gilg, was substituted by Lignum
aquilariae resinatum (Aquilaria agallocha (Lour.) Roxb.)
[11]. According to Zhonghua Bencao, A. sinensis and A.
agallocha have similar active constituents and the same
Fig. 1 Historic herbal materials used in this study. a Display cabinets of historical herbal medicine in Utrecht Botanical Gardens. b Historic
collections of Zingiber officinale (Sample ID Z.o_1900) in Utrecht Botanical Gardens. c Historic Curcuma aromatica collection (Sample ID C.a_1900)
in Utrecht Botanical Gardens. d Historic Alpinia officinarum rhizome (Sample ID A.o_1900) at Utrecht Botanical Gardens. e Historic Glycyrrhiza glabra
root (Sample ID G.g_1929) in Naturalis Biodiversity Center
Jia et al. Chin Med
(2020) 15:6
clinical effect [12]. In addition, historic local names of
herbs have changed or are confused with different plant
species with similar names elsewhere in China [11]. All
these variations and changes in names and species over
time may lead to mistakes in recipes and the use of the
wrong herbal materials with potential risks to consumers [13]. Most previous studies that evaluate historical changes in TCM have largely focused on literature
research [14], but many of their conclusions have not
yet been confirmed by the revision of physical samples
from premodern collections of Chinese herbal materials.
Therefore, it is critical to have an objective analytic tool
for the assessment of these ancient collections of herbal
materials [14].
The identification of historic collections of TCM is
challenging, as the amount of stored material per species is often very small and fragile. Due to the distinctiveness of these historic collections, we are constrained to
perform analytic studies with limited amounts of herbal
materials. Instead of destructive methods such as DNA
analysis and chemical profiling studies, non-destructive
techniques are preferred to identify ancient and very valuable TCM collections. Delayed luminescence (DL) was
developed as a rapid, direct, systemic tool to measure
the decaying ultra-weak luminescence (up to seconds or
minutes) exhibited by materials after being illuminated
with light. As a sample loss-free technique, DL is a sensitive approach and widely applied in determining food
quality [15], seed germination [16] and cancerous cells
[17]. DL is a photo-induced ultra-weak photon emission
[18], of which the properties are influenced by molecular structures and interactions [19], in particular the long
chain molecules [20, 21]. The molecular absorption of
excitation energy determines the dynamics of the subsequent DL emission [19]. Compared with other existing
methods, DL has the following advantages: (1) Simplicity. Herbal material only needs be ground to powder,
no other complicated treatment is required; (2) Sample
loss-free. Powdered medicinal material is exposed to
light for 10 s, then the photon released from the sample
is recorded by the instrument. There will be no chemical or biological changes in the samples, and they can
be used again for other analytical research; (3) Rapidity. The whole experiment process is simple and fast: it
only takes several seconds for the measurement and ca.
10 min from sample pre-processing to obtaining the data
for one sample; (4) Reduced costs. Compared to chromatography and DNA-barcoding, DL experimental instruments are very cheap and no kits are required for sample
preparation.
Recently, various researchers have successfully applied
DL in herbal medicine to identify specific properties of
herbal materials or to detect variations in the material
Page 3 of 12
due to variation in environmental growth conditions [22],
different processing methods [23, 24] and determination
of authenticity [25]. Differences detected by DL in herbal
materials are also reflected by their chemical profiling
[24, 26] as well as therapeutic activities [27]. The ability
of DL to rapidly distinguish between herbal material with
different growth conditions, processing states, taxonomic
identity or therapeutic properties, all of which are linked
to differences in chemical compositions in the materials, suggest that DL is a promising technology for further
evaluation of the quality of herbal material [24, 27].
In this work, we have tested whether DL can also be
used to detect changes in herbal medicine over time.
