J Nat Med DOI 10.1007/s11418-013-0807-7
ORIGINAL PAPER
Aim for production of Glycyrrhizae Radix in Japan (3): development of a new licorice cultivar Kazuo Ozaki • Makio Shibano
Received: 19 June 2013 / Accepted: 17 October 2013 Ó The Japanese Society of Pharmacognosy and Springer Japan 2013
Abstract The development of cultivars is indispensable for the establishment of a method aimed at producing licorice in Japan. The cultivar should have the following attributes: (1) the underground parts should grow vigorously; (2) the glycyrrhizin (GL) content must be 2.5 % or greater; and (3) the architecture of the aerial parts should be erect. A new cultivar suitable for the domestic production of licorice was developed by crossbreeding between strain A-19 (with a high GL content) as the mother and strain G-6 (with vigorous growth) as the father. After 2 years of cultivation, strain C-2 exhibited vigorous growth; the fresh weight and stem diameter were 148.8 g and 0.89 mm, respectively. Moreover, the dry-weight GL and total flavonoid contents of the new cultivar (strain C-2) from cultured plants were 3.61 and 1.365 %, respectively. Keywords Glycyrrhiza uralensis Cultivar Plastic tube Glycyrrhizin content Total flavonoid content
Introduction Licorice (Glycyrrhizae Radix) is mainly the root and stolon of Glycyrrhiza uralensis Fischer or Glycyrrhiza glabra Linne. It has long been used as a flavoring and natural
K. Ozaki (&) Takeda Garden for Medicinal Plant Conservation, Kyoto, Takeda Pharmaceutical Company, Ltd., Ichijoji, Sakyo-ku, Kyoto 606-8134, Japan e-mail: [email protected] M. Shibano (&) Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki 569-1094, Japan e-mail: [email protected]
sweetener worldwide, including Asia, Europe, and North America. In addition, licorice is one of the most important crude drugs in Asia, because it is the most frequently used in traditional Chinese medicine and Japanese kampo medicine prescriptions. In Japan, G. uralensis was cultivated for Japanese kampo medicine in Yamanashi prefecture during the Edo period about 300 years ago. Plants of the genus Glycyrrhiza are distributed in the People’s Republic of China (PR China), Mongolia, Russia, the Middle East, and along the shores of the Mediterranean. The large amount of licorice used in traditional medicines and as food additives is dependent on the harvest of wild G. uralensis, mainly the 3,500 tons exported annually from PR China. Recently, the Chinese government has begun to regulate exports of crude drugs from wild plants, including licorice and ephedra, to conserve resources and prevent desertification. Wild Glycyrrhiza plants growing in desert areas help stabilize the sand and retain moisture in the soil. Therefore, some groups have initiated cultivation programs of G. uralensis in PR China [1–3]. Our group has also started a G. uralensis cultivation program to ensure the stable supply of high-quality cultivated licorice in Japan [4–6]. However, it is a common problem that the glycyrrhizin (GL) content of cultivated licorice is low. The Pharmacopoeia of Japan, 16th edition, requires that licorice should contain greater than 2.5 % GL as the main active principle. In a previous study, strain A-19 (with a high GL content) and strain G-6 (with vigorous growth) were selected from among various strains of G. uralensis seedling plants as pharmaceutically promising for commercial cultivation [5]. This paper introduces a new cultivar (strain C-2) suitable for the domestic production of licorice, which was developed by crossbreeding strain A-19 as the mother and strain G-6 as the father. In addition, the total flavonoid and
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J Nat Med
GL contents from various strains of commercial wild licorice and the new cultivar were investigated to evaluate the chemical quality of cultivated licorice.
