The constituents of Urtica cannabina used in Uighur medicine

Abstract

Urtica cannabina L. (Urticaceae) is a perennial herb that grows in Xinjiang Uighur Autonomous Region (northwest China). Two megastigmanes (1, 2) and five flavonoid glycosides (3-7) were isolated from its fruit. Compound 1 was determined by spectroscopic analysis to be (+)-blumenol A, and its absolute stereochemistry was determined in detail using chemical conversion and a modification of Mosher’s method. Other compounds were identified as (+)-dehydrovomifoliol (2), isovitexin (3), isoquercitrin (4), astragalin (5), afzelin (6), and quercitrin (7) using spectroscopic (NMR, HMBC, MS) and physical methods (melting point and optical rotation). Compounds 1-7 were isolated from this plant for the first time, while this is the first report of megastigmanes in the Urticaceae family. The chemotaxonomic significance of the isolation of these megastigmanes and flavonoid glycosides from Urtica species is discussed.

Keywords::

• Flavonoid glycoside
• megastigmane (+)-blumenol A
• Urtica cannabina

Introduction

“The genus Urtica (Urticaceae) contains about 50 taxa distributed from temperate to subtropical regions, e. g. Europe, North Africa, Northwest China, Japan and North America. In China, there are 23 taxa (16 species, 6 subspecies, and 1 variety). In China there are 23 taxa (16 species, 6 subspecies, and 1 variety), including seven species that are native to the Xinjiang Uighur Autonomous Region (northwest China) (Wang et al., 2002) (Urtica cannabina L., U. angustfolia Fisch., U. laetevirens Maxi., U. dioica L., U. kunlunshanica Y. Yang., U. thunbergiana Sieb. et Zucc., and U. urens L.).

The leaves of Urtica are nutritious and rich in micronutrients; however, they need to be steamed or otherwise cooked before ingestion to destroy their stinging hairs, which contain histamine, formic acid, acetylcholine, acetic acid, butyric acid, leukotrienes, 5-hydroxytryptamine, and other irritants (Wagner et al., 1994; Emmelin & Feldberg, 1949). Contact with the hairs leads to a mildly painful sting, development of an erythematous macule, and itching or numbness for a period lasting from minutes to days.

Plants of the genus Urtica are commonly used in the traditional medicine of Turkey (Baser et al., 1986), various countries in Europe (Cai, 2001), and Japan (Namba, 1980) for the treatment of rheumatism, internal disease, skin conditions such as rashes, bleeding due to wounds, and benign prostatic hyperplasia (BPH) (Iida et al., 1994).

The hydrophilic components of Urtica, including polysaccharides and lectins, appear to be important medicine, and particularly inhibit prostatic hyperplasia (Lichius et al., 1999). The importance of Urtica root lignans, such as (-)-3,4-divanillyltetrahydrofuran, to improve benign prostatic hyperplasia (BPH) and other androgen- and estrogen-sensitive conditions, may be due to interference with the binding of sex hormone binding globulin (SHBG) to testosterone, the testosterone receptor, and/or the SHBG receptor (Schottner et al., 1997; Hryb et al., 1995). The steroidal compounds stigmasterol, stigmast-4-en-3-one, and campesterol inhibit the prostatic sodium/potassium pump, which may contribute to the effects of Urtica on BPH (Hirano et al., 1994). However, the small quantity of β-sitosterol contained in Urtica root (<0.01% of the total mass) is unlikely to have an effect on BPH, given that 60 mg β-sitosterol daily is the usual amount necessary to reduce symptoms (Berges et al., 1995). U. dioica agglutinin (UDA) is a heat- and acid-resistant lectin found in stinging Urtica, primarily in the root (Galelli & Truffa, 1993). UDA activates the cell-mediated immunity, not observed for any other known plant lectin (Galelli & Truffa, 1993). UDA appears to prevent formation of a systemic lupus erythematosus-like condition in mice, and has diverse antiviral effects in vitro (Musette et al., 1996; Balzarini et al., 1992). It also antagonizes the epidermal growth factor receptor that may inhibit the formation of BPH (Wagner et al., 1995).