We performed DL analysis on historic TCM collections
of the species Glycyrrhiza glabra L., Glycyrrhiza inflata
Batalin, Glycyrrhiza uralensis Fisch., Curcuma aromatica
Salisb., Zingiber officinale Roscoe, Alpinia officinarum
Hance and Acorus calamus L. and contemporary materials of their corresponding species. The results obtained
from our DL measurements show that DL properties
indicate differences between aged and contemporary
herbal materials, and that DL may be further used to
verify the storage time of herbal materials. Therefore, DL
can provide new insights into the quality and safety of
herbal medicines.
Materials and methods
Herbal materials
Historic herbal material was sampled from the collections of Chinese medicine that are housed in the Economic Botany collection of Naturalis Biodiversity Center
(Leiden, the Netherlands), which were obtained in the
1950s in Indonesia and from collections of the Utrecht
University Museum, stored in the Wachendorffzaal of
Utrecht Botanical Gardens (Utrecht, the Netherlands).
Due to the small amount of herbal material stored per
species, only a limited amount of samples could be taken
from the museum collections for this study. Contemporary herbal materials were obtained from the Institute of
Chinese Materia Medica, the Beijing Institute of Chinese
Medicine, National Institutes for Food and Drug Control
and TongRenTang Co., Ltd., all located in Beijing, China.
All samples were verified for the correct taxonomic identification by Dr. Mei Wang and Dr. Yuhua Shi and later
deposited at the European Center for Chinese Medicine
and Natural Compounds of Leiden University (Leiden,
the Netherlands).
Sample preparation and DL measurement
Herbal materials were milled by a grinder (model
QE-100, Yili Company, Zhejiang Province, China) and
passed through a sieve to obtain 150 μm particles. The
powdered herbal material was kept in light-proof boxes
Jia et al. Chin Med
(2020) 15:6
Page 4 of 12
containing some 3–5 mm silica gel (BoomBV, Meppel,
the Netherlands) at room temperature for 16 h before the
DL measurements [23].
DL assays were performed according to the published
protocol [23]. The instrument used in our measurements
was development by Meluna Research (Geldermalsen,
the Netherlands) and included a photomultiplier tube
(PMT) (type 9558QB; Electron Tubes Enterprises Ltd.,
Ruislip, UK), vertically positioned on a dark sample
chamber kept at 22 °C. The PMT contained a cathode
end (51 mm diameter) with sensitivity at 300–800 nm.
The PMT was cooled to − 25 °C in order to reduce the
dark count rate to 10 counts per second. A fast preamplifier (model 9301, ORTEC, Oak Ridge, TN) was used to
amplify the photon emission signal. Data were extracted
by a computer with a model 6602 counting card (National
Instruments, Austin, TX). For each sample, 1 g powder
was taken and put into a petri dish (diameter: 35 mm),
then exposed to light for 10 s using a model 284–2812
white halogen excitation source (Philips, Germany). The
DLs of these samples were successively measured three
times. The data obtained from the three measurements
were used to analyze the DL properties of each sample.
The DL decay signature was obtained by recording the
number of photon counts in consecutive 0.05-s periods
for a total of 60 s, yielding a total of 1200 data points.
Statistical analysis of DL properties
In order to calculate the DL properties of the samples,
all the photon counts measured during the 60 s of each
decay curve were used according to the following hyperbolic function [24]:
I(t) =
I0
1+
Beta
t
Tau
1
T = e Beta − 1 × Tau
This hyperbolic function is a general formula for fitting
the DL decay curve of samples. The four parameters (I0,
Beta, Tau and T) obtained by this hyperbolic function
can well express the characteristics of the DL decay curve
[24, 28]. I0 is the initial intensity of the DL curve, Beta is
an index factor associated with the rate of DL decay, and
Tau and T represent the DL characteristics and decay
time, respectively. The parameters of the repeated measurements (at least three times) were averaged and used
to represent the DL properties of each sample. Principal
components analysis (PCA) scores were used to indicate
the level of discrimination between DL properties by
tools provided in the MetaboAnalyst software package
(http://www.metaboanalyst.ca). A two-tailed, unpaired
Student’s t test and One-way analysis of variance
(ANOVA) with least significant difference (LSD) post hoc
analysis (SPSS version 23.0) were used to compare the DL
properties between herbal samples; differences were considered significant at p < 0.05.