Materials and methods Plant materials Two pharmaceutically promising strains, A-19 and G-6, were used in this experiment. In addition, strains C-1 and C-2 were developed by crossbreeding A-19 and G-6, and strains S-1–S-10 were obtained from self-pollination of A-19. Strains A-19 and G-6 were identified using DNA analysis by Prof. Hiroaki Hayashi (Iwate Medical University), and voucher specimens are filed in the Takeda Garden for Medicinal Plant Conservation (Kyoto, Japan) [7]. Fig. 1 Dehiscence of anthers and condition of stigma
Cultivation conditions Glycyrrhiza uralensis (strains A-19 and G-6) were cultivated in a plastic tube (10 cm i.d., 55–100 cm long) or circular planters (55 cm i.d., 45 L) at Takeda Garden for Medicinal Plant Conservation. The potting soil (pH 6.2–6.3) was made from a mix of humus-rich soil, cow manure, and calcium carbonate at a ratio of 40:2:1. The cultivation conditions were based on the results of previous studies [5, 6]. The circular planters were used to grow seedling plants (SPs). The seeds, which were obtained by crossbreeding between strains A-19 and G-6, were germinated in 90-mm Petri dishes containing distilled sterile water for 24 h in a warm area (25 °C). The germinated seeds were planted into a growing pot in May 2005. After 1 month, they were transplanted into a planter and then placed in a greenhouse to grow outside. In contrast, a plant from the stolon (RP) and a cultured plant (CP) of strains C-1 and C-2 was processed as follows. Each stolon was cut into pieces of about 5 cm and planted in a plastic tube (50 cm) in April 2007. The CP was transplanted into a plastic tube (50 cm) after habituation in April 2009. Node culture CPs of strains C-1 and C-2 were obtained through node culture. The shoots of G. uralensis (strains C-1 and C-2) were cut and sterilized in 70 % ethanol and 1 % hypochlorite. The sterilized shoots were rinsed several times in sterile water, and then the growing points were removed under a stereomicroscope. The apexes were cultured on 1/3 Murashige and Skoog (MS) medium supplemented with 1 % sugar, 0.2 % Gelrite, indole-3-acetic acid (0.1 mg/L), and 6-benzylaminopurine (0.3 mg/L). The sprouted shoots were cut into sections with one axillary bud. The nodes were transferred
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onto the 1/3 MS medium supplemented with indol-3-butyric acid (IBA) (0.1 mg/L) and they elongated and took root after 1–2 weeks of culture. The culture was maintained at 25 °C under a 16-h light photo-period of 4,000 lux provided by fluorescent tubes. These plants were transferred to potting soil with vermiculite and then acclimatized. Crossbreeding between strains A-19 and G-6 Prepollination steps were begun just before the flower opened (Fig. 1). The stamens of the buds were removed to prevent any self-pollination from the seed parent (strain A-19), and then each flower was covered with a paper bag. After 4 days, the pollen from a flower of the pollen parent (strain G-6) was painted onto the stigma of emasculated flowers. The flowers were again covered with paper bags. After 1 month, in July 2007, five seeds from cross-pollination were obtained (Fig. 2), in addition to 15 seeds from self-pollination. Commercial licorice samples Commercial samples of licorice were purchased from Shinwa Bussan Co. Ltd. (Osaka, Japan), Yamada Yakken Co. Ltd. (Osaka, Japan), Uchida Wakanyaku Co. Ltd. (Tokyo, Japan), Tochimoto Tenkaido Co. Ltd. (Osaka, Japan), Fukuda Syouten (Nara, Japan), and Mikuni Co. Ltd. (Osaka, Japan). Total flavonoid analysis [8] Sample solution preparation Dried G. uralensis roots were powdered with a mill and mortar. The powdered sample (300 mg) was extracted and
J Nat Med
with CH2Cl2–MeOH (50:1?1:1) to afford 51 fractions (fr.). Each fraction was subjected to HPLC separation [Develosil phA 10 i.d. 9 250 mm (Nomura Chemical Co., Ltd., Seto, Japan) or Cosmosil 5PE-MS 10 i.d. 9 250 mm (Nacalai Tesque), CH3CN-1 % AcOH (33:67 or 30:70)]. From fr. 22–31, ISO (65 mg) and FOR (6 mg) were obtained as yellow and colorless powders, respectively. From fr. 32–44, LIQ (151 mg) was obtained as a colorless powder. Their purities were confirmed by spectroscopic analyses such as 1H-NMR spectra. GL content analysis
Fig. 2 Fructification by cross-pollination (strain A-19 9 strain G-6)
hydrolyzed with 8 mL of 35 % HCl–H2O–MeOH (1:2:2) under reflux for 2 h. After cooling, the solution was neutralized with 3.5 mL of 28 % ammonia water–MeOH (1:2). The filtrate was transferred to a 20-mL volumetric flask. The residual powder was washed twice with 1.0 mL of methanol. The respective solutions were combined, and then the final volume was adjusted to 20 mL. All samples were filtered through a 0.45-lm membrane filter prior to use.
The powdered sample (100 mg) was extracted with 50 % ethanol (10 mL) for 20 min by sonication, and the solution was filtered through a 0.45-lm membrane filter. An aliquot (10 lL) of the filtrate was analyzed using HPLC. The conditions for HPLC were: column, Nacalai Tesque (Kyoto, Japan) Cosmosil 2.5C18-MS-II (100 9 2.0 mm i.d.; 2.5 lm); mobile phase, 1 % acetic acid in water:acetonitrile (72:28); flow rate, 0.4 mL/min; column oven temperature, 40 °C; and detection, UV 254 nm. The quantitative determination was carried out using an external standard for calibration. The concentrations of standard solutions of GL (Japanese pharmacopoeia reference standard) were 8.0 mg/ 10 mL, 4.0 mg/10 mL, 2.0 mg/10 mL, and 0.4 mg/10 mL. The retention time of GL was 5.62 min.