In Xinjiang Uighur Autonomous Region (northwest China), U. cannabina aerial portion and fruit are medicinal plants used in Uighur traditional medicine (Liu et al., 1999; Yasen et al., 1999). The fruit as a component of a preparation has been used for chronic bronchitis (Yasen et al., 1999). However, there have been few studies of the constituent chemicals of the fruit. Zhang et al. (2005) isolated scutellarein-7-O-α-l-rhamnoside and vicenin-2 from the leaves, while Ma et al. (2004) isolated β-sitosterol, scopoletin, caprylic alcohol, daucosterol, p-coumaric acid methyl ester, and p-coumaric acid from the whole herb. In the present study, we attempted to identify useful components of the fruit of U. cannabina to support its development as a Uighur traditional medicinal resource.

Herein, we report the isolation of the megastigmanes 1 and 2 from a methanol (MeOH) extract of U. cannabina fruit. To the best of our knowledge, this is the first report on the Urticaceae family. Compounds 3-7 were also isolated from this plant for the first time. In addition, the chemotaxonomic significance of these compounds was evaluated and compared among Urtica species.

Materials and methods

General experimental procedures

Melting points (mp) were determined using a Yanaco micro melting point apparatus, and were uncorrected. UV spectra were obtained using a Shimadzu UV 1600 spectrophotometer and optical rotation data were obtained using a Horiba SEPA-300 digital polarimeter. Electron Ionization Mass Spectrometry (EI-MS) and Fast Atom Bombardment Mass Spectrometry (FAB-MS) data were obtained using a JEOL JMS-700 mass spectrometer. Circular dichroism (CD) spectra were obtained in MeOH using a JASCO J-820 spectrometer. ¹H- and ¹³C-NMR spectra were obtained using a JEOL JNM-AL 400 spectrometer with tetramethylsilane as an internal standard. TLC was performed using silica gel 60 F₂₅₄ TLC plates (Merck) and RP-18 F_254S TLC plates (Merck). Silica gel 60 N (particle size: 100-200 μm, Kanto Chemical) was used for silica gel open column chromatography. Separation was carried out using normal and reverse phase HPLC with an SPD-20A detector (Shimadzu) at 254 nm.

Plant materials

Dried fruits of U. cannabina were purchased from Xinjiang Hospital of Uighur Medicine (Urumqi, Xinjiang, China), for use as an herbal medicine in May 2006. A voucher specimen was identified by Kuang Hai Xue (Heilongjiang University of Traditional Chinese Medicine) and deposited at the Department of Natural Medicine and Phytochemistry, Meiji Pharmaceutical University (Tokyo) (Accession No. 06UC001).

Extraction and isolation

Dried fruits of U. cannabina (8.5 kg) were extracted with 13 L of MeOH under reflux for 4 h (3 times). The MeOH extract was concentrated under reduced pressure to yield a sample of 416.6 g, of which 410 g was suspended in water and partitioned with ethyl acetate (AcOEt). The AcOEt-soluble portion (10 g) was subjected to column chromatography on silica gel and eluted stepwise using a series of n-hexane/AcOEt mixtures (20:1, 10:1, 5:1, 2:1, 1:1, 1:2, and 1:5), AcOEt, and MeOH to yield 10 fractions (A1-A10). Fraction A7 (1034.6 mg) was further separated by chromatography on a Sephadex LH-20 column (φ 40 × 1200 mm, GE Healthcare) using MeOH into 7 subfractions, of which subfraction A7-2 (147.4 mg) was subjected to preparative reverse phase HPLC (COSMOSIL 5C₁₈-ARII column of φ 10 × 250 mm, at 1 mL/min with UV detection at 254 nm) using MeOH/H₂O (5:4) to yield compound 1 (31.2 mg). Fraction A6 (1072.6 mg) was purified by normal phase HPLC (COSMOSIL 5SL-II column of φ 20 × 250 mm, at 2 mL/min, with UV detection at 254 nm) using n-hexane/AcOEt (1:2) to give 4 subfractions, of which subfraction A6-2 (32.3 mg) was purified by reverse phase HPLC on the COSMOSIL 5C₁₈-ARII column as above using MeOH/H₂O (2:3) to yield compound 2 (5.8 mg). Fraction A10 (2.6 g) was further separated by chromatography on the Sephadex LH-20 column (φ 40 × 1200 mm, GE Healthcare) using MeOH to yield 8 subfractions, of which subfraction A10-7 (100.5 mg) was purified by reverse phase HPLC on the COSMOSIL 5C₁₈-ARII column as above using MeOH/H₂O (5:4) to yield compounds 3 (2.9 mg) and 4 (7.7 mg). Fraction A9 (2.2 g) was further separated by chromatography on the Sephadex LH-20 column (φ 40 × 1200 mm, GE Healthcare) using MeOH to yield 9 corresponding fractions, of which subfraction A9-7 (38 mg) was purified by reverse phase HPLC on the COSMOSIL 5C₂₂-ARII column using MeOH/H₂O (5:4) to yield compounds 5 (6.3 mg) and 6 (5.0 mg). Subfraction A9-8 (9.4 mg) was purified by reverse phase HPLC on the COSMOSIL 5C₂₂-ARII column using MeOH/H₂O (5:4) to yield compound 7 (2.7 mg).

