Time-Course Accumulation of Main Bioactive Components in the Rhizome of Ligusticum chuanxiong

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

Fresh rhizomes of Ligusticum chuanxiong, a commonly used traditional Chinese medicinal herb, were collected monthly from a cultivating base in China practicing good agriculture practice (GAP). These samples were analyzed by HPLC-UV for their main chemical ingredients. Senkyunolide A (6), coniferyl ferulate (7) and Z-ligustilide (8) were identified as the major ingredients. The accumulation of the main ingredients with time in the herb was elucidated. Both individual and total contents of all main components gradually increased from the beginning of October to the middle of next April. The weight of a single rhizome reached a plateau at the end of May, whilst the content of the major ingredients peaked in the middle of April. Based on these results, it is recommended that Rhizoma Chuanxiong be harvested between the middle of April and the end of May.

The dried form of Rhizoma Chuanxiong, the rhizome of Ligusticum chuanxiong Hort. (Umbelliferae), is one of the traditional Chinese medicinal (TCM) herbs frequently used in TCM prescriptions for the treatment of cerebro- and cardio-vascular diseases. L. chuanxiong grown in Sichuan Province, China, has been traditionally recognized as the authentic and superior plant source for the herbal Rhizoma Chuanxiong [1]. To date, a variety of chemical compounds have been isolated and identified from Rhizoma Chuanxiong, including vanillin (1), ferulic acid (2), tetramethylpyrazine (3), senkyunolide I (4), senkyunolide H (5), senkyunolide A (6), coniferyl ferulate (7), Z-ligustilide (8), 3-butylidenephthalide (9), riligustilide (10) and levistolide A (11) [2], [3], [4], [5], [6], [7], [8] (Fig. [1]). Pharmacological studies have revealed that most of the aforementioned ingredients produce multiple biological activities which may contribute to the therapeutic effects of the herb [3], [9], [10], [11], [12]. Furthermore, our previous studies showed that the contents of these components varied significantly in different commercial Rhizoma Chuanxiong samples, indicating the importance of quality control of the herb [13], [14].

TCM herb producers are encouraged to farm under good agriculture practice (GAP) to ensure better quality control. Currently, several GAP cultivating bases for Rhizoma Chuanxiong are under development in Dujiangyan County, Sichuan Province, China. Amongst many factors affecting the quality of TCM herbs, harvest time is one of the most important factors that can be controlled by GAP cultivation. However, there is no documented evidence to support the traditional harvest time for Rhizoma Chuanxiong. This study aims to identify the optimal time for harvesting Rhizoma Chuanxiong.

L. chuanxiong is an annual herb. As illustrated in Table [1], its growing period can be divided into six stages. In the present study, rhizomes of L. chuanxiong grown in a GAP cultivating base, were collected monthly from the stem emergence and growth stage (beginning of October) to the tillering stage (middle of the following April). At the final stage of growth, the rhizome expanded rapidly. Samples were collected weekly until the herb was harvested at the end of May. Fresh herbal samples were analyzed using our previously developed HPLC-UV method [14]. As shown in Fig. [2] A, all samples contain five compounds with 6 - 8 as the major and 2 and 9 as the minor components, while only trace amounts of components 10 and 11 were found in some of the samples. Furthermore, both individual and total contents of the major components increased gradually with time. The peak was achieved in the middle of April. This was followed by a decline before it increased again from the middle of May until the harvest time (the end of May).

It was noted that compounds 4 and 5 were determined in commercial dried and/or processed Rhizoma Chuanxiong [13], [14], [15], but were not detected in fresh herb in the present study. Our preliminary study (unpublished data) suggests that hydroxylated phthalides, such as compounds 4 and 5 present in the commercial dried Chuanxiong herb, may be generated from the chemical conversion of the major phthalide, compound 8, during processing and/or storage. Studies on such chemical reactions during herbal processing are currently in progress.