HPLC analysis A Shimadzu (Kyoto, Japan) Shim-pack XR-ODS III column (150 9 2.0 mm i.d.; 2.2 lm) was used for HPLC analysis. The mobile phase consisted of 1 % acetic acid in water and acetonitrile at a ratio of 77:23. The flow rate was 0.4 mL/min, the column oven temperature was 40 °C, and detection was at 254 nm. The injection volume was 5 lL. The quantitative determination was carried out using an external standard for calibration. The standard solutions for liquiritigenin (LIQ), isoliquiritigenin (ISO), and formononetin (FOR) were prepared as follows: LIQ, 1.49, 0.596, 0.298, and 0.149 mg/mL; ISO, 0.45, 0.18, 0.09, and 0.045 mg/mL; and FOR, 0.45, 0.09, 0.045, and 0.025 mg/ mL. Total flavonoid contents were calculated as the (LIQ ? ISO ? FOR) contents. LIQ, ISO, and FOR were isolated and purified as follows: dried licorice (300 g) was refluxed three times with MeOH (3 L) for 3 h. The solution was concentrated under reduced pressure to give an extract (78.5 g). The extract was hydrolyzed in 35 % HCl–H2O–MeOH (50:100:100) under reflux for 2 h. After cooling, the mixture was neutralized with 10 % NaOH and extracted with ethyl acetate. The ethyl acetate phase was concentrated in vacuo to give a flavonoid mixture. This mixture (19 g) was chromatographed on a silica gel (400 g) column (5.3 i.d. 9 50 cm)
Results Comparison between growth and GL content of roots from SPs (C-1, C-2, and S1–S10) Strain A-19 (high GL content; plant architecture: semiprostrate) and strain G-6 (vigorous growth; plant architecture: erect) were selected from among the SPs as pharmaceutically promising for commercial cultivation [5]. To develop a novel cultivar suitable for the production of licorice in Japan, strains A-19 and G-6 were crossbred. The seeds were sown in a growing pot, and 12 seedling plants (strains C-1, C-2, and S1–S10) were obtained. The growth and GL content were investigated after allowing these 12 SPs to grow for 2 years (Table 1). The diameter of the taproot head of both strains C-1 and C-2 was 2.5 cm, and their fresh weights were 67.2 and 89.0 g, respectively. Of 10 plants obtained by self-pollination, the growth of 9 plants was defective; however, the taproot head diameter and fresh weight of strain S8 were 2.2 cm and 46.1 g, respectively. Defective growth resulting from genetic deterioration represents a serious problem for seed production in Japan. On the other hand, the GL contents of strains C-1 and C-2 were 2.97 and 3.45 %, respectively.
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These values were higher than the mean GL value of 1.48 ± 0.54 % (n = 30) from the SPs (from Mongolia) investigated in the preliminary study. Table 1 Comparison of growth and GL content of the root from seedling plants (C-1, C-2, and S1–S10) Strain
Taproots (cm)
No. of stolons
Weight (g) Fresh
Dry
GL content (%)
Head diameter
Length
C-l
2.5
29
6
67.2
33.3
2.97
C-2
2.5
38
6
89.0
41.6
3.45
S-l
1.3
30
0
34.2
17.0
3.18
S-2
0.9
10
0
4.3
1.8
2.43
S-3
1.8
22
1
22.8
10.7
2.62
S-4
1.9
28
1
39.5
19.5
3.65
S-5
0.4
8
0
0.8
–
–
S-6
1.2
25
0
23.7
9.4
2.87
S-7
1.2
20
0
19.8
9.1
2.97
S-8
2.2
32
3
46.1
22.9
S-9
0.4
8
0
0.7
–
S-10
1.1
30
0
23.1
10.8
3.72 – 3.80
Table 2 Comparison of aerial parts of strains A-19, G-6, C-1, and C-2 Strain
No. of samples
Stems Height (cm)
No.