Results
In order to assess the applicability of DL techniques in
detecting differences between aged and contemporary
herbal material, five different types of herbal products,
each with historical and corresponding recent materials
were used in this research. The samples measured in this
study are shown in Table 1. The four parameters of the
Table 1 Sample information
Pharmaceutical name
Glycyrrhizae Radix et Rhizoma
Curcumae Radix
Zingiberis Rhizoma
Sample id
Scientific name
Sample source
Sampling time
G.g_1900
Glycyrrhiza glabra L.
Utrecht Botanic Gardens
1900
G.g_1929
Glycyrrhiza glabra L.
Naturalis Biodiversity Center
1929
G.g_2018
Glycyrrhiza glabra L.
Institute of Chinese Materia Medica
2018
G.i_2018
Glycyrrhiza inflata Batalin
Institute of Chinese Materia Medica
2018
G.u_2018
Glycyrrhiza uralensis Fisch.
Institute of Chinese Materia Medica
2018
C.a_1900
Curcuma aromatica Salisb.
Utrecht Botanic Gardens
1900
C.a_1957
Curcuma aromatica Salisb.
Naturalis Biodiversity Center
1957
C.a_2018
Curcuma aromatica Salisb.
National Institutes for Food and Drug Control
2018
Z.o_1900
Zingiber officinale Roscoe
Utrecht Botanic Gardens
1900
Z.o_1952
Zingiber officinale Roscoe
Naturalis Biodiversity Center
1952
Z.o_2017
Zingiber officinale Roscoe
TongRenTang Co., Ltd.
2017
Alpiniae Officinarum Rhizoma
A.o_1900
Alpinia officinarum Hance
Utrecht Botanic Gardens
1900
A.o_2017
Alpinia officinarum Hance
TongRenTang Co., Ltd.
2017
Acori Tatarinowii Rhizoma
A.c_1900
Acorus calamus L.
Utrecht Botanic Gardens
1900
A.c_2017
Acorus calamus L.
TongRenTang Co., Ltd.
2017
Jia et al. Chin Med
(2020) 15:6
DL decay curves were calculated by a hyperbolic function, which was used to fit the observed decay curves.
The differences in four separate parameters were visualized by the PCA, which allowed us to achieve a focused
view of the variance in the four properties. The correlation of each parameters to the different samples was illustrated in a PCA biplot.
Alpinia officinarum and Acorus calamus were firstly
analyzed. For these two species we had only samples
from two different points in time: historical samples from
Page 5 of 12
around 1900 and modern samples from 2017. The DL
decay curves of A. officinarum (Fig. 2a), clearly show differences between samples from 1900 and 2017. The four
parameters of the DL decay curve that were compared all
differed significantly between the recent and aged samples (Fig. 2b). Figure 2c displays the PCA results in the
form of a score plot, in which the A. officinarum samples were clustered into two age groups. The PCA biplot
(Fig. 2d) reveals that parameters I0, Beta, Tau and T are
responsible for distinguishing between the two groups.
Fig. 2 DL analysis of Alpinia officinarum Hance samples. a DL decay curves comparison among Alpinia officinarum Hance samples.
BG = background. b Comparison of DL properties among the Alpinia officinarum Hance samples. I0 is the initial intensity of the DL curve, Beta is an
index factor associated with the rate of DL decay, and Tau and T represent the DL characteristics and decay time, respectively. *p < 0.05. c PCA score
plots of the DL properties obtained from Alpinia officinarum Hance samples. d PCA biplot indicating how each parameter influences the similarity of
DL decay curves
Jia et al. Chin Med
(2020) 15:6
The DL decay curve of Acorus calamus (Fig. 3a) also
shows that samples of different ages have different
curves. The DL parameters analysis proved that the initial intensity (I0), curve rate (Beta), DL curve characteristics (Tau) and decay time (T) are significantly different
between these two samples (Fig. 3b). The PCA score plot
shows that samples of different age clustered into separate groups (Fig. 3c). The PCA biplot indicates that all
four parameters contribute to separate samples of different ages (Fig. 3d). The results of our DL analysis of A.
officinarum and A. calamus show that DL technology is
Page 6 of 12
capable of discriminating between historic and recently
collected herbal materials. All four parameters exhibited
significant differences and the PCA clustered samples
of unequal ages into different groups. However, as the
tested materials were collected in time periods 117 years
apart, we wondered whether specimens with less extreme
age differences would also differ in their DL properties.
