Characterization of a Glycyrrhizin-Deficient Strain of Glycyrrhiza uralensis

Abstract

A triterpene saponin, glucoglycyrrhizin, was isolated from a glycyrrhizin-deficient strain 83-555 of Glycyrrhiza uralensis (Leguminosae), and the structure was determined by chemical and spectral data to be 3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]-glycyrrhetinic acid. Since this saponin has a 2′-O-β-D-glucopyranosyl moiety instead of the 2′-O-β-D-glucuronopyranosyl moiety of glycyrrhizin, the glucuronidation of 3-O-β-D-glucuronopyranosyl-glycyrrhetinic acid leading to glycyrrhizin is inhibited in this strain. All 4 offspring of the 83-555 strain produced glucoglycyrrhizin. Interestingly, 2 of the offspring produced both glycyrrhizin and glucoglycyrrhizin, and sequence analysis of the pkr gene suggested that these 2 offspring were hybrids of 83-555 strain and glycyrrhizin-producing strains.

Glycyrrhizin (GL) is a sweet-tasting triterpene diglucuronide isolated from the roots and stolons of the Glycyrrhiza plant (licorice), which belongs to the family Leguminosae. Licorice is one of the most frequently used components in traditional medicine in Japan, and Glycyrrhiza uralensis FISCH. is mainly used for this purpose.^1,2) Since G. uralensis is the most important Glycyrrhiza plant used for traditional medicine in Japan, it is important to elucidate the variations in the G. uralensis strains from the viewpoint of the medicinal resources related to licorice. In our previous study, comparative analysis of G. uralensis strains was undertaken to characterize variations in G. uralensis.³⁾ During this process, we found a unique G. uralensis strain, 83-555, producing only a trace amount of glycyrrhizin. Therefore, in the present study, chemical characterization of the GL-deficient strain was examined, and a major triterpene saponin, glucoglycyrrhizin (GGL), was isolated and characterized from this strain. Furthermore, offspring of the strain 83-555 were analyzed to select for a high-GGL-producing strain.

Materials and Methods

General Methods

¹H-NMR and ¹³C-NMR spectra were recorded using a Unity Inova-500 (Varian) spectrometer. Chemical shifts are given on a δ (ppm) scale with tetramethylsilane as the internal standard. Specific rotation was measured on a SEPA-300 (Horiba, Japan) spectrometer. Quantitative HPLC analysis and negative electrospray ionization (ESI)-MS were measured on an LCMS-IT-TOF (Shimadzu, Kyoto, Japan) spectrometer.

Plant Materials

G. uralensis strain 83-555 was derived from a seed obtained from the former East Germany in 1983 and was cultivated in the Kyoto Herbal Garden, Takeda Pharmaceutical Co., Ltd. G. uralensis strain 01A26-6 was derived from a seed collected in Kazakhstan, as reported previously.⁴⁾ Strain 83-555 was also cultivated in the Herbal Garden at the Osaka University of Pharmaceutical Sciences, and the harvested roots were used for the isolation of saponin. Seeds of strain 83-555 were collected from the Kyoto Herbal Garden, Takeda Pharmaceutical Co., Ltd., and 4 offspring derived from the seeds were cultivated at the Herbal Garden of Iwate Medical University. Voucher specimens of these strains were deposited at the herbarium of Tohoku University.

HPLC Analysis

Dried samples were powdered with a mortar and pestle, and then 50 mg of each powdered sample was extracted with 5 mL of 80% methanol at 60°C for 2 h. An aliquot (2 µL) of the extract was analyzed by HPLC (column: Inertsil ODS-SP [3 µm, 2.1 mm i.d. × 100 mm, GL Sciences, Japan]; solvent: MeCN/H₂O [0.1% formic acid] gradient of 15% MeCN to 100% MeCN in 50 min). The quantities of the constituents were determined on the basis of their peak area of UV absorption at 254 nm. Each constituent was identified by comparing its retention time, UV spectrum, and negative ESI-MS with its respective authentic sample.

