1　Materials and methods

1.1　Animals

C57BL/6 mice (8-10 weeks) were used for behavioral testing (Experimental Animal Center, Nanjing University of Chinese Medicine, Nanjing, China). Mice were housed, and behavior experiments were performed in a controlled environment of 20-24℃, humidity of 50%-60% with a 12-h day/night cycle. GCaMP3 and neomycin resistance genes were inserted into the Pirt locus using targeted homologous recombination^[13]. Pirt-GCaMP3 heterozygotes were used in all Ca²⁺ imaging experiments.

1.2　Behavior tests

The neck of the mice was clipped and depilated with electric hair clippers 48 h before experiments. Mice were placed in a box for approximately 15 min for acclimatization. Subcutaneous injection of allantoin or saline into the neck back of experimental mice was adopted. Behavior of the testing mice was collected by camera for 1 h. A bout of scratching was defined as a continuous scratching movement with a hindpaw directed at drug injection site^[14].

1.3　Calcium imaging and cell culture

DRG neurons, isolated from Pirt-GCaMP3 heterozygotes, were collected in DH10 medium on ice (90% DMEM/F-12, 10% FBS, 100 U/ml penicillin, 100 g/L streptomycin). Dissected DRGs were digested for 30 min at 37℃ in a protease solution (5 g/L dispase, 1 g/L collagenase type II in HBSS) DRG neurons were then triturated to free neurons and pelleted by centrifugation. Pelleted neurons were re-suspended in DH10 medium supplemented with NGF (20 µg/L) and plated onto glass coverslips (8 mm) coated with poly-D-lysine (0.5 g/L, Sigma) and laminin (10 g/L, Sigma). Neurons were cultured in an incubator (95% O₂ and 5% CO₂) for 24 h before they were used for calcium imaging.

1.4　Patch clamp

In whole recordings, inward currents were recorded with an Axon 700B amplifier and the pCLAMP 10.1 software package (Axon Instruments). DRG neurons were cultured in normal solution: 140 mmol/L NaCl, 4 mmol/L KCl, 2 mmol/L CaCl₂, 2 mmol/L MgCl₂, 10 mmol/L HEPES, 5 mmol/L Glucose, pH 7.4 in NaOH to adjust. Pipette resistance ranged from 3-4 MΩ. The internal solution was 35 mmol/L KCl, 3 mmol/L MgATP, 0.5 mmol/L Na₂ATP, 1.1 mmol/L CaCl₂, 2 mmol/L EGTA, 5 mmol/L Glucose, pH 7.4 in KOH to adjust, and osmolarity was adjusted to 300 mOSmol/L with sucrose. Electrodes were pulled (Sutter, model P-97) from borosilicate glass. All experiments were performed at room temperature.

2　Results

2.1　Purification and crystallization of allantoin

We speculated that the compounds in yam could induce itch. Followed work was finding the pruritic compounds in yam. Multidimensional chromatographic separation methods including membrane filtration, ion exchange chromatography, C18 reverse phase chromatography, silica column and preparation liquid phase, mass spectrometry analysis were used to purify the active compounds. Fresh Chinese yam was extracted by 85% ethanol (material liquid ratio 1∶2). Ethanol extract was obtained by filtration of extract from gauze. It was concentrated and dried at 55℃ using rotary evaporator. Behavior tests and calcium imaging in vitro were used to test the activation of the ethanol extract.

Ethanol extract contained saccharides or glycol- proteins and polar secondary metabolites^[15]. To separate these complex ingredients, technics were adopted including membrane separation technology, C18 reverse phase chromatography, silica column, preparative liquid phase for fast preparation and purification of interested targets. The ethanol extract was separated by molecular weights of 10 ku and 100 ku and brought to three fractions with low molecular weight (LMW, <10 ku), middle (MMW,10-100 ku) molecular weight and high molecular weight (HMW, >100 ku). LMW fraction was separated into five fractions by preparative C18 column in a 30-min cycle. Each fraction was accurately collected and tracking detected by HPLC. Five fractions were dried in room temperature or freeze-dried before volatilizing the methanol. The third fraction of the five extracts grows out crystallization (Figure 1a). Crystals were cultured by suspension drop method with water as crystallizing solvent. Crystallization temperature was controlled at 20-25℃. The crystal growth under polarizing microscope was observed. Dual-wavelength single crystal diffractometer was used to confirm the structure of allantoin. X-ray crystal diffraction pattern of allantoin showed in Figure 1b. The crystal data of allantoin was showed in Table 1.

Fig. 1 Allantoin crystal and X-ray crystal diffraction pattern

NOTE: (a)Allantoin crystal in 0.2 g discolourant silica gel. (b)X