To answer this question, we used samples of Curcuma
aromatica and Zingiber officinale, which were approximately stored for 60 and 120 years, and compared them
to contemporary samples of corresponding species. We
Fig. 3 DL analysis of Acorus calamus L. samples. a DL decay curves comparison among Acorus calamus L. samples. BG = background. b DL
properties comparison among Acorus calamus L. samples. I0 is the initial intensity of the DL curve, Beta is an index factor associated with the rate
of DL decay, and Tau and T represent the DL characteristics and decay time, respectively. *p < 0.05. c PCA score plots of the DL properties obtained
from Acorus calamus L. samples. d PCA biplot shown how strongly each parameter influence the similarity of DL decay curves
Jia et al. Chin Med
(2020) 15:6
subjected the samples to DL analysis to verify whether
this technology was able to distinguish three different
storage times that were not so far apart. The DL analysis results of C. aromatica are presented in Fig. 4. Samples from 1957 and 2018 had similar DL decay curves,
while the curve of the sample from 1900 was quite distinctive (Fig. 4a). To compare the four parameters of
the DL decay curves, a one-way ANOVA test was used.
Results revealed that for four parameters, the sample
from 1900 was significantly different from other two
Page 7 of 12
more recent samples, while the samples from 1957 and
2018 did not significantly differ except for parameter T
(Fig. 4b). The PCA results (Fig. 4c) show that all tested
samples were divided into three clusters based on storage time and that the sample from 1900 clustered far
from the other two groups. The PCA biplot indicates
that parameter I0 and T were heavily responsible for
identifying the clusters (Fig. 4d). Together these results
suggest that DL technology is able to distinguish samples of C. aromatica of 120 years old from 60 year-old
Fig. 4 DL analysis of Curcuma aromatica Salisb. samples. a DL decay curves comparison among Curcuma aromatica Salisb. samples.
BG = background. b DL properties comparison among Curcuma aromatica Salisb. samples. I0 is the initial intensity of the DL curve, Beta is an index
factor associated with the rate of DL decay, and Tau and T represent the DL characteristics and decay time, respectively. *p < 0.05. c PCA score plots
of the DL properties obtained from Curcuma aromatica Salisb. samples. d PCA biplot shown how strongly each parameter influence the similarity of
DL decay curves
Jia et al. Chin Med
(2020) 15:6
samples, but not between 60 year-old and contemporary samples.
The DL decay curves for Zingiber officinale are shown
in Fig. 5, The sample from 2017 was strikingly different than the other two (Fig. 5a). The parameters of the
DL decay curves suggest that only parameters Beta and
T differ significantly between the samples (Fig. 5b). The
PCA results illustrate that samples from 2017 formed a
quite distinct cluster, and that the samples from 1900 and
1952 formed two relatively close clusters (Fig. 5c). The
PCA biplot demonstrates that the parameters Beta and
Page 8 of 12
T were responsible for dividing the samples from each
other. These results indicate that DL technology is able to
distinguish ginger roots of 120 and 60 years old from relevant contemporary samples, but not between samples of
120 and 60 years old.