Isolation of a Major Saponin from Underground Parts of the GL-Deficient Strain 83-555

Dried underground parts (50 g) of strain 83-555 were extracted twice with 500 mL of ethyl acetate at 60°C for 1 h. The dried residue was further extracted twice with 500 mL of methanol at 60°C for 1 h. The methanol extract was evaporated in vacuo, and the dried extract (3 g) was applied on a Sephadex LH-20 (200 mL) column and eluted with 80% methanol-water containing 0.1% acetic acid in 15-mL fractions (fractions 1–20). Fractions 5–12 were repeatedly chromatographed using preparative HPLC to isolate compound 1 (202 mg). The conditions used for preparative HPLC were as follows: column, Intersil PREP-ODS (20 mm i.d. × 250 mm, GL Sciences, Japan); solvent, 40% acetnitrile–water containing 0.1% formic acid; and flow rate, 5 mL/min.

Glucoglycyrrhizin (1): Colorless amorphous powder: [α]_D²⁵ +67.2° (c=0.21, MeOH); neg. High resolution (HR)-ESI-MS: Calcd for C₄₂H₆₃O₁₅ [M−H]⁻: m/z 807.4168. Found: m/z 807.4153 [M−H]⁻ (Calcd for C₄₂H₆₃O₁₅); ¹H- and ¹³C-NMR spectra (in pyridine-d₅) (Table 1).

Table 1. ¹H- and ¹³C-NMR Data for Glucoglycyrrhizin (in Pyridine-d₅)

Position	δC, mult.		δH (J in Hz)		Position		δC, mult.		δH (J in Hz)
1	39.5	CH2		1.05 m, 3.07 m	GlcUA	1′	105.4	CH	5.04 d	(7.5)
2	26.8	CH2	2.11 a), 2.30 a)			2′	82.9	CH	4.39 a) br t
3	89.0	CH	3.35 dd	(11.5, 4.5)		3′	77.8	CH	4.43 t	(9.0)
4	40.0	C				4′	73.2	CH	4.59 t	(9.0)
5	55.4	CH	0.75 br d	(11.5)		5′	77.5	CH	4.63 d	(9.0)
6	17.7	CH2	1.32 a), 1.56 a)			6′	172.5	C
7	33.0	CH2	1.30 a), 1.58 a)
8	45.6	C			Glc	1″	106.1	CH	5.45 d	(7.5)
9	62.2	CH	2.47 br s			2″	77.3	CH	4.17 dd	(9.0, 7.5)
10	37.3	C				3″	78.0	CH	4.30 t	(9.0)
11	199.6	C				4″	71.7	CH	4.38 a) br t
12	128.7	CH	5.99 s			5″	78.4	CH	3.97 ddd	(9.0, 3.5, 3.5)
13	169.7	C				6″	62.8	CH2	4.53 m
14	43.5	C
15	26.9	CH2	1.10 a), 1.73 a)
16	26.7	CH2	0.96 m
17	32.2	C
18	48.8	CH	2.55 br dd	(12.0, 3.5)
19	41.7	CH2	1.76 a), 2.15 a)
20	44.2	C
21	31.6	CH2	1.48 a), 2.30  a)
22	38.5	CH2	1.42 a), 1.71 a)
23	28.2	CH3	1.34 s
24	16.8 b)	CH3	1.17 s
25	16.8 b)	CH3	1.26 s
26	18.8	CH3	1.10 s
27	23.6	CH3	1.45 s
28	28.7	CH3	0.80 s
29	28.8	CH3	1.36 s
30	179.2	C

a), b): overlapped signals.

Acid Hydrolysis of Compound 1

Compound 1 (5 mg) was dissolved in 5 mL of 5 M HCl–1,4-dioxane (1 : 3), and the solution was refluxed for 4 h. After the reaction mixture was cooled, it was passed through an Amberlite IRA-67 (OH⁻ form) column to neutralize it. Sugars were analyzed by TLC (n-propanol–H₂O, 17 : 3).