Diameter (mm)
A-19
9
62.3 ± 3.7
3.4 ± 0.6
0.62 ± 0.02
G-6
8
82.3 ± 2.1
3.5 ± 0.4
0.63 ± 0.02
C-1
8
36.0 ± 3.1
1.9 ± 0.4
0.46 ± 0.03
C-2
7
72.9 ± 2.5
1.7 ± 0.3
0.89 ± 0.06
Each value represents the mean ± standard error
Comparison of aerial parts of strains A-19, G-6, C-1, and C-2 Each RP (plant from stolon) from strains A-19, G-6, C-1, and C-2 was grown in a plastic tube (10 cm i.d.; length 55–100 cm) from one stock for 15 months. In August 2008, the plant architecture, mean plant height, mean stem diameter at ground level, and number of stems of each RP were examined (Table 2). The mean plant height of strain G-6 was the greatest (82.3 cm) among the four strains, followed by strain C-2 at 72.9 cm. The plant height of strain C-1 was the lowest (36.0 cm). Strains A-19 and G-6 had a mean 3.4 and 3.5 stems, respectively, while strain C-2 had only 1.7. The mean stem diameter of strain C-2 was 0.89 mm, and thus its architecture was erect. In largescale cultivation of licorice, intertillage between ridges is necessary. This plant characteristic could make it suitable for agricultural mechanization (cultivator) in licorice cultivation. In addition, effective weed control could be achieved. The growth and GL content of the underground parts of strains C-1 and C-2 from RPs were examined (Table 3). The growth of the root of strain C-2 was excellent, and the GL content (3.61 %) of strain C-2 was greater than that of strain C-1 (2.61 %). GL and total flavonoid contents of strain C-2 from CPs as a candidate licorice cultivar Licorice contains flavonoids and triterpenoids as the major bioactive components. Most of the flavonoids are the glycosidic form of LIQ, ISO, and FOR. This analytical method did not give the separate glycoside contents of each but gave the aglycone contents using acid hydrolysis. Thus, for the chemical evaluation of licorice, total flavonoid (LIQ ? ISO ? FOR) and GL contents in strain C-2 (as 8-
Table 3 Comparison of growth and GL content of the root from RPs (plants from stolons) (C-1 and C-2) Strain
Head diameter (cm)
Maximum length of roots (cm)
No. of lateral roots
Weight (g) Fresh
GL content (%) Dry
C-1
1.8 ± 0.1
54.8 ± 2.9
1.0 ± 0.4
107.4 ± 11.6
48.7 ± 4.8
2.61 ± 0.18
C-2
2.0 ± 0.1
58.6 ± 3.3
1.0 ± 0.3
148.8 ± 19.6
64.6 ± 8.9
3.61 ± 0.24
Each value represents the mean ± standard error (n = 5) Table 4 Growth and GL content of the root from cultured plants (strain C-2) Strain
C-2
Cultivation period (months)
Head diameter (cm)
Maximum length of roots (cm)
No. of lateral roots
8
1.0 ± 0.1
57.8 ± 1.9
0.4 ± 0.2
34.8 ± 5.5
15.5 ± 2.4 3.56 ± 0.32
1.051 ± 0.039
20
1.7 ± 0.1
56.9 ± 2.4
0.9 ± 0.3
123.4 ± 19.2
49.9 ± 8.1 3.83 ± 0.20
1.365 ± 0.032
Each value represents the mean ± standard error (n = 10)
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Weight (g) Fresh
Dry
GL content (%)
Total flavonoid content (%)
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protection system of Japan. The roots of Miyako No. 1 plants (from RP) cultivated in the field for 2 years had a mean GL content of 3.25 ± 0.24 % (n = 12) and a total mean flavonoid content of 1.983 ± 0.173 % (n = 12). Research on the cultivation of G. uralensis for the stable supply of high-quality cultivated licorice is continuing.
References
Fig. 3 GL and total flavonoid contents of commercial licorice and strain C-2
or 20-month-old CP plants) and commercial wild licorice were investigated. Table 4 shows the GL and total flavonoid contents measured in a candidate licorice cultivar (strain C-2). The constituents analyzed in the roots were also compared between 20-month-old CP plants and commercial wild licorice, and the results are shown in Fig. 3. The candidate licorice cultivar showed higher GL content (3.83 ± 0.20 %) than the average content (3.29 ± 0.75 %) of commercial licorice. The total flavonoid (1.365 ± 0.032 %) content might be influenced by the cultivation years and conditions.
