Field Survey of Glycyrrhiza Plants in Central Asia (4). Characterization of G. glabra and G. bucharica Collected in Tajikistan

Abstract

The characteristics of 2 Glycyrrhiza plants, G. glabra and G. bucharica (=Meristotropis bucharica), were investigated in Tajikistan. The glycyrrhizin content in the underground parts of G. glabra varied from 2.56 to 9.29% of the dry weight, and the content of glabridin, a species-specific flavonoid of G. glabra, varied from 0.09 to 0.92% of the dry weight. Seeds of G. glabra plants from Tajikistan were cultivated for 3 years in Japan, and the glycyrrhizin content of the harvested roots ranged from 0.75 to 1.82% of the dry weight. In addition, HPLC analysis of leaf extracts indicated that the G. glabra plants collected in Tajikistan could be divided into various types, according to the flavonoid contents of the leaves. The endemic G. bucharica was also collected. A phylogenetic tree of rbcL nucleotide sequences from various Glycyrrhiza plants indicated that G. bucharica was closely related to the three glycyrrhizin-producing Glycyrrhiza spp. (G. uralensis, G. inflata, and G. glabra), even though G. bucharica does not produce glycyrrhizin.

Licorice, the roots and stolons of Glycyrrhiza glabra L., G. uralensis FISCH., and G. inflata BATAL. (Leguminosae), is one of the most important crude drugs in the world. Its main constituent, glycyrrhizin, is used both as a natural sweetener and as a pharmaceutical agent, owing to its anti-inflammatory and hepatoprotective properties.^1,2) Licorice extracts are also used as cosmetics, food additives, tobacco flavorings, and confectionery foods.³⁾ Extensive chemical studies revealed that underground parts of Glycyrrhiza plants contain not only glycyrrhizin, a triterpene saponin, but also a variety of flavonoids,⁴⁾ including species-specific flavonoids, such as glabridin, glycycoumarin, and licochalcone A, which are specific to G. glabra, G. uralensis, and G. inflata, respectively.^4–6) Notably, glabridin-containing licorice extracts are used in cosmetic preparations for their skin-whitening properties⁷⁾ and as an ingredient in functional foods for reducing body fat in Japan.⁸⁾ In addition, flavonoid variation in the leaves of Glycyrrhiza plants has also been reported,^5,9–11) and variations in the flavonoid contents of leaves are useful for identifying unique species and strains. Glycyrrhiza plants could be divided into two types, according to the major flavonol glycoside, isoquercitrin (IQ-type) or rutin (RT-type).⁹⁾ However, the relationship between their geographical distribution and chemical constituents has not been fully elucidated.

Tajikistan is a landlocked country in Central Asia, and six Glycyrrhiza species (G. glabra, G. uralensis, G. aspera, G. kulabensis, G. gontscharovii, G. bucharica) are distributed in Tajikistan.¹²⁾ Notably, a licorice-extracting factory of Avalin, a Tajik–Japanese joint company, has been operating since 2011 in Kubodiyon, Tajikistan, which is near the border with Afghanistan. Although Tajikistan is a globally important production center of licorice, the characteristics of Glycyrrhiza plants in Tajikistan were not elucidated in detail. Thus, in the present study, field surveys were performed to characterize Glycyrrhiza plants in Tajikistan.

MATERIALS AND METHODS

Plant Materials

Field surveys of Glycyrrhiza plants were conducted in the southern part of Tajikistan (Fig. 1). During our field survey, we collected and identified two Glycyrrhiza plants: G. glabra L. (Fig. 2A) and G. bucharica Rgl. (Fig. 2B). These two Glycyrrhiza plants were identified based on phenotypic change of fruits and leaves by Hayashi.¹²⁾ Roots and stolons of G. glabra were collected from collection sites 1, 3, and 6, and they were also obtained from the heap of licorice at the extraction factory in Kubodiyon. Roots and stolons of G. bucharica were collected from collection sites 7 and 8. The leaves, seeds and plant specimens of Glycyrrhiza plants were also collected from several sites in Tajikistan. All plant specimens collected in the present study were deposited into the Herbarium of the Institute of Botany, Plant Physiology and Genetics, Academy of Science of Tajikistan. Seeds of G. glabra collected in Tajikistan were germinated after treatment with conc. H₂SO₄ for 30 min. The germinated seeds were planted in pots containing vermiculite, fertilized with liquid nutrients, and grown indoors under artificial light. After 6 months (August 2012), the plants were transferred to Wagner pots (1/2000a) that contained a mixture of sand and composted bark (80 : 20, v/v) and were grown outdoors at the Herbal Garden of Iwate Medical University. Roots were harvested for HPLC analysis in October 2015, and leaves were collected for HPLC analysis in June 2016.