Previous studies reported that the average dried weight of a single fresh rhizome of L. chuanxiong increased rapidly in the rhizome expansion stage and reached the highest value at the end of May [17], [18]. When the weight of the rhizome was taken into consideration, a continuous accumulation pattern of the total amount of the main bioactive ingredients per rhizome was obtained (Fig. [2] B). It was found that both individual and total amounts of the main components per rhizome increased rapidly from the end of October to the middle of the following April. This trend leveled off for a period leading to the tillering stage, the middle of May, before it rose again until harvest time at the end of May.

From ancient time until today Rhizoma Chuanxiong has being harvested in late May of each year. Our results showed that late May might be the optimal harvest time for the producers to obtain the highest herbal yield and thus have more benefits based on the weight of the herb sold. However, the results also suggest that from the pharmaceutical manufacturer’s point of view, it would be more cost-effective to harvest this herb in the middle of April each year, when the total content of the main bioactive components reaches its highest level. Therefore, the suitable harvest time for rhizome Chuanxiong is in the period from the middle of April to the end of May, depending upon the commercial interest of herbal producers or herbal pharmaceutical manufacturers.

Materials and Methods

All fresh rhizomes of L. chuanxiong were supplied by Tianfu Chuanxiong Pharmaceutical Development Company Ltd. in Dujiangyan County, Sichuan Province, China. Herbal samples were collected monthly from October 8, 2002 to April 11, 2003 followed by weekly sampling until May 20, 2003. At each collecting time at least three herbal plants were collected and analyzed individually. The voucher specimens were deposited at Department of Pharmacology, The Chinese University of Hong Kong. Fresh rhizomes were cut into pieces, lyophilized, and ground into powder (40 mesh) in the dark. The dried powders (0.1 g) were extracted with 5.0 mL of methanol with continuous shaking at room temperature for 30 min twice. After centrifugation, the supernatants were combined, filtered, and subjected to the HPLC-UV analysis in the same manner as described in our previous work [14]. Each herbal sample was analyzed in triplicate.

Acknowledgements

The authors gratefully acknowledge the Innovative Technology Commission of Hong Kong for the financial support of ITF fund (UIM/034) and the Tianfu Chuanxiong Pharmaceutical Development Company Ltd. for their continuous supply of fresh samples of Rhizoma Chuanxiong.

References

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• 16 Peking Institute of Pharmaceutical I ndustries. Structure elucidation and synthesis of tetramethylpyrazine.  Zhong Cao Yao. 1977;  8 150-2
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  PubMedSearch in Google Scholar