For Curcuma aromatica, DL analysis showed no significant differences between samples from 1957 and 2018,
but both of them significantly differed from the one from
1900. We speculate that 60 years of storage time does not
change its DL properties, but 120 years is long enough
to do so. Conversely, the DL results of Zingiber officinale
Fig. 5 DL analysis of Zingiber officinale Roscoe samples. a DL decay curves of Zingiber officinale Roscoe samples of different ages. BG = background.
b DL properties of the three different Zingiber officinale Roscoe samples. I0 is the initial intensity of the DL curve, Beta is an index factor associated
with the rate of DL decay, and Tau and T represent the DL characteristics and decay time, respectively. *p < 0.05. c PCA score plot of the DL
properties obtained from Zingiber officinale Roscoe samples. d PCA biplot showing how each parameter influences the similarity of DL decay curves
Jia et al. Chin Med
(2020) 15:6
had no significant differences between samples from
1900 and 1952, but both of them significantly differed
from the one from 2017.
Apparently, the DL properties of ginger roots change
after 60 years of storage time, but after that they remain
stable. The opposite is true for curcuma roots, which
apparently start to change in DL properties after ca.
50 years. These findings suggest clearly that timedependent changes in DL properties of herbal products
occur, but that these changes are also species-dependent. Further research with more samples of multiple
storage times and more species is needed to verify our
speculation.
Apart from changes in DL values over time, we were
also interested in the performance of DL technology in
distinguishing closely related taxonomic species. Therefore, we compared contemporary and historic samples
of Glycyrrhizae Radix et Rhizoma, known as liquorice
root, and widely used as medicine, food supplement and
flavoring agent. The botanical identity of our historical
samples of Glycyrrhizae Radix et Rhizoma was Glycyrrhiza glabra L. However, according to the 2015 edition
of the Chinese Pharmacopoeia, three different, botanically related species (G. glabra, G. inflata Batalin and
G. uralensis Fisch.) can be used as Glycyrrhizae Radix
et Rhizoma [29]. Therefore, in our study, two differently aged samples of Glycyrrhizae Radix et Rhizoma (G.
glabra) and several contemporary samples of Glycyrrhizae Radix et Rhizoma of three different species were used
for DL analysis. With samples collected at three distinct
points in time and three different species, we continued
to verify the capability of DL technology to distinguish
among samples of different age and among closely related
species.
Figure 6 presents the results obtained from the DL
analysis of the various samples of liquorice roots. The
DL decay curves of G.g_1900 and G.i_2018 are clearly
different from the other samples (Fig. 6a), which are all
above the background level. Four parameters of the DL
decay curves were compared between all samples using a
one-way ANOVA test (Fig. 6b). For parameter I0, sample
G.g_1900 and G.i_2018 are significantly different from
the others. For parameter Beta, sample G.g_1929 differs
significantly from the other samples. The Tau parameter
of sample G.g_1900 is significantly different as well as
between the G.g_1929 and G.i_2018. The T parameter of
sample G.g_1900 is significantly different from the other
samples. The PCA score plot shows that the Glycyrrhizae
Radix et Rhizoma samples are divided into three groups:
samples collected in 1900, 1929 and 2018. Interestingly,
despite the different species and batches, samples collected in 2018 are clustered into one group. The PCA
biplot reveals that parameters I0, Tau and T are mainly
Page 9 of 12
responsible for distinguishing group 1900 from the other
two groups. Parameter Beta is responsible for distinguishing between group 1929 and group 2018 (Fig. 6d).
Our PCA data illustrate that different aged samples of
the same species clustered into different groups, while
distinctive species with same collection time showed
no significant differences in DL values. Four parameters
showed no significant differences, except I0 in sample
G.i_2018. PCA clusters were mostly overlapping. Taken
together, these results suggest that the DL technique
is capable of discriminating samples of different ages of
Glycyrrhizae Radix et Rhizoma (at least for G. glabra),
but not able to discriminate between closely-related species with same collection time.
Glycyrrhizae Radix et Rhizoma is one of the most
frequently used herbal products in traditional Chinese
medicine. On account of similar active ingredients (glycyrrhizic acid and liquiritin) and clinical effect, the three
different species G. glabra, G. inflata and G. uralensis
are grouped under the same pharmacological term [29].