Conclusion An application for registration of strain C-2 as licorice cultivar Miyako No. 1 was filed with the plant variety
1. Yamamoto Y, Tani T (2002) Growth and glycyrrhizin contents in Glycyrrhiza uralensis roots cultivated for four years in eastern NeiMeng-gu of China. J Trad Med 19:87–92 2. Yamamoto Y, Majima T, Sasaki I, Tani T (2003) Pharmaceutical evaluation of Glycyrrhiza uralensis roots cultivated in Eastern NeiMeng-Gu of China. Biol Pharm Bull 26:1144–1149 3. Toda N, Sasaki S, Takeda O, Wei SL, Wang WQ, Wang YG, Shi H, Li G (2012) Cultivation of licorice (Glycyrrhiza uralensis) in China (Part 1): Glycyrrhizin contents in root influenced by stem cutting. Shoyakugaku Zasshi 66:65–70 4. Ozaki K, Shibano M, Kusano G, Watanabe H (2007) Aim for production of Glycyrrhizae Radix in Japan (1): a novel cultivation method of Glycyrrhiza uralensis Fisher. Shoyakugaku Zasshi 61:89–92 5. Ozaki K, Shibano M, Kusano G, Watanabe H (2010) Aim for production of Glycyrrhizae Radix in Japan (2): selection of pharmaceutically fine strains from Glycyrrhiza uralensis Fisher. Shoyakugaku Zasshi 64:76–82 6. Shibano M, Kawase S, Murakami R, Kita S, Kusano G, Shibata T, Hatakeyama Y, Agata I (2000) Basic studies for cultivation of Glycyrrhiza uralensis (1). Nat Med 54:70–74 7. Hayashi H, Inoue K, Ozaki K, Watanabe H (2005) Comparative analysis of ten strains of Glycyrrhiza uralensis cultivated in Japan. Biol Pharm Bull 26:1113–1116 8. Shibano M, Ozaki K, Watanabe H, Tabata A, Taniguchi M, Baba K (2010) Determination of flavonoids in licorice using acid hydrolysis and reversed-phase HPLC and evaluation of the chemical quality of cultivated licorice. Planta Med 76:729–733
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ORIGINAL PAPER
Aim for production of Glycyrrhizae Radix in Japan (3): development of a new licorice cultivar Kazuo Ozaki • Makio Shibano
Received: 19 June 2013 / Accepted: 17 October 2013 Ó The Japanese Society of Pharmacognosy and Springer Japan 2013
Abstract The development of cultivars is indispensable for the establishment of a method aimed at producing licorice in Japan. The cultivar should have the following attributes: (1) the underground parts should grow vigorously; (2) the glycyrrhizin (GL) content must be 2.5 % or greater; and (3) the architecture of the aerial parts should be erect. A new cultivar suitable for the domestic production of licorice was developed by crossbreeding between strain A-19 (with a high GL content) as the mother and strain G-6 (with vigorous growth) as the father. After 2 years of cultivation, strain C-2 exhibited vigorous growth; the fresh weight and stem diameter were 148.8 g and 0.89 mm, respectively. Moreover, the dry-weight GL and total flavonoid contents of the new cultivar (strain C-2) from cultured plants were 3.61 and 1.365 %, respectively. Keywords Glycyrrhiza uralensis Cultivar Plastic tube Glycyrrhizin content Total flavonoid content
Introduction Licorice (Glycyrrhizae Radix) is mainly the root and stolon of Glycyrrhiza uralensis Fischer or Glycyrrhiza glabra Linne. It has long been used as a flavoring and natural
K. Ozaki (&) Takeda Garden for Medicinal Plant Conservation, Kyoto, Takeda Pharmaceutical Company, Ltd., Ichijoji, Sakyo-ku, Kyoto 606-8134, Japan e-mail: [email protected] M. Shibano (&) Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki 569-1094, Japan e-mail: [email protected]
sweetener worldwide, including Asia, Europe, and North America. In addition, licorice is one of the most important crude drugs in Asia, because it is the most frequently used in traditional Chinese medicine and Japanese kampo medicine prescriptions. In Japan, G. uralensis was cultivated for Japanese kampo medicine in Yamanashi prefecture during the Edo period about 300 years ago. Plants of the genus Glycyrrhiza are distributed in the People’s Republic of China (PR China), Mongolia, Russia, the Middle East, and along the shores of the Mediterranean. The large amount of licorice used in traditional medicines and as food additives is dependent on the harvest of wild G. uralensis, mainly the 3,500 tons exported annually from PR China. Recently, the Chinese government has begun to regulate exports of crude drugs from wild plants, including licorice and ephedra, to conserve resources and prevent desertification. Wild Glycyrrhiza plants growing in desert areas help stabilize the sand and retain moisture in the soil. Therefore, some groups have initiated cultivation programs of G. uralensis in PR China [1–3]. Our group has also started a G. uralensis cultivation program to ensure the stable supply of high-quality cultivated licorice in Japan [4–6]. However, it is a common problem that the glycyrrhizin (GL) content of cultivated licorice is low. The Pharmacopoeia of Japan, 16th edition, requires that licorice should contain greater than 2.5 % GL as the main active principle. In a previous study, strain A-19 (with a high GL content) and strain G-6 (with vigorous growth) were selected from among various strains of G. uralensis seedling plants as pharmaceutically promising for commercial cultivation [5]. This paper introduces a new cultivar (strain C-2) suitable for the domestic production of licorice, which was developed by crossbreeding strain A-19 as the mother and strain G-6 as the father. In addition, the total flavonoid and
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GL contents from various strains of commercial wild licorice and the new cultivar were investigated to evaluate the chemical quality of cultivated licorice.