Compound 1

(+)-Blumenol A (1): white crystal, mp 108-109°C. [α]D²⁶ + 245.2° (c = 0.69, MeOH). UV λ_max (MeOH) nm (log ϵ) 237 (4.06). CD (c = 1.34 × 10⁻⁴ M, MeOH) Δϵ (nm): +14.89 (241.6), -0.85 (320.2). Positive-mode FAB-MS m/z 225 (M+H)⁺, 247 (M+Na)⁺. Negative-mode FAB-MS m/z 223 (M-H)⁻. NMR (Tables 1 and 2).

Table 1.  ¹H-NMR data of compounds 1 and 2.

Proton	1^a)	2^b)
2a	2.41 (1H, d, J =17.0 Hz)	2.28 (1H, d, J =17.0 Hz)
2b	2.20 (1H, d, J =17.0 Hz)	2.60 (1H, d, J =17.0 Hz)
4	5.87 (1H, s)	5.93 (1H, s)
7	5.75 (1H, d, J =15.6 Hz)	6.99 (1H, d, J =15.9 Hz)
8	5.82 (1H, dd, J =15.6, 5.1 Hz)	6.43 (1H, d, J =15.9 Hz)
9	4.37 (1H, m)
10	1.26 (3H, d, J =6.6 Hz)	2.30 (3H, s)
11	1.05 (3H, s)	1.02 (3H, s)
12	0.98 (3H, s)	1.06 (3H, s)
13	1.86 (3H, d, J =1.5 Hz)	1.90 (3H, d, J =1.5 Hz)

ppm from TMS, in CDCl_3^a), CD₃OD^b), 400 Hz, at room temperature.

Table 2.  ¹³C-NMR data of compounds 1 and 2.

Carbon	1^a)	2^b)
1	41.24	42.66
2	49.74	50.52
3	197.83	199.99
4	126.68	127.82
5	162.80	164.34
6	79.01	79.90
7	128.84	148.05
8	135.54	131.49
9	68.02	200.29
10	23.84	27.71
11	23.02	23.58
12	24.15	24.81
13	19.07	19.24

ppm from TMS, in CDCl_3^a), CD₃OD^b), 100 Hz, at room temperature.

Preparation of (R)- and (S)-MTPA esters 1a and 1b from compound 1

A working solution of (R)-MTPA [(R)-MTPA (Sigma-Aldrich) 51.5 mg and DCC (Nacalai) 36 mg in 0.5 mL of dry CH₂Cl₂] was prepared. Compound 1 (2.9 mg, 13 μmol) and DMAP (Wako) (16.4 mg) were dissolved in dry CH₂Cl₂ (0.5 mL) and stirred at room temperature for 30 min. The (R)-MTPA working solution was then added and the mixture was stirred at room temperature for 1 h. After the addition of 5 mL each of water and CH₂Cl₂, the solution was washed successively with 5% HCl (5 mL), saturated NaHCO₃ (5 mL) and brine (5 mL). The organic soluble part was dried with Na₂SO₄ and concentrated in vacuo to yield a residue (Kai et al., 2007). 1a (2.9 mg, 50.8%) was purified on a silica gel (2 mL) column using n-hexane/AcOEt. Using a similar procedure, 1b (2.3 mg, 40%) was prepared from compound 1 (2.9 mg, 13 μmol) with (S)-MTPA (49.7 mg), DCC (36 mg) and DMAP (17.3 mg) (Figure 1).

Figure 1.  Preparation of (R)- and (S)-MTPA esters 1a and 1b from compound 1.