Moreover, genetic studies show that the gene sequences
of these three species are highly similar [30]. This may be
the reason that the DL values of recent samples of these
three species are not significantly different. But when taking storage time into account, we do not know whether
age has the same impact on DL properties for the three
different species. As we only had access to historic samples of G. glabra, and not for the other species, we do not
know whether DL can distinguish different species of
Glycyrrhiza in historic collections.
Discussion
Investigation of ancient Chinese herbal materials will
help us not only to understand the origin and historical use of Chinese herbal medicine, but also to clarify
the confusion on botanical identity, nomenclature and
changes in species and plant parts over time. Studying historical samples with DL technology gives us new
insight into the possible changes in chemical properties
of herbal materials over time. Especially when there is a
limited amount of herbal material available for testing,
DL technique gives us support and solution from an analytic point of view. As DL has already applied to identify
different processing methods [24] and determination of
authenticity [25], the novelty of the present study is the
analysis of aged herbal materials, which provides unique
opportunities to understand the effect of long term storage on herbal materials.
In this research, we found that DL can provide sensitive measurements that reflect differences between historic and contemporary herbal materials. DL properties
can be affected by changes of molecular conformations
and interactions such as forming of hydrogen bonds and
Jia et al. Chin Med
(2020) 15:6
Page 10 of 12
Fig. 6 DL analysis of Glycyrrhizae Radix et Rhizoma samples. a DL decay curves comparison among Glycyrrhizae Radix et Rhizoma samples.
BG = background. b DL properties comparison among Glycyrrhizae Radix et Rhizoma samples. I0 is the initial intensity of the DL curve. All five
samples’ I0 parameter were significantly different (p < 0.05) from each other, except G.g_2018–G.g_1929 group and G.g_2018–G.u_2018 group.
Beta is an index factor associated with the rate of DL decay. The Beta parameter of G.g_1929 was significantly different (p < 0.05) from the other
four samples. Tau represent the DL characteristics. The Tau parameter of G.g_1900 and G.g_1929–G.i_2018 group and G.g_1929–G.u_2018 group
were significantly different (p < 0.05). T describes the DL decay time. T parameter of G.g_1900 was significantly different (p < 0.05) from the other
four samples. c PCA clustering of DL properties obtained from Glycyrrhizae Radix et Rhizoma samples. d PCA biplot showing how each parameter
influences the similarity of DL decay curves
carbon-to-nitrogen ratio, resulting in the radiant transfer
of energy from one excited molecule to another, causing
a change in the material’s DL kinetics. Recently, Grasso
et al. [31–33] reported significantly different DL kinetics
of intensities and decay time intervals between amylose
and cellulose, which share the same glucose-based repeat
units, but have differently molecular structures, and the
different DL properties of these two compounds may be
related to the soliton mechanism. Amylose and cellulose
are polysaccharides, which occur widely in herbal material as bioactive components, such as liquorice roots
(Glycyrrhiza spp.) [34]. With increasing storage time, the
polysaccharide content in plants may change, causing a
change of molecular structure [35]. Large time intervals
may cause significant changes in the chemical composition of polysaccharides. This may be the reason that DL
Jia et al. Chin Med
(2020) 15:6
distinguished historic and modern samples of Glycyrrhizae Radix et Rhizoma, but not the three botanically different Glycyrrhiza samples collected in similar periods.
The bioactive components in the other medicinal species used in this research are mainly volatile oils [29]. The
volatile oil content of herbal material also changes during the storage time [36], which may be the reason why
the DL characteristics of our long-term stored samples
were so different from recently harvested samples. Future
studies should focus on the chemical differences between
historical and contemporary herbal material to investigate whether DL could characterize the chemical changes
caused by storage time.
Storage time is an important factor in the stability of
plant products and thus the quality of herbal medicine.