Amplification and Sequencing of the pkr Gene

DNA was extracted from dry leaves by using the DNeasy Plant Mini Kit (Qiagen). The DNA fragment covering two simple sequence repeats (SSRs) and several single nucleotide polymorphisms (SNPs) in the pkr gene⁵⁾ was amplified by polymerase chain reaction (PCR) using the template DNA from leaves, Taq DNA polymerase (New England Biolabs), anti-Taq high (Toyobo) and 2 primers (i.e., 5′-GCG AAT TCC TTA ACT TCC TCT TCC C-3′ and 5′-GTC TCG AGA GAT TTG CGG AGA G-3′). After initial denaturation (2 min at 95°C), 30 cycles each of 30 s at 95°C, 30 s at 60°C and 1 min at 72°C were performed in a thermocycler. The amplified fragment was treated with ExoSAP-It (Affymetrix) to remove the primers. The purified fragments were sequenced directly by the dideoxy chain termination method in a DNA sequencer (3130xl Genetic Analyzer, Applied Biosystems). The 2 primers used for PCR amplification and an additional primer, 5′-GCT GCT ATT GAG ATC CCA ACA AAG G-3′ (for plants with mixed SSRs), were used for the sequencing of the Glycyrrhiza pkr gene. If the pkr genotype contained mixed SSRs and SNPs, the PCR fragment was cloned with the pGEM-T Easy Vector System (Promega), and sequenced using the T7 promoter primer and SP6 promoter primer. DNA sequences of the haplotypes in the present study have been deposited in the DDBJ/EMBL/GenBank under the accession numbers AB823670 (7-3-GGGAGCT), AB823671 (18-3-GAAAGCT), AB823672 (4-5-AGATATC), and AB823673 (8-3-GGGAGCT).

Results and Discussion

A GL-Deficient Strain of G. uralensis

The G. uralensis strain 83-555 was introduced from the former East Germany to the Kyoto Herbal Garden, Takeda Pharmaceutical Co., Ltd., as a seed of G. glabra var. glandulifera in 1983. However, the cultivated strain derived from the seed was identified as G. uralensis based on the morphologic characteristics of the falcate fruits, ovate leaflets (4–8 paired), and dense and compact racemes.⁶⁾

The underground parts of strain 83-555 were analyzed by HPLC. Figure 1 shows the HPLC profiles of the methanol extracts of the underground parts of strains 83-555 and 01A26-6 (a GL-producing strain). Although a small peak corresponding to GL was detected at 18.7 min in the HPLC profile of strain 83-555, this peak was found to be a mixture of an unidentified saponin and trace amounts of GL by LC-MS analysis. Instead of GL, compound 1 showing an ion [M−H]⁻ at m/z 807.4153 in the negative ESI-MS was detected at 19.3 min in the HPLC profile of strain 83-555. Compound 1 was not detected in the HPLC profile of the GL-producing G. uralensis strain (i.e., strain 01A26-6). The UV spectrum of compound 1 was identical to that of GL, suggesting that compound 1 was an analogue of GL. Therefore, isolation of this saponin from strain 83-555 was undertaken (see below).

Fig. 1. HPLC Profiles of Methanol Extracts of Underground Parts of G. uralensis Strains 83-555 (GL-Deficient Strain) and 01A26-6 (GL-Producing Strain)

Absorbance at 254 nm. GL, glycyrrhizin; 1, glucoglycyrrhizin.

Isolation and Characterization of a Triterpene Saponin in the Underground Parts of the GL-Deficient Strain 83-555

Dried roots and stolons of strain 83-555 were extracted with ethyl acetate, and the dried residue was further extracted with methanol. The methanol extract was purified by a Sephadex LH-20 column and preparative HPLC to isolate compound 1 (202 mg).