Fig. 1. Collection Sites of Glycyrrhiza Plants in Tajikistan

1: Kubodiyon (37°09′11″N/68°09′01″E); 2: Muminabad (38°14′15″N/70°05′35″E); 3: Kubodiyon (36°57′03″N/68°02′23″E); 4: Kulyab (37°52′49″N/69°57′58″E); 5: Dashtidzhum (37°57′50″N/70°12′04″E); 6: Muminabad (38°18′43″N/70°08′38″E); 7: Dangara (38°19′38″N/69°25′31″E); 8: Khuroson (38°11′17″N/68°39′30″E). Solid lines in the map indicate the route of field surveys in Tajikistan.

Fig. 2. Glycyrrhiza glabra (A) and G. bucharica (B) in Tajikistan

Chemicals

The glycyrrhizin standard for HPLC analysis was purchased from Wako Pure Chemical Industries, Ltd., Japan; authentic glabridin was obtained from Maruzen Pharmaceuticals, Japan; and rutin and isoquercitrin were purchased from Extrasynthese, France. Meanwhile, pinocembrin and glabranin were isolated from leaves of G. glabra, as described previously.^10,11)

HPLC Analysis of Glycyrrhizin and Glabridin in Underground Parts

HPLC analysis of field grown and cultivated roots and stolons was performed to determine the glycyrrhizin and glabridin contents, which are of economic importance. Dried roots and stolons were powdered using a mortar and pestle, and 50 mg of each powdered sample was extracted using 5 mL 80% methanol at 60°C for 2 h. Subsequently, 2-µL aliquots of each extract were 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] linear gradient of 15% MeCN to 100% MeCN in 50 min; flow rate: 0.2 mL/min; column temp.: 40°C; detector: photodiode array detector). The quantities of glycyrrhizin and glabridin were determined on the basis of their peak areas of UV absorption at 254 and 280 nm, respectively. The identity of each constituent was verified by comparing its retention time and UV spectrum to that of its respective authentic sample.

HPLC Analysis of Flavonoids in Leaves

HPLC analysis was performed to examine the chemical compositions of the leaves of G. glabra collected in Tajikistan. Fifty milligrams of powdered dry leaves of field grown and cultivated plants was extracted using 5 mL 80% methanol at 60°C for 2 h. Subsequently, 2-µL aliquots of each extract were analyzed by HPLC (column: Inertsil ODS-SP [3 µm, 2.1 mm i.d.×250 mm, GL Sciences, Japan]; solvent: MeCN/H₂O [0.1% formic acid] linear gradient of 15% MeCN to 100% MeCN in 40 min; flow rate: 0.2 mL/min; column temp.: 40°C; detector: photodiode array detector). The quantities of pinocembrin and glabranin, as well as rutin and isoquercitrin, were determined on the basis of their peak areas of UV absorption at 292 and 350 nm, respectively. The identity of each constituent was verified by comparing its retention time and UV spectrum to that of its respective authentic sample.