Storage usually modifies the composition of herbal medicines, directly affecting safety and therapeutic value [35,
36]. Most methods used to study composition changes
caused by storage time are HPLC or GC–MS [37], but
these require expensive analytical tools and sample loss is
inevitable. Compared to chromatographic methods, DL
is a direct, rapid approach, which is quite affordable and
does not imply sample loss. Moreover, DL can provide a
comprehensive perspective for the sample’s overall features, rather than only measuring the amount of certain
components [27]. Therefore, DL maybe a suitable technology to detect changes in herbal material influenced
by storage time. In addition, due to distinctiveness and
limited amounts of ancient herbal materials, DL is suitable to study the differences between ancient and contemporary herbal material of the same species. Because
of DL’s sample loss-free nature, it is suitable as the first
analytic method, so it can be decided afterwards if it is
necessary to employ further destructive methods such
as DNA analysis [38] and chemical profiling studies. Our
study has shown that DL has the potential as a practical
approach to verify the applicability of herbal material
for clinical use. However, we need to establish a significant database for this purpose. In general, herbal material that has been stored for decades or centuries is no
longer suitable for clinical use, but may provide valuable
information to understand (changes in) properties of the
material. To examine this possibility, using herbal materials with different storage time (e.g., from 1 to 5 years) and
combining DL, chemical study and bioactivity analysis
will provide a better understanding of changes in herbal
material over time.
Conclusions
In this study, we used historical Chinese herbal medicine as research object to verify whether Delayed
Luminescence was a suitable technology to analyze the
Page 11 of 12
differences between aged herbal material and corresponding contemporary herbal material. Our findings
suggest that DL is a promising approach tool to study
historical herbal material, as it is able to identify different properties among samples with different storage
time. Our study also showed that patterns of properties changes are likely to be plant species dependent.
Our study contributes to the lack in scientific data on
the effects of storage time on herbal material. More
research should focus on expanding the number of
herbal species for DL analysis, data accumulation and
mining to better understand DL technology’s potential on assessment of quality related aspects of herbal
material.
Abbreviations
DL: delayed luminescence; PCA: principal component analysis; ANOVA:
analysis of variance.
Acknowledgements
This research was supported by “single cell foundation”; We are grateful to
Paul Lambers (Utrecht University Museum), Hans Persoon (Utrecht Botanical
Garden) and Christel Scholaardt (Naturalis Biodiversity Center) for giving us
access to the historical collections of Chinese herbal medicine; The authors
acknowledge “the Fundamental Research Funds for the Central public welfare
research institutes” (GH2017-01-01) for support this research.
Authors’ contributions
YJ, MW and TA designed the study. YJ, EW and RW conducted the statistical
analyses and prepared figures. YJ and MS performed DL measurements. YS
and ZZ carried identification of herbal samples. YJ and MW drafted the manuscript. All authors read and approved the final manuscript.
Funding
Not applicable.
Availability of data and materials
The datasets used in this study are available from the corresponding author
upon reasonable request.
Ethics approval and consent to participate
Not applicable.
Consent for publication
The manuscript is approved by all authors for publication.
Competing interests
The authors declare that they have no competing interests.
Author details
1
LU-European Center for Chinese Medicine and Natural Compounds, Institute
of Biology, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands.
2
Institute of Biology, Leiden University, Sylviusweg 72, 2333 BE Leiden, The
Netherlands. 3 Naturalis Biodiversity Centre, Darwinweg 2, 2333 CR Leiden,
The Netherlands. 4 Changchun University of Chinese Medicine, No. 1035,
Boshuo Rd, Jingyue Economic Development District, 130117 Changchun,
China. 5 Institute of Chinese Materia Medica, China Academy of Chinese
Medical Sciences, Beijing 100700, China. 6 Meluna Research, Koppelsedijk 1-a,
4191 LC Geldermalsen, The Netherlands. 7 SU BioMedicine, Post Bus 546, 2300
AM Leiden, The Netherlands. 8 Shenzhen Huakai Traditional Chinese Medicine
and Natural Medicine Research Center, Shenzhen 518114, China. 9 State Key
Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, N22 Avenida da Universidade, Taipa, Macau.
Jia et al. Chin Med
(2020) 15:6
Page 12 of 12
Received: 22 November 2019 Accepted: 9 January 2020
22.
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