Study on genetic polymorphism and essential oil composition of Asian Curcuma species and crude drugs for standardization

概要

論
論 文 題 目

文

要

約

Study on genetic polymorphism and essential oil composition of Asian
Curcuma species and crude drugs for standardization

課程・専攻名:
氏

博士後期課程・薬科学専攻

名： 劉 群棟

Introduction
Genus Curcuma (Zingiberaceae) comprises approximately 120 species and approximately 30
species’ rhizomes have been used as traditional medicines, spices, dyes and cosmetics. Recently, with
the increasing popularity of foods with health claims and so-called health food in Japan and other
countries, the Curcuma rhizomes are frequently used worldwide. However, quality assessment
including correct identification has not been conducted. Because of the wide distribution and
morphological similarities of Curcuma species, classification of some species is debated and
nomenclature is inconsistent among countries, especially for C. aromatica and C. zedoaria. This
situation leads to confusion in the use of Curcuma crude drugs. The medicinal properties of C. longa
are mainly attributed to its content of curcuminoids which have been reported to possess antiinflammatory, anticancer activities, etc. However, other Curcuma drugs that contain no or few
curcuminoids but characteristic essential oil (EO) also have pharmacological effects, such as antiinflammatory, antioxidant and antiobesity activities, etc. Among literatures, inconsistent reports on
EO compositions of the same species were not rare, and such inconsistence might be attributed to
misidentification of botanic origin, difference of cultivation condition or processing method.
Therefore, comparative data on the EO compositions from Curcuma species which are identified
correctly and processed with same method is necessary.
In our laboratory, molecular analysis based on the trnK intron sequences of chloroplast DNA
was performed to identify Chinese and Japanese Curcuma crude drugs, however the sequence
comparison of this region was not enough to determine the botanical origin of Asian ones. Recently,
the intron length polymorphism (ILP) markers in two intron regions of genes encoding diketide-CoA
synthase (DCS) and one intron region of genes encoding curcumin synthase (CURS) were found to
have potential for discrimination of Chinese and Japanese Curcuma plants and crude drugs.
In this study, to elucidate molecular markers for discriminating Curcuma species in Asia, and to
solve the confusion on the botanical origin of Curcuma crude drugs, molecular analysis based on ILP
markers as well as trnK intron sequences was conducted using a number of Curcuma specimens and
crude drug samples from nine countries. Then, subcloning coupled with sequencing analysis was also
performed on DCS intron Ⅰ and CURS intron regions of representative species. Furthermore, to find
out the species-specific EO compositions, GC-MS analysis was conducted on 12 species.

1.

Molecular analyses based on ILP markers in DCS and CURS genes and trnK intron
sequences [1]

ILP patterns and trnK intron sequences were determined for 59 plant specimens and 42 crude
drug samples of 13 Curcuma species obtained from Asian countries. These plant specimens were
collected from several medicinal plant gardens in Japan; most of them were introduced from China,
Thailand, Indonesia, India, Malaysia and Nepal; all of them were identified based on detailed
observation and comparison of their morphology with the taxonomic literatures. The ILP patterns of
the respective species revealed high consistency within the same species in C. aromatica (group JA),
C. zedoaria (Ze), C. phaeocaulis (P), C. aeruginosa (Ae), C. wenyujin (W) and C. zanthorrhiza (Za),
but showed intraspecies polymorphism in C. longa (L), C. kwangsiensis (K), C. amada (A/M), C.
mangga (A/M) and C. comosa (C). The similarities of the ILP patterns enabled them to be divided
into the corresponding groups in the Neighbor-Joining tree (Fig. 1). Groups Pe (C. petiolata) and C
formed a clade, separated from the large clade. Group L formed one subclade and was further divided
into three subgroups and this grouping was highly consistent with the geographical origins of the
included samples; thus, they were tentatively assigned as China-Japan (L1), Thailand (L2) and IndiaIndonesia (L3) groups. Another subclade comprising the other species was further divided into two
branches: one composed of groups JA, Ze, Ae, P, W and K; and the other composed of Za and A/M.
Based on the combined data of the ILP markers and the trnK intron sequences, the botanical origins
of some crude drugs from Thailand and India were correctly determined, and some crude drug
samples from India were clarified to have hybrid origin. Moreover, morphological and molecular data
showed that C. aromatica and C. zedoaria cultivated in Japan had close relations with C. aromatica
from China and Thailand, and C. zedoaria from Indonesia and India, respectively.
Subcloning and sequencing analysis for DCS intron Ⅰ and CURS intron regions [2]
Six plant specimens from five Curcuma species, including C. longa, C. zedoaria, C. phaeocaulis,
C. aromatica and C. zanthorrhiza which showed distinct ILP patterns were subjected to subcloning
coupled with sequencing analysis for the DCS intron Ⅰ and CURS intron regions. More than 30
sequences of each region from each specimen were grouped into genes DCS1, DCS2 or CURS1-3
2.

and subsequently the sequences of the same genes were compared. Sequences belonging to the same
gene showed inter-species similarity, and thus these intron sequences were less informative within
each single gene region. The determined sequences from each specimen showed 3-5 kinds of
sequence lengths in DCS intron I region, and 5-7 kinds of sequence lengths in CURS intron region.
These were in accordance with the fragment numbers and lengths in the corresponding ILP patterns,
explaining well the origin of ILP pattern of Curcuma species.
3.