Ge Lin

Department of Pharmacology

Faculty of Medicine

The Chinese University of Hong Kong

Shatin

N. T.

Hong Kong

SAR

Phone: +852-2609-6824

Fax: +852-2603-5139

Email: linge@cuhk.edu.hk

References

• 1 Hu S L, Chi Q, Zhao Z Z. The authentic and superior medicinal herbals in China. Haerbin; Heilongjiang Press of Science and Technology 1989: p. 133-7
  Search in Google Scholar
• 2 Wang P S, Gao Y X, Wang Y X, Yoshiyasu F, Iwao M, Michiharu S. Phthalides from the rhizome of Ligusticum wallichi .  Phytochemistry. 1984;  23 2033-8
  CrossrefPubMedSearch in Google Scholar
• 3 Wang P S. Survey of investigation on chemical principles and biological activities of Ligusticum wallichi .  Zhongguo Yiyao Gongye. 1988;  19 553-9
  PubMedSearch in Google Scholar
• 4 Kobayashi M, Fujita M, Mitsuhashi H. Studies on the constituents of Umbelliferae plants. XV. Constituents of Cnidium officinale: Occurrence of pregnenolone, coniferyl ferulate and hydroxyphthalides.  Chem Pharm Bull. 1987;  35 1427-33
  CrossrefPubMedSearch in Google Scholar
• 5 Tsuchida T, Kobayashi M, Kaneko K, Mitsuhashi H. Studies on the constituents of Umbelliferae plants. XVI. Isolation and structures of three new ligustilide derivatives from Angelica acutiloba .  Chem Pharm Bull. 1987;  35 4460-4
  CrossrefPubMedSearch in Google Scholar
• 6 Kobayashi M, Mitsuhashi H. Studies on the constituents of Umbelliferae plants. XVII. Structures of three new ligustilide derivatives from Ligusticum wallichi .  Chem Pharm Bull. 1987;  35 4789-92
  CrossrefPubMedSearch in Google Scholar
• 7 Kaouadji M, Pachtere F, Pouget C, Chulia A J. Three additional phthalide derivatives, an epoxymonomer and two dimers, from Ligusticum wallichi rhizomes.  J Nat Prod. 1986;  49 872-7
  CrossrefPubMedSearch in Google Scholar
• 8 Naito T, Katsuhara T, Niitsu K, Ikeya Y, Okada M, Mitsuhashi H. Phthalide dimers from Ligusticum chuanxiong Hort.  Heterocycles. 1991;  32 2433-42
  PubMedSearch in Google Scholar
• 9 Naito T, Kubota K, Shimoda Y, Sato T, Ikeya Y, Okada M. Effects of constituents in a Chinese crude drug, Ligustici Chuanxiong Rhizoma on vasocontracton and blood viscosity.  Nat Med. 1995;  49 288-92
  PubMedSearch in Google Scholar
• 10 Kobayashi S, Mimura Y, Notoya K, Kimura I, Kimura M. Antiproliferative effects of the traditional Chinese medicine Shimotsu-To, its component Cnidium rhizome and derived compounds on primary cultures of mouse aorta smooth muscle cells.  Jap J Pharmacol. 1992;  60 397-401
  PubMedSearch in Google Scholar
• 11 Kobayashi S, Mimura Y, Naito Y, Kimura I, Kimura M. Chemical structure-activity of Cnidium rhizome-derived phthalides for the competence inhibition of proliferation in primary cultures of mouse aorta smooth muscle cells.  Jap J Pharmacol. 1993;  63 353-9
  PubMedSearch in Google Scholar
• 12 Foye W O, Wang H P, Wang H F. Synthesis and platelet aggregation inhibitory effects of harman and phthalide derivatives related to Ligusticum chuanxiong Hort. constituents.  Med Chem Res. 1997;  7 180-91
  PubMedSearch in Google Scholar
• 13 Li S L, Chan S SK, Lin G, Ling L, Yan R, Chung H S. et al . Simultaneous analysis of seventeen chemical ingredients of Ligusticum chuanxiong by on-line high performance liquid chromatography-diode array detector-mass spectrometry.  Planta Med. 2003;  69 445-51
  Thieme ConnectPubMedSearch in Google Scholar
• 14 Yan R, Li S L, Chung H S, Tam Y K, Lin G. Simultaneous quantification of 12 bioactive components of Ligusticum chuanxiong Hort. by high-performance liquid chromatography.  J Pharm Biomed Anal. 2005;  37 87-95
  CrossrefPubMedSearch in Google Scholar
• 15 Li S L, Lin G, Chung H S, Tam Y K. Study on fingerprint of Rhizoma Chuanxiong by HPLC-DAD-MS.  Acta Pharm Sin. 2004;  39 621-6
  PubMedSearch in Google Scholar
• 16 Peking Institute of Pharmaceutical I ndustries. Structure elucidation and synthesis of tetramethylpyrazine.  Zhong Cao Yao. 1977;  8 150-2
  PubMedSearch in Google Scholar
• 17 Chen X F, Ding D R, Liu S R, Huang W X, Liu S X. Studies on biological characters of Ligusticum chuanxiong Hort.  Zhongguo Zhongyao Zazhi. 1994;  19 463-6
  PubMedSearch in Google Scholar
• 18 Chen X F, Ding D R, Huang W X, Liu S R, Liu S X. Investigations into biological characters of Ligusticum chuanxiong Hort. during growth.  Zhongguo Zhongyao Zazhi. 1997;  22 527-9
  PubMedSearch in Google Scholar

Ge Lin

Department of Pharmacology

Faculty of Medicine

The Chinese University of Hong Kong

Shatin

N. T.

Hong Kong

SAR

Phone: +852-2609-6824

Fax: +852-2603-5139

Email: linge@cuhk.edu.hk