Compound 1 was obtained as a colorless amorphous solid and showed an ion [M−H]⁻ at m/z 807.4153 in high-resolution negative ESI-MS, which corresponds to the molecular formula C₄₂H₆₃O₁₅. Acid hydrolysis with 5 M HCl–1,4-dioxane (1 : 3) of compound 1 yielded glucuronic acid and glucose. The ¹H- and ¹³C-NMR spectra (pyridine-d₅, Table 1) were similar to those of GL, except that compound 1 had a glucopyranosyl moiety instead of a glucuronopyranosyl moiety in GL. The oligoglycoside structure and its connectivity with aglycone in compound 1 were confirmed by an heteronuclear multiple bond connectivity (HMBC) experiment. Long-range correlations were observed between the signals of H-1′ and C-3 and of H-1″ and C-2′. Therefore, the structure of compound 1, called glucoglycyrrhizin (GGL), was determined to be 3-O-[β-D-glucopyranosyl(1→2)-β-D-glucuronopyranosyl]-glycyrrhetinic acid. Thus, GGL was isolated for the first time from higher plants; however, it has been previously reported to be synthesized from glycyrrhetic acid by stepwise glycosidation.⁷⁾ GGL has a sweet taste and has been reported to have a potent cytoprotective effect on CCl₄-induced hepatotoxicity.⁷⁾

Figure 2 shows hypothetical biosynthetic pathways leading to saponins in G. uralensis. Since both GL and GGL share a possible intermediate 3-O-[β-D-glucuronopyranosyl]-glycyrrhetinic acid (monoglucuronide glycyrrhetinic acid, MGGA), it is suggested that the glucuronidation of MGGA leading to GL is inhibited in strain 83-555. Many diglucuronide saponins have been isolated from licorice roots,^8–10) and 2 monoglucuronide saponins, apioglycyrrhizin and araboglycyrrhizin, were isolated from the underground parts of G. inflata.¹⁰⁾

Fig. 2. Biosynthetic Pathways Leading to Glycyrrhizin and Glucoglycyrrhizin in G. uralensis

Characterization of the GL-Deficient Strain 83-555 and Its Offspring

Since the GL-deficient strain 83-555 produces the unique saponin GGL, this strain is an important source for the breeding of Glycyrrhiza plants. Thus, seeds of strain 83-555 (collected from the Kyoto Herbal Garden, Takeda Pharmaceutical Co., Ltd.) were germinated, and 4 offspring derived from the seeds were cultivated outdoors for 2 years.

Table 2 shows the GL and GGL content in the stolons of strain 83-555 and its offspring. All the 4 offspring of strain 83-555 produced GGL. Interestingly, 2 offspring, 83-555-2 and 83-555-3, produced both GL and GGL, although their mother plant, 83-555, produced only a trace amount of GL.

Table 2. Glucoglycyrrhizin and Glycyrrhizin Content in the Stolons of the Strains 83-555

Strain No.	Diameter(mm)	Content (% of dry weight)
		Glucoglycyrrhizin	Glycyrrhizin
83-555	10.6	1.07	< 0.13 *
83-555-2	6.5	0.79	1.56
83-555-3	10.3	0.30	0.37
83-555-5	9.6	1.04	< 0.16 *
83-555-7	7.3	0.88	< 0.13 *

* Mixture with an unidentified major saponin

Figure 3 shows the fruits of strain 83-555 and its offspring. Although the 3 offspring (i.e., 83-555-3, 83-555-5, and 83-555-7) had falcate fruits identical to those of strain 83-555, offspring 83-555-2 had a slightly falcate fruit, which was identical to that of the intermediate-type plants between G. uralensis and G. glabra seen in Kazakhstan.¹¹⁾ Figure 4 shows the shapes of the leaves from strain 83-555 and its offspring. The shapes of the leaves of the 3 offspring, 83-555-3, 83-555-5, and 83-555-7, were identical to those of the leaves of strain 83-555, whereas those of the offspring 83-555-2 were different from those of the mother plant. The shapes of the leaves of 83-555-2 were similar to those of the intermediate-type plants observed in Kazakhstan,¹¹⁾ These morphological data suggest that 83-555-2 is a hybrid of strain 83-555 with a glycyrrhizin-producing G. glabra plant.