Essential oil composition analyzed by headspace solid-phase microextraction coupled with
gas chromatography-mass spectrometry (HS-SPME-GC-MS) [3]
The EO compositions of genetically identified 47 plant specimens belonging to 11 Curcuma

species as well as 20 crude drug samples were analyzed by HS-SPME-GC-MS. Plant specimens of
the same species showed similar EO patterns, even those were introduced from different areas. Based

on the similarity of EO patterns, all the plant specimens and C. comosa crude drug samples were
separated into eight main groups: L; Ze-P-Ae; Za; JA-W; K; Am(C. amada)-M(C. mangga); Pe; C.
From all the plant rhizomes and crude drug samples, 54 major sesquiterpene and monoterpene
compounds with relative higher content ( ＞ 1%) were identified. The eight groups contained
characteristic sesquiterpenes belonging to bisabolane type (L); curzerene, germacrane types (Ze-PAe); cedrane, bisabolane types (Za); germacrane, curzerene types (JA-W); curzerene, germacrane
types (K); caryophyllane type (Am-M; Pe); santalene, bisabolane types (C), respectively. Most of
the major compounds of group L, Ze, P, Ae and Za plant specimens were detected in their respective
crude drug samples correspondently; while some compounds, such as turmerone and α-cedrene or
curcumenol, dramatically decreased or increased in crude drug samples, which was probably due to
the processing or long-time storage. The genetically-deducing hybrid sample “Khamin oi” from
Thailand contained major compounds similar to C. longa and a C. comosa sample. OPLS-DA clearly
differed the groups L, Za and JA from the others with characteristic compounds turmerone,
xanthorrhizol and neocurdione, respectively. Groups Ze, P and Ae showed similar EO compositions
with common major compounds curzerenone and 4,5-epoxygermacrone (Fig. 3).
Conclusion
The ILP patterns successfully discriminated 13 Curcuma species and served as useful genetic
marker to identify the related crude drug samples. Based on the ILP markers and the trnK intron
sequences, the botanical origins of some confused crude drug samples in Asian countries were
correctly determined and the sources of C. aromatica and C. zedoaria cultivated in Japan were
inferred. The determined sequences of DCS intron Ⅰ and CURS intron regions well explained the
origin of ILP patterns. The comparative data of EO composition from 11 Curcuma specimens
revealed the major compounds in each species and several groups possessing chemical similarities
were detected. In summary, molecular method based on the ILP markers in DCS and CURS genes
and trnK intron sequences, as well as EO composition analysis were demonstrated to be useful for
taxonomic arrangement of Asian Curcuma species and standardization of Curcuma crude drugs.
References
1. Liu Q, Zhu S, Hayashi S, Iida O, Takano A, Miyake K, Sukrong S, Agil M, Balachandran I,
Nakamura N, Kawahara N, Komatsu K (2021) Discrimination of Curcuma species from Asia
using intron length polymorphism markers in genes encoding diketide-CoA synthase and
curcumin synthase. J Nat Med, published online, https://doi.org/10.1007/s11418-021-01558-2
2. Liu Q, Zhu S, Hayashi S, Anjiki N, Takano A, Kawahara N, Komatsu K (2021) Genetic analysis
of Curcuma species from Asia based on intron regions of genes encoding diketide-CoA synthase
and curcumin synthase. J Nat Med, published online, https://doi.org/10.1007/s11418-021-01563
-5
3.

Liu Q, Komatsu K, Toume K, Zhu S, Hayashi S, Anjiki N, Kawahara N, Takano A, Miyake K,
Nakamura N. Essential oil composition of Curcuma species and drugs from Asia analyzed by
headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry
(HS-SPME-GC-MS). (in preparation)

Fig. 1 Dendrogram constructed by neighbor–joining method based on similarity of the ILP patterns.
The scale under the tree indicates branch length.

Fig 2. Representative GC-MS chromatograms of Curcuma rhizomes
A, C. longa (L1), Q32; B, C. longa (L2), 94009; C, C. phaeocaulis (P), Q38; D, C. aeruginosa (Ae), Q41; E,
C. zedoaria (Ze), 91014; F, C. zanthorrhiza (Za), Q48; G, C. aromatica (JA), 01005; H, C. wenyujin (W),
Q49; I, C. amada (Am), 00591; J, C. mangga (M), 00959; K, C. kwangsiensis (K), Q63; L, C. petiolata (Pe),
94008. 2, β-pinene; 3, β-myrcene; 5, eucalyptol; 9, δ-elemene; 10, camphor; 14, β-elemene; 15, caryophyllene;
21, germacrene D; 23, zingiberene; 26, α-cedrene; 28, β-sesquiphellandrene; 29, aR-curcumene; 30, γelemene; 32, curzerene; 36, β-elemenone; 39, turmerone; 40, curzerenone; 42, germacrone; 43, β-turmerone;
44, aR-turmerone; 46, neocurdione; 52, 4,5-epoxygermacrone; 53, curcumenone; 54, xanthorrhizol.

Fig 3. OPLS-DA of EOs from Curcuma rhizomes
A, Score plot of all the plant specimens of 11 species; B, Loading plot, showing compounds contributed to the
discrimination; C, Score plot of the specimens of C. kwangsiensis, C. amada, C. mangga, C. zedoaria, C.
phaeocaulis and C. aeruginosa; D, Loading plot, showing compounds contributed to the discrimination.

この論文で使われている画像