Materials and methods Plant materials Two pharmaceutically promising strains, A-19 and G-6, were used in this experiment. In addition, strains C-1 and C-2 were developed by crossbreeding A-19 and G-6, and strains S-1–S-10 were obtained from self-pollination of A-19. Strains A-19 and G-6 were identified using DNA analysis by Prof. Hiroaki Hayashi (Iwate Medical University), and voucher specimens are filed in the Takeda Garden for Medicinal Plant Conservation (Kyoto, Japan) [7]. Fig. 1 Dehiscence of anthers and condition of stigma
Cultivation conditions Glycyrrhiza uralensis (strains A-19 and G-6) were cultivated in a plastic tube (10 cm i.d., 55–100 cm long) or circular planters (55 cm i.d., 45 L) at Takeda Garden for Medicinal Plant Conservation. The potting soil (pH 6.2–6.3) was made from a mix of humus-rich soil, cow manure, and calcium carbonate at a ratio of 40:2:1. The cultivation conditions were based on the results of previous studies [5, 6]. The circular planters were used to grow seedling plants (SPs). The seeds, which were obtained by crossbreeding between strains A-19 and G-6, were germinated in 90-mm Petri dishes containing distilled sterile water for 24 h in a warm area (25 °C). The germinated seeds were planted into a growing pot in May 2005. After 1 month, they were transplanted into a planter and then placed in a greenhouse to grow outside. In contrast, a plant from the stolon (RP) and a cultured plant (CP) of strains C-1 and C-2 was processed as follows. Each stolon was cut into pieces of about 5 cm and planted in a plastic tube (50 cm) in April 2007. The CP was transplanted into a plastic tube (50 cm) after habituation in April 2009. Node culture CPs of strains C-1 and C-2 were obtained through node culture. The shoots of G. uralensis (strains C-1 and C-2) were cut and sterilized in 70 % ethanol and 1 % hypochlorite. The sterilized shoots were rinsed several times in sterile water, and then the growing points were removed under a stereomicroscope. The apexes were cultured on 1/3 Murashige and Skoog (MS) medium supplemented with 1 % sugar, 0.2 % Gelrite, indole-3-acetic acid (0.1 mg/L), and 6-benzylaminopurine (0.3 mg/L). The sprouted shoots were cut into sections with one axillary bud. The nodes were transferred
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onto the 1/3 MS medium supplemented with indol-3-butyric acid (IBA) (0.1 mg/L) and they elongated and took root after 1–2 weeks of culture. The culture was maintained at 25 °C under a 16-h light photo-period of 4,000 lux provided by fluorescent tubes. These plants were transferred to potting soil with vermiculite and then acclimatized. Crossbreeding between strains A-19 and G-6 Prepollination steps were begun just before the flower opened (Fig. 1). The stamens of the buds were removed to prevent any self-pollination from the seed parent (strain A-19), and then each flower was covered with a paper bag. After 4 days, the pollen from a flower of the pollen parent (strain G-6) was painted onto the stigma of emasculated flowers. The flowers were again covered with paper bags. After 1 month, in July 2007, five seeds from cross-pollination were obtained (Fig. 2), in addition to 15 seeds from self-pollination. Commercial licorice samples Commercial samples of licorice were purchased from Shinwa Bussan Co. Ltd. (Osaka, Japan), Yamada Yakken Co. Ltd. (Osaka, Japan), Uchida Wakanyaku Co. Ltd. (Tokyo, Japan), Tochimoto Tenkaido Co. Ltd. (Osaka, Japan), Fukuda Syouten (Nara, Japan), and Mikuni Co. Ltd. (Osaka, Japan). Total flavonoid analysis [8] Sample solution preparation Dried G. uralensis roots were powdered with a mill and mortar. The powdered sample (300 mg) was extracted and
J Nat Med
with CH2Cl2–MeOH (50:1?1:1) to afford 51 fractions (fr.). Each fraction was subjected to HPLC separation [Develosil phA 10 i.d. 9 250 mm (Nomura Chemical Co., Ltd., Seto, Japan) or Cosmosil 5PE-MS 10 i.d. 9 250 mm (Nacalai Tesque), CH3CN-1 % AcOH (33:67 or 30:70)]. From fr. 22–31, ISO (65 mg) and FOR (6 mg) were obtained as yellow and colorless powders, respectively. From fr. 32–44, LIQ (151 mg) was obtained as a colorless powder. Their purities were confirmed by spectroscopic analyses such as 1H-NMR spectra. GL content analysis
Fig. 2 Fructification by cross-pollination (strain A-19 9 strain G-6)
hydrolyzed with 8 mL of 35 % HCl–H2O–MeOH (1:2:2) under reflux for 2 h. After cooling, the solution was neutralized with 3.5 mL of 28 % ammonia water–MeOH (1:2). The filtrate was transferred to a 20-mL volumetric flask. The residual powder was washed twice with 1.0 mL of methanol. The respective solutions were combined, and then the final volume was adjusted to 20 mL. All samples were filtered through a 0.45-lm membrane filter prior to use.