9-O-(R)-MTPA ester (1a)

Colorless oil, [α]D²⁹ + 189.3° (c = 0.097, CHCl₃). UV λ_max (CHCl₃) nm (log ϵ) 239.5 (4.24). EI-MS m/z (rel. int. %) 440 (1) (M)⁺, 206 (26), 189 (77), 150 (100), ¹H-NMR (Table 3).

Table 3.  ¹H-NMR spectral data of compound 1 and derivatives 1a and 1b.

	1	1b	1a	Δ(δ1b-δ1a)
				ppm	Hz
2a	2.41 (1H, d, J =17.0 Hz)	2.31 (1H, d, J =17.0 Hz)	2.38 (1H, d, J =17.0 Hz)	−0.072	−28.8
2b	2.20 (1H, d, J =17.0 Hz)	2.21 (1H, d, J =17.0 Hz)	2.24 (1H, d, J =17.0 Hz)	−0.034	−13.6
4	5.87 (1H, s)	5.88 (1H, brs)	5.90 (1H, brs)	−0.019	−7.6
7	5.75 (1H, d, J =15.6 Hz)	5.75 (1H, d, J =15.4 Hz)	5.85 (1H, d, J =15.4 Hz)	−0.093	−37.2
8	5.82 (1H, dd, J =15.6, 5.1 Hz)	5.80 (1H, d, J =15.4 Hz)	5.90 (1H, d, J =15.4 Hz)	−0.097	−38.8
9	4.37 (1H, m)	5.64 (1H, m)	5.61 (1H, m)	+0.023	+9.2
10	1.26 (3H, d, J =6.6 Hz)	1.44 (3H, d, J =6.6 Hz)	1.39 (3H, d, J =6.6 Hz)	+0.045	+18.0
11	1.05 (3H, s)	1.04 (3H, s)	1.06 (3H, s)	−0.015	−6.0
12	0.98 (3H, s)	0.92 (3H, s)	0.94 (3H, s)	−0.019	−7.6
13	1.86 (3H, d, J =1.5 Hz)	1.82 (3H, d, J =1.5 Hz)	1.85 (3H, d, J =1.5 Hz)	−0.031	−12.4
Ph		7.50-7.34 (5H, m)	7.50-7.34 (5H, m)
OCH3		3.56 (3H, d, J =1.2 Hz)	3.51 (3H, d, J =1.2 Hz)	+0.052	+20.8

ppm from TMS, in CDCl₃, 400 Hz, at room temperature.

9-O-(S)-MTPA ester (1b)

Colorless oil, [α]D²⁸ + 159.1° (c = 0.067, CHCl₃). UV λ_max (CHCl₃) nm (log ϵ) 239.5 (4.32). EI-MS m/z (rel. int. %) 440 (1) (M)⁺, 206 (25), 189 (77), 150 (100), ¹H-NMR (Table 3).

Compound 2

(+)-Dehydrovomifoliol (2): yellow oil, [α]D²⁶ + 119.9° (c = 0.48, MeOH). UV λ_max (MeOH) nm (log ϵ) 239 (4.04). CD (c = 1.26 × 10⁻⁴ M, MeOH) Δϵ (nm) +20.44 (243). EI-MS m/z (rel. int. %) 222 (1) (M)⁺, 166 (32), 149 (10), 124 (100), NMR (Tables 1 and 2).

Compound 3

Isovitexin (3): yellow amorphous solid, mp 210°-215°C. [α]D²⁷ + 40.8° (c = 0.21, MeOH). UV λ_max (MeOH) nm (log ϵ) 207 (3.06), 271.0 (2.73), 333.5 (2.74). FAB-MS m/z (neg.) 431 (M-H)⁻. ¹H-NMR (400 MHz, DMSO-d₆) δ 6.77 (1H, s, H-3), 6.5 (1H, s, H-8), 7.93 (2H, d, J = 8.8, H-2′, 6′), 6.93 (2H, d, J = 8.8, H-3′, 5′), 4.59 (1H, d, J = 9.8, H-1″ of Glc), 13.55 (1H, brs, OH-5), ¹³C-NMR (Table 4).

Table 4.  ¹³C-NMR data of compounds 3-7.