Amplification and Sequencing of the rbcL Gene

To elucidate the phylogenetic relationship of G. bucharica to other Glycyrrhiza plants, the 1374-bp DNA fragment covering most of the chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene (rbcL) was amplified by PCR using the total DNA isolated from Glycyrrhiza plants collected in Tajikistan. DNA was extracted from dry leaves using a DNeasy Plant Mini Kit (Qiagen, Germany), and a fragment of rbcL was amplified from the DNA template by PCR using Taq DNA polymerase (New England Biolabs, U.S.A.), anti-Taq high (Toyobo, Japan), and rbcL-specific primers (5′-ATG TCA CCA CAA ACA GAA ACT AAA GC-3′ and 5′-AGC AGC AGC TAA TTC AGG ACT CCA-3′), as previously reported.⁵⁾ The amplified fragments were treated with ExoSAP-It (Affymetrix/USB, U.S.A.) to remove primers, and the purified fragments were directly sequenced using the dideoxy chain termination method (3130xl Genetic Analyzer, Applied Biosystems, U.S.A.), with the two primers used for PCR amplification and two additional primers (5′-TTT ATG CGT TGG AGA GAC CG-3′ and 5′-AAG TAG ACC ATT ATC TCG GC-3′). Finally, a phylogenetic tree was constructed using Genetyx-Mac software (Genetyx Co., Japan).

RESULTS AND DISCUSSION

Field Surveys of Glycyrrhiza Plants in Tajikistan

Although habitats of G. glabra, which is an economically important Glycyrrhiza plant, were observed in many places in Tajikistan, large G. glabra habitats were observed in 2 districts, Kubodiyon and Muminabad (Fig. 1). Both G. glabra with eglandular fruits (var. glabra; 10A02, 12A04) and G. glabra with echinulate fruits (var. glandulifera; 10A01, 10A03, 12A02, 12A03) grew together at collection site 1 in Kubodiyon (Fig. 1), as previously observed in Turkey,¹³⁾ Kazakhstan,¹⁴⁾ and Uzbekistan,⁹⁾ whereas only G. glabra with eglandular fruits (var. glabra) grew at collection site 2 in Muminabad (Fig. 1). Kubodiyon (Collection sites 1 and 3; Fig. 1) is the location of an extracting factory, owned by the joint Tajik-Japanese company Avalin. This habitat is located on the riverside plain of the Amu Darya. The climate of this region is very hot and dry, and water for the growth of G. glabra plants seems to be supplied by rivers. The other big habitat was Muminabad, where many G. glabra plants were observed on top of a hill (Collection sites 2, 6; Fig. 1).

Meanwhile, the endemic G. bucharica was collected on foothills in the southern part of Tajikistan (Collection sites 4, 5, 7, and 8; Fig. 1). G. bucharica is alternatively classified as Meristotropis bucharica KRUG.¹⁵⁾ Furthermore, an intermediate (12A28) of G. glabra and G. bucharica was also collected at collection site 5 (Fig. 3), along with G. glabra (12A29) and G. bucharica (12A30). However, since only the aerial part of the intermediate plant was collected and the plant was not in fruit, its morphological identification to species was not successful.

Fig. 3. Leaves from G. glabra (12A29), an Intermediate Plant (12A28), and G. bucharica (12A30) from Collection Site 5

Glycyrrhizin and Glabridin Contents of Underground Parts from Collected G. glabra

The glycyrrhizin content of the underground parts from G. glabra varied from 2.56 to 9.29% of the dry weight (Table 1), which was higher than the lower limit of Japanese Pharmacopoeia (JP17, 2% dry weight).¹⁶⁾ The maximum content (9.29% dry weight) was higher than the glycyrrhizin contents of Glycyrrhiza plants collected in Turkey (1.1–8.0%),¹³⁾ Spain (0.7–4.4%),¹⁷⁾ Uzbekistan (3.33–6.13%),⁹⁾ and Kazakhstan (2.14–5.72%).¹⁴⁾

Table 1. Glycyrrhizin and Glabridin Contents of Roots and Stolons from Glycyrrhiza glabra Collected in Tajikistan

Date	Collection site	Plant number	Root/Stolon	Diameter (mm)	Contents (% of dry weight) of
					Glycyrrhizin	Glabridin
March 8, 2012	③Kubodiyon	—	Stolon	6.1–8.1	4.77	0.51
		—	Stolon	8.7–11.7	4.89	0.53
		—	Stolon	8.2–10.9	5.11	0.57
		Mean±S.D.			4.92±0.17	0.54±0.03
June 1, 2012	①Kubodiyon	12A02	Stolon	6.0–8.1	4.92	0.50
		12A02	Stolon	9.4–11.0	2.56	0.92
		12A02	Stolon	8.1–9.1	4.90	0.58
		Mean±S.D.			3.74±1.67	0.71±0.30
		12A03	Stolon	9.9–11.0	6.37	0.33
		12A03	Stolon	10.1–10.8	4.21	0.22
		12A03	Stolon	13.8–15.7	3.11	0.25
		Mean±S.D.			4.56±1.66	0.27±0.06
October 8, 2012	⑥Muminabad	12A40	Stolon	19.2–21.5	4.81	0.53
		12A40	Stolon	10.6–12.1	6.25	0.10
		12A40	Stolon	20.9–22.6	7.50	0.09
		Mean±S.D.			5.53±1.02	0.32±0.30
March 20, 2012	Avalin (Kubodiyon)	—	Root	19.7–22.1	7.68	0.37
(Collected from the licorice heap)		—	Stolon	11.0–14.5	4.10	0.62
		—	Root	13.5–15.8	9.29	0.09
		Mean±S.D.			7.02±2.66	0.36±0.27

Meanwhile, the content of glabridin, a species-specific flavonoid for G. glabra, varied from 0.09 to 0.92% of the dry weight, and the maximum content (0.92% dry weight) was also higher than the glabridin contents of G. glabra collected in Turkey (0.15–0.70%),¹³⁾ Spain (0.07–0.80%),¹⁷⁾ Uzbekistan (0.03–0.35%),⁹⁾ and Kazakhstan (0.28–0.34%).¹⁴⁾

Glycyrrhizin and Glabridin Contents of Underground Parts from Cultivated G. glabra

Since the glycyrrhizin and glabridin contents were very high in the underground parts of G. glabra collected in Tajikistan, we also investigated the contents of roots from plants grown from seeds of G. glabra from Tajikistan (Table 2). The glycyrrhizin content of the harvested roots ranged from 0.75 to 1.82% of the dry weight, which is lower than the lower limit of Japanese Pharmacopoeia (2% dry weight).¹⁶⁾ Since these values were similar to those observed for plants cultivated from G. glabra seeds from Turkey,¹⁸⁾ it seems that environmental conditions in Japan are not suitable for the accumulation of glycyrrhizin in the underground parts of G. glabra. However, in contrast to the glycyrrhizin content, the glabridin content of the cultivated roots ranged from 0.13 to 0.43% of the dry weight, which was almost the same as the glabridin content of plants collected in Tajikistan and elsewhere.^9,13,14,17)

Table 2. Glycyrrhizin and Glabridin Contents of Roots from Glycyrrhiza glabra Cultivated in Iwate, Japan

Plant number	Collection site of seeds	Diameter (mm)	Contents (% of dry weight) of
			Glycyrrhizin	Glabridin
10A02-1	①Kubodiyon	10.1–12.0	1.45	0.17
10A02-3		11.7–12.6	0.90	0.13
10A03-1		10.3–10.9	1.82	0.18
10A03-2		10.1–11.1	1.39	0.43
10A03-4		9.1–10.7	0.75	0.30
Mean±S.D.			1.26±0.43	0.24±0.12
11A04-3	②Muminabad	13.2–14.5	1.77	0.16
11A04-4		7.3–8.3	0.80	0.19
11A05-1		9.9–11.5	1.57	0.27
11A05-2		7.5–8.8	1.49	0.36
11A05-3		8.9–9.4	1.08	0.28
Mean±S.D.			1.34±0.39	0.25±0.08

Flavonoid Contents of Leaves from Collected G. glabra

HPLC analysis indicated that G. glabra plants from Tajikistan could be divided into various types according to their HPLC profiles (Table 3). At collection site 1 in Kubodiyon, G. glabra plants were divided into two types according to the major flavonol glycoside, isoquercitrin (IQ-type) or rutin (RT-type), as previously reported for a collection site at Yangiyer, Uzbekistan.⁹⁾ The offspring of 10A02 (RT-type), which were cultivated in Japan, were IQ-type and RT-type, whereas the offspring of 10A03 (IQ-type) were only IQ-type.

Table 3. Contents of Four Flavonoids in the Leaves of Glycyrrhiza glabra

Collection date	Collection site	Plant number	Type of HPLC profile	Contents (% of dry weight) of
				Rutin	Isoquercitrin	Pinocembrin	Glabranin
August 25, 2010	①Kubodiyon	10A01	IQ-type	0.14±0.10	0.30±0.08	1.80±0.82	0.21±0.08
		10A02	RT-type	0.47±0.10	0.08±0.01	1.69±0.31	0.43±0.18
		10A03	IQ-type	0.10±0.05	0.17±0.07	0.86±0.25	0.46±0.29
June 1, 2012	①Kubodiyon	12A02	IQ-type	0.30±0.02	0.54±0.09	1.62±0.36	0.39±0.09
		12A03	IQ-type	0.23±0.06	0.43±0.13	1.07±0.58	0.25±0.13
		12A04	IQ-type	0.17±0.04	0.43±0.03	2.52±0.44	0.40±0.08
October 12, 2011	②Muminabad	11A03	RT/IQ-type	1.46±0.03	0.77±0.05	0.45±0.09	0.07±0.01
		11A04	RT/IQ-type	1.14±0.04	0.63±0.05	0.53±0.09	0.07±0.01
		11A05	RT/IQ-type	1.22±0.09	0.67±0.02	0.49±0.30	0.06±0.03
October 8, 2012	②Muminabad	12A36	RT/IQ-type	1.08±0.02	0.51±0.09	0.31±0.03	0.06±0.00
		12A37	RT/IQ-type	0.88±0.09	0.66±0.10	0.53±0.22	0.19±0.06
		12A38	RT/IQ-type	1.14±0.16	0.58±0.06	0.34±0.06	0.07±0.02
October 8, 2012	⑥Muminabad	12A40	RT-type	1.24±0.08	0.17±0.03	0.69±0.03	0.26±0.04
June 12, 2016	Cultivated at Iwate, Japan	10A02-1	IQ-type	0.43±0.04	0.78±0.06	0.79±0.53	0.39±0.11
(Collection site of seeds: ①Kubodiyon)		10A02-3	RT-type	1.18±0.05	0.21±0.03	0.83±0.73	0.62±0.30
		10A03-1	IQ-type	0.37±0.01	0.49±0.05	0.99±0.57	1.00±0.37
		10A03-2	IQ-type	0.35±0.07	0.85±0.10	0.58±0.38	0.70±0.26
		10A03-4	IQ-type	0.26±0.03	0.57±0.04	1.15±0.79	1.20±0.45
June 12, 2016	Cultivated at Iwate, Japan	11A04-3	IQ/RT-type	0.54±0.03	0.68±0.08	0.68±0.08	0.54±0.06
(Collection site of seeds: ②Muminabad)		11A04-4	IQ/RT-type	0.74±0.04	0.92±0.08	0.19±0.15	0.79±0.48
		11A05-1	RT/IQ-type	0.94±0.11	0.81±0.04	0.22±0.04	0.51±0.05
		11A05-2	RT/IQ-type	0.94±0.06	0.70±0.12	0.13±0.08	0.38±0.13
		11A05-3	RT/IQ-type	1.22±0.07	0.76±0.07	0.10±0.09	0.14±0.04

Values indicate means±S.D. (n=3).

In contrast to collection site 1 in Kubodiyon, all six G. glabra plants at collection site 2 in Muminabad were RT/IQ-type, in which the contents of both isoquercitrin and rutin were greater than 0.5% of the dry weight, and the rutin content (ca. 1%) was higher than the isoquercitrin content. Notably, the rutin content of the RT/IQ-type was much higher than that of the RT-types from collection site 1 in Kubodiyon, Kazakhstan,¹⁷⁾ and Uzbekistan.⁹⁾ Interestingly, the offspring of 11A04 (RT/IQ-type) were IQ/RT-type, in which contents of both isoquercitrin and rutin were higher than 0.5% of the dry weight and the isoquercitrin content (ca. 1%) was higher than the rutin content, whereas the offspring of 11A05 (RT/IQ-type) were RT/IQ-type. Furthermore, the glabranin content of leaves from G. glabra plants cultivated in Japan was higher than that observed in Tajikistan.

In the present study, the flavonoid profiles of some offspring of 10A03 and 11A04 were different from those of the mother plants. These results suggested that both genetic background and environmental conditions (heat or humidity) might affect the flavonoid variations of the leaves of G. glabra. Therefore, further experiments are necessary to fully understand the variation of flavonoids in G. glabra.