The powdered sample (100 mg) was extracted with 50 % ethanol (10 mL) for 20 min by sonication, and the solution was filtered through a 0.45-lm membrane filter. An aliquot (10 lL) of the filtrate was analyzed using HPLC. The conditions for HPLC were: column, Nacalai Tesque (Kyoto, Japan) Cosmosil 2.5C18-MS-II (100 9 2.0 mm i.d.; 2.5 lm); mobile phase, 1 % acetic acid in water:acetonitrile (72:28); flow rate, 0.4 mL/min; column oven temperature, 40 °C; and detection, UV 254 nm. The quantitative determination was carried out using an external standard for calibration. The concentrations of standard solutions of GL (Japanese pharmacopoeia reference standard) were 8.0 mg/ 10 mL, 4.0 mg/10 mL, 2.0 mg/10 mL, and 0.4 mg/10 mL. The retention time of GL was 5.62 min.
HPLC analysis A Shimadzu (Kyoto, Japan) Shim-pack XR-ODS III column (150 9 2.0 mm i.d.; 2.2 lm) was used for HPLC analysis. The mobile phase consisted of 1 % acetic acid in water and acetonitrile at a ratio of 77:23. The flow rate was 0.4 mL/min, the column oven temperature was 40 °C, and detection was at 254 nm. The injection volume was 5 lL. The quantitative determination was carried out using an external standard for calibration. The standard solutions for liquiritigenin (LIQ), isoliquiritigenin (ISO), and formononetin (FOR) were prepared as follows: LIQ, 1.49, 0.596, 0.298, and 0.149 mg/mL; ISO, 0.45, 0.18, 0.09, and 0.045 mg/mL; and FOR, 0.45, 0.09, 0.045, and 0.025 mg/ mL. Total flavonoid contents were calculated as the (LIQ ? ISO ? FOR) contents. LIQ, ISO, and FOR were isolated and purified as follows: dried licorice (300 g) was refluxed three times with MeOH (3 L) for 3 h. The solution was concentrated under reduced pressure to give an extract (78.5 g). The extract was hydrolyzed in 35 % HCl–H2O–MeOH (50:100:100) under reflux for 2 h. After cooling, the mixture was neutralized with 10 % NaOH and extracted with ethyl acetate. The ethyl acetate phase was concentrated in vacuo to give a flavonoid mixture. This mixture (19 g) was chromatographed on a silica gel (400 g) column (5.3 i.d. 9 50 cm)
Results Comparison between growth and GL content of roots from SPs (C-1, C-2, and S1–S10) Strain A-19 (high GL content; plant architecture: semiprostrate) and strain G-6 (vigorous growth; plant architecture: erect) were selected from among the SPs as pharmaceutically promising for commercial cultivation [5]. To develop a novel cultivar suitable for the production of licorice in Japan, strains A-19 and G-6 were crossbred. The seeds were sown in a growing pot, and 12 seedling plants (strains C-1, C-2, and S1–S10) were obtained. The growth and GL content were investigated after allowing these 12 SPs to grow for 2 years (Table 1). The diameter of the taproot head of both strains C-1 and C-2 was 2.5 cm, and their fresh weights were 67.2 and 89.0 g, respectively. Of 10 plants obtained by self-pollination, the growth of 9 plants was defective; however, the taproot head diameter and fresh weight of strain S8 were 2.2 cm and 46.1 g, respectively. Defective growth resulting from genetic deterioration represents a serious problem for seed production in Japan. On the other hand, the GL contents of strains C-1 and C-2 were 2.97 and 3.45 %, respectively.
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These values were higher than the mean GL value of 1.48 ± 0.54 % (n = 30) from the SPs (from Mongolia) investigated in the preliminary study. Table 1 Comparison of growth and GL content of the root from seedling plants (C-1, C-2, and S1–S10) Strain
Taproots (cm)
No. of stolons
Weight (g) Fresh
Dry
GL content (%)
Head diameter
Length
C-l
2.5
29
6
67.2
33.3
2.97
C-2
2.5
38
6
89.0
41.6
3.45
S-l
1.3
30
0
34.2
17.0
3.18
S-2
0.9
10
0
4.3
1.8
2.43
S-3
1.8
22
1
22.8
10.7
2.62
S-4
1.9
28
1
39.5
19.5
3.65
S-5
0.4
8
0
0.8
–
–
S-6
1.2
25
0
23.7
9.4
2.87
S-7
1.2
20
0
19.8
9.1
2.97
S-8
2.2
32
3
46.1
22.9
S-9
0.4
8
0
0.7
–
S-10
1.1
30
0
23.1
10.8
3.72 – 3.80
Table 2 Comparison of aerial parts of strains A-19, G-6, C-1, and C-2 Strain
No. of samples
Stems Height (cm)
No.