Carbon	3^a)	4^b)	5^b)	6^a)	7^a)
2	163.50	158.73	158.93	156.84	156.85
3	102.51	135.40	135.33	133.90	134.00
4	181.42	179.13	179.23	177.27	177.30
5	160.29	162.73	162.79	160.91	161.03
6	108.71	99.77	99.82	98.57	98.80
7	163.04	165.73	165.67	164.01	164.69
8	93.52	94.60	94.69	93.57	93.64
9	155.91	158.17	158.26	156.16	156.27
10	102.98	105.53	105.67	103.85	103.70
1′	120.83	122.99	122.69	120.26	120.54
2′	128.14	117.39	132.05	130.30	115.32
3′	115.75	145.63	115.94	115.15	145.05
4′	160.86	149.57	161.30	159.63	148.39
5′	115.75	115.82	115.94	115.15	115.47
6′	128.14	122.88	132.05	130.30	120.93
1″	72.98	104.20	104.13	101.59	101.78
2″	70.10	75.65	75.70	70.20	70.51
3″	78.82	78.04	78.04	70.49	70.42
4″	70.50	71.16	71.42	70.99	71.27
5″	81.39	78.29	78.32	69.95	70.07
6″	61.38	62.53	62.71	17.48	17.60

ppm from TMS, in DMSO-d₆, CD₃OD^b), 100 Hz, at room temperature.

Compound 4

Isoquercitrin (4): yellow powder, mp 175°-177°C. [α]D²³ -5.2° (c = 0.10, MeOH). UV λ_max (MeOH) nm (log ϵ) 202.5 (4.59), 257 (4.29), 356.5 (4.16). FAB-MS m/z (neg.) 463 (M-H)⁻. ¹H-NMR (400 MHz, CD₃OD) δ 6.2 (1H, d, J = 2.2, H-6), 6.39 (1H, d, J = 2, H-8), 7.71 (1H, brs, H-2′), 6.87 (1H, d, J = 8.2, H-5′), 7.58 (1H, dd, J = 8.2, H-6′), 5.25 (1H, d, J = 7.6, H-1″ of Glc), ¹³C-NMR (Table 4).

Compound 5

Astragalin (5): yellow powder, mp 174°-178°C. [α]D²⁷ -11.3° (c = 0.32, MeOH). UV λ_max (MeOH) nm (log ϵ) 266 (4.26), 350 (4.22). FAB-MS m/z (neg.) 447 (M-H)⁻. ¹H-NMR (400 MHz, CD₃OD) δ 6.22 (1H, d, J = 2, H-6), 6.41 (1H, d, J = 2, H-8), 8.05 (2H, d, J = 8.9, H-2′, 6′), 6.89 (2H, d, J = 8.9, H-3′, 5′), 5.22 (1H, d, J = 7.6, H-1″ of Glc), ¹³C-NMR (Table 4).

Compound 6

Afzelin (6): yellow powder, mp 173°-175°C. [α]D²⁶ -104° (c = 0.1, MeOH). UV λ_max (MeOH) nm (log ϵ) 265 (3.98), 342 (3.81). FAB-MS m/z (neg.) 431 (M-H)⁻. ¹H-NMR (400 MHz, DMSO-d₆) δ 6.21 (1H, d, J = 2, H-6), 6.40 (1H, d, J = 2, H-8), 7.75 (2H, d, J = 8.8, H-2′, 6′), 6.91 (2H, d, J = 8.8, H-3′, 5′), 5.3 (1H, d, J = 1.5, H-1″ of Rha), 12.6 (1H, brs, OH-5), ¹³C-NMR (Table 4).

Compound 7

Quercitrin (7): yellow powder, mp 178°-180°C. [α]D²⁷ -96° (c = 0.1, MeOH). UV λ_max (MeOH) nm (log ϵ) 206 (4.64), 256.5 (4.19), 351.5 (4.06). FAB-MS m/z (neg.) 447 (M-H)⁻. ¹H-NMR (400 MHz, DMSO-d₆) δ 6.18 (1H, d, J = 2.2, H-6), 6.37 (1H, d, J = 2.2, H-8), 7.26 (1H, d, J = 2.2, H-2′), 6.85 (1H, d, J = 8.3, H-5′), 7.22 (1H, d, J = 8.3, 2.2, H-6′), 5.2 (1H, d, J = 1.2, H-1″ of Rha), 12.58 (1H, brs, OH-5), ¹³C-NMR (Table 4).

Results and discussion

Here we report the first isolation of megastigmanes 1 and 2 and flavonoid glycosides 3-7 (Figure 2) from U. cannabina as well as the first isolation of megastigmanes from plants of the Urticaceae family.

Figure 2.  Structures of compounds 1-7.

The absolute configuration of 1 at C-6 was determined by application of the circular dichroism (CD) helicity rule (Weiss et al., 1973) based on the known compound (+)-dehydrovomifoliol (2). The CD spectrum of 2 showed a positive Cotton effect (Δϵ +20.44) at 243 nm (Figure 3), so the 6-position was determined to have the S configuration. On the basis of this evidence, because the CD spectrum of 1 exhibited a positive Cotton effect (Δϵ +14.89) at 241.6 nm (Figure 3), we also concluded that the 6-position of 1 has the S configuration. In order to clarify the absolute stereostructure at the 9-position of compound 1, a modified Mosher’s method was used. Compound 1 was treated with either (R)- or (S)-α-methoxy-α-(trifluoromethyl)phenylacetic acid (MTPA), N,N-dicyclohexylcarbodiimide (DCC), and 4-(dimethylamino)pyridine in dry dichloromethane at room temperature to yield the 9-O-(R)-MTPA ester 1a (50.8%), and the 9-O-(S)-MTPA ester 1b (40.0%), respectively (Figure 1). As shown in Figure 4, the NMR signals due to protons attached at the 2a, 2b, 4, 7, 8, 11, 12, and 13-positions in 1a were observed at a lower field than in 1b (negative Δδ), while the signal of the protons at the 10-position in 1a were observed at a higher field than in 1b (positive Δδ). Consequently, the absolute configuration at the 9-position was elucidated to be R. On the basis of this evidence, the structure of 1 was determined to be (+)-blumenol A.

Figure 3.  CD curves of compounds 1 and 2.

Figure 4.  Results of modified Mosher’s method of 2a and 2b.

The other five compounds were identified as isovitexin (3), isoquercitrin (4), astragalin (5), afzelin (6), and quercitrin (7) by comparison of their spectroscopic data (NMR, UV, MS, melting points and optical rotation) with reported values.

(+)-Blumenol A (1) has been previously isolated from the leaves of Podocarpus blumei Endl. (Podocarpaceae) (Galbraith & Horn, 1972), while (+)-dehydrovomifoliol (2) has been isolated from the aerial parts of Beta vulgaris L. var. cicla L. (Chenopodiaceae) (Kim et al., 2004). However, this is the first report of the isolation of megastigmanes from plants of the Urticaceae family. Afzelin (6) and quercitrin (7) have been isolated from the fruits of Ocotea velloziana Meisn. (Lauraceae) (Garcez et al., 1995) and Juniperus communis L. (Cupressaceae) (Hiermann et al., 1996). Isovitexin (3) has been isolated from other Urticaceae species, including the leaves of Phenax angustifolius Wedd. (Piccinelli et al., 2005). Isoquercitrin (4) and astragalin (5) have been isolated from flowers of U. dioica (Wang et al., 2006), and roots of U. fissa (Chaurasia & Wichtl, 1987).

The presence of megastigmanes 1 and 2, afzelin (6) and quercitrin (7) has chemotaxonomic significance for Urticaceae family. Isovitexin (3) does not appear to have a significant chemotaxonomic presence among Urtica species, although it has been reported in Phenax, another member of the Urticaceae family. As isovitexin has not previously been reported in Urtica species, its presence in U. cannabina is also of chemotaxonomic significance.

Naturally occurring flavonoids have been isolated from more than 6000 species, and several have been reported to exhibit anti-allergic, anti-inflammatory, anticancer and antiviral activities. Megastigmanes have been reported to exhibit radical scavenging activity (Matsunami et al., 2006). In future studies, the compounds identified in this plant may be investigated for activity against chronic bronchitis, which this plant used for in Uighur traditional medicine.

Acknowledgement

Authors are grateful to Professor Dr. Uper Hamurat of Xinjiang Medical University in China, and Dr. Hisahiro KAI of Kyushu University of Health and Welfare in Japan. A part of this work was supported by Japan China Medical Association in 2007.

Declaration of interest

The authors report no conflict of interest. The authors are responsible for the content and writing of the paper.