Phylogenetic Relationship of Glycyrrhiza Plants Collected in Tajikistan Based on rbcL Sequences

Four G. bucharica plants analyzed in the present study yielded identical rbcL sequences (G-A-A type; Table 4), and rbcL sequences of three G. glabra plants were identical to those reported for G. glabra previously (A-T-G type; Table 4).⁵⁾ Three nucleotide substitutions were observed between the sequences of G. bucharica and G. glabra, and only one substitution was observed between G. bucharica and the two other glycyrrhizin-producing Glycyrrhiza species: G. uralensis and G. inflata (G-A-G type). A phylogenetic tree (Fig. 4) constructed from the rbcL sequences also indicated that G. bucharica is closely related to G. uralensis and G. inflata.

Table 4. Origin and the rbcL Genotypes of Glycyrrhiza glabra and G. bucharica Specimens Collected in Tajikistan

Plant number	Collection date	Collection site	Species	rbcL Genotype
10A01	Aug. 25, 2010	①Kubodiyon	G. glabra	A-T-G Type
11A03	Oct. 12, 2011	②Muminabad	G. glabra	A-T-G Type
12A24	Oct. 7, 2012	④Kulyab	G. bucharica	G-A-A Type
12A28	Oct. 7, 2012	⑤Dashtidzhum	G. sp.	G-A-A Type
12A29	Oct. 7, 2012	⑤Dashtidzhum	G. glabra	A-T-G Type
12A30	Oct. 7, 2012	⑤Dashtidzhum	G. bucharica	G-A-A Type
13A05	Jul. 20, 2013	⑦Dangara	G. bucharica	G-A-A Type
13A24	Oct. 3, 2013	⑧Khuroson	G. bucharica	G-A-A Type

Fig. 4. Phylogenetic Tree of rbcL Sequences from G. bucharica and Seven Other Glycyrrhiza Species

The phylogenetic tree was constructed using the UPGMA Method¹⁹⁾ with Genetyx-Mac software (Genetyx Corporation, Japan). Accession numbers are shown on the right.

In addition, the rbcL sequence of the intermediate plant (12A28) was identical to that of G. bucharica (G-A-A type). Interestingly, G. bucharica is known to hybridize with G. glabra, and another endemic species of Tajikistan, G. gontscharovii T. MASL., is suggested to be a hybrid between G. glabra and G. bucharica.¹²⁾ Therefore, we suggest that the intermediate plant is a hybrid, like G. gontscharovii, although further morphological identification is necessary to confirm its identity.

Since G. bucharica was shown to be closely related to glycyrrhizin-producing Glycyrrhiza plants, HPLC analysis was performed to examine the chemical composition of the underground parts of G. bucharica collected in Tajikistan. Neither glycyrrhizin nor macedonoside C, the major triterpene saponin of three non-glycyrrhizin-producing species (G. macedonica, G. echinata, and G. pallidiflora),⁵⁾ were detected in the underground parts of G. bucharica. Further experiments are underway to isolate the chemical components of G. bucharica.

A rbcL nucleotide sequence from G. bucharica has been deposited in DDBJ, EMBL, and GenBank under the accession number LC128586.

Acknowledgments

This research was supported by the Japan International Cooperation Agency (JICA) and the Japan Society for the Promotion of Science (JSPS) Program, Dispatch of Science and Technology Researchers. This work was also supported by the Takeda Science Foundation and JSPS KAKENHI (Grant Numbers JP19590121, JP15K07999). We are grateful to Mr. Manuchehr Gadoev, the CEO of Avalin, for his assistance in visiting the extracting plants in Kubodiyon and to Prof. Hikmat Hisoriev for depositing the plant specimens in the herbarium, as well as to Dr. Musavvara Shukurova, Ms. Zumrad Sharopova, Mr. Shoh Sharipov, Mr. Husniddin Kuziboev, Ms. Shakhnoza Negmatova, Mr. Mirzokarim Sobirov, Mr. Yukichi Goto, and Mr. Masakazu Kanamoto for their assistance in the field surveys. We would also like to thank Editage (www.editage.com) for English language editing.

Conflict of Interest

The authors declare no conflict of interest.

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