Diameter (mm)
A-19
9
62.3 ± 3.7
3.4 ± 0.6
0.62 ± 0.02
G-6
8
82.3 ± 2.1
3.5 ± 0.4
0.63 ± 0.02
C-1
8
36.0 ± 3.1
1.9 ± 0.4
0.46 ± 0.03
C-2
7
72.9 ± 2.5
1.7 ± 0.3
0.89 ± 0.06
Each value represents the mean ± standard error
Comparison of aerial parts of strains A-19, G-6, C-1, and C-2 Each RP (plant from stolon) from strains A-19, G-6, C-1, and C-2 was grown in a plastic tube (10 cm i.d.; length 55–100 cm) from one stock for 15 months. In August 2008, the plant architecture, mean plant height, mean stem diameter at ground level, and number of stems of each RP were examined (Table 2). The mean plant height of strain G-6 was the greatest (82.3 cm) among the four strains, followed by strain C-2 at 72.9 cm. The plant height of strain C-1 was the lowest (36.0 cm). Strains A-19 and G-6 had a mean 3.4 and 3.5 stems, respectively, while strain C-2 had only 1.7. The mean stem diameter of strain C-2 was 0.89 mm, and thus its architecture was erect. In largescale cultivation of licorice, intertillage between ridges is necessary. This plant characteristic could make it suitable for agricultural mechanization (cultivator) in licorice cultivation. In addition, effective weed control could be achieved. The growth and GL content of the underground parts of strains C-1 and C-2 from RPs were examined (Table 3). The growth of the root of strain C-2 was excellent, and the GL content (3.61 %) of strain C-2 was greater than that of strain C-1 (2.61 %). GL and total flavonoid contents of strain C-2 from CPs as a candidate licorice cultivar Licorice contains flavonoids and triterpenoids as the major bioactive components. Most of the flavonoids are the glycosidic form of LIQ, ISO, and FOR. This analytical method did not give the separate glycoside contents of each but gave the aglycone contents using acid hydrolysis. Thus, for the chemical evaluation of licorice, total flavonoid (LIQ ? ISO ? FOR) and GL contents in strain C-2 (as 8-
Table 3 Comparison of growth and GL content of the root from RPs (plants from stolons) (C-1 and C-2) Strain
Head diameter (cm)
Maximum length of roots (cm)
No. of lateral roots
Weight (g) Fresh
GL content (%) Dry
C-1
1.8 ± 0.1
54.8 ± 2.9
1.0 ± 0.4
107.4 ± 11.6
48.7 ± 4.8
2.61 ± 0.18
C-2
2.0 ± 0.1
58.6 ± 3.3
1.0 ± 0.3
148.8 ± 19.6
64.6 ± 8.9
3.61 ± 0.24
Each value represents the mean ± standard error (n = 5) Table 4 Growth and GL content of the root from cultured plants (strain C-2) Strain
C-2
Cultivation period (months)
Head diameter (cm)
Maximum length of roots (cm)
No. of lateral roots
8
1.0 ± 0.1
57.8 ± 1.9
0.4 ± 0.2
34.8 ± 5.5
15.5 ± 2.4 3.56 ± 0.32
1.051 ± 0.039
20
1.7 ± 0.1
56.9 ± 2.4
0.9 ± 0.3
123.4 ± 19.2
49.9 ± 8.1 3.83 ± 0.20
1.365 ± 0.032
Each value represents the mean ± standard error (n = 10)
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Weight (g) Fresh
Dry
GL content (%)
Total flavonoid content (%)
J Nat Med
protection system of Japan. The roots of Miyako No. 1 plants (from RP) cultivated in the field for 2 years had a mean GL content of 3.25 ± 0.24 % (n = 12) and a total mean flavonoid content of 1.983 ± 0.173 % (n = 12). Research on the cultivation of G. uralensis for the stable supply of high-quality cultivated licorice is continuing.
References
Fig. 3 GL and total flavonoid contents of commercial licorice and strain C-2
or 20-month-old CP plants) and commercial wild licorice were investigated. Table 4 shows the GL and total flavonoid contents measured in a candidate licorice cultivar (strain C-2). The constituents analyzed in the roots were also compared between 20-month-old CP plants and commercial wild licorice, and the results are shown in Fig. 3. The candidate licorice cultivar showed higher GL content (3.83 ± 0.20 %) than the average content (3.29 ± 0.75 %) of commercial licorice. The total flavonoid (1.365 ± 0.032 %) content might be influenced by the cultivation years and conditions.
Conclusion An application for registration of strain C-2 as licorice cultivar Miyako No. 1 was filed with the plant variety
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