Fig. 3. Comparison of Fruits of the Strain 83-555 and Its Offspring

Fig. 4. Comparison of Leaves of the Strain 83-555 and Its Offspring

To characterize the relationships of these strains, we determined the nucleotide sequence of a nuclear gene for polyketide reductase (pkr).⁵⁾ The partial sequence of the pkr gene of Glycyrrhiza plants has 2 SSRs and several SNPs, which are very useful in determining the relationships among Glycyrrhiza plants. Table 3 shows the genotypes of the partial pkr sequences of the 83-555 strain and its offspring. The pkr genotype of the 83-555 strain is a heterozygote of 2 pkr haplotypes, 18-3-GAAAGCT and 7-3-GGGAGCT, and the genotypes of its offspring 83-555-5 and 83-555-7 are homozygotes of one of their mother plant’s haplotypes, suggesting that these plants are self-pollinated offspring of 83-555. In contrast, the offspring 83-555-2 and 83-555-3 have a haplotype that does not exist in their mother plant. Offspring 83-555-2 may be an interspecific hybrid between the 83-555 strain (G. uralensis) and a GL-producing G. glabra with the haplotype 4-5-GGGAGCT, and offspring 83-555-3 may be an intraspecific offspring between the 83-555 and a G. uralensis strain with the haplotype 8-3-GGGAGCT. As shown in the present study, the pkr gene is a good marker for elucidating the relationship between strain 83-555 and its offspring. More extensive analyses of the pkr gene of various Glycyrrhiza plants will be reported elsewhere.

Table 3. The Pkr Genotype of Glycyrrhiza uralensis Strain 83-555 and Its Offspring

Strain No.	Voucher No.	Number of SSRs		SNPs							Genotype
		(AAC)n	(GCT)n	147	159	180	201	240	315	318
83-555	TUS421027	7/18	3	G	R	R	A	G	C	T	7-3-GGGAGCT/18-3-GAAAGCT
83-555-2	TUS421028	7/4	3/5	R	G	R	W	R	Y	Y	7-3-GGGAGCT/4-5-AGATATC
83-555-3	TUS421029	7/8	3	G	G	G	A	G	C	T	7-3-GGGAGCT/8-3-GGGAGCT
83-555-5	TUS421030	18	3	G	A	A	A	G	C	T	18-3-GAAAGCT
83-555-7	TUS421031	7	3	G	G	G	A	G	C	T	7-3-GGGAGCT

R=A and G, W=A and T, Y=C and T. Numerals above sequence are nucleotide positions of haplotype 7-3-GGGAGCT (AB823670).

GGL, the main saponin of the 83-555 strain, has a long-lasting sweetness like GL. GL is used extensively as a sweetener as well as a medicine because of its anti-inflammatory, hepatoprotective, and anti-viral activities.^1,2,12) The pharmacological activities of GGL, including the quality of sweetness, might be different from those of GL. Further experimentation is necessary to confirm this possibility.

It is noteworthy that the content of GGL in the stolons of strain 83-555 was lower than that of GL in the strain 01A26-6. This difference might be due to a mutation in the glycosidation step of GL biosynthesis. Not only GL but also various flavonoids have been isolated from licorice, and these flavonoids have several pharmacological activities, including antioxidative, antimicrobial and anticancer activities.^1,2,12) Licorice is regarded as a health-promoting food that may reduce the risk of cardiovascular disease and cancer,¹³⁾ however, overuse of licorice results in pseudoaldosteronism, hypokalemia, and hypertension, which are the adverse effects related to GL.¹²⁾ Thus, a G. uralensis strain that produces various flavonoids but a low level of saponin might provide promising material for health-promoting foods.

Although offspring 83-555-5 and 83-555-7, which seem to be self-pollinated, did not grow well, offspring 83-555-3 grew vigorously, probably due to heterosis. Thus, offspring 83-555-3 is a candidate for further breeding of G. uralensis. Further experiments are underway to select high-GGL-producing as well as low-GGL-producing strains from the offspring of 83-555-3.

Acknowledgment

This work was supported in part by the Takeda Science Foundation and a Grant-in-Aid for Scientific Research (No. 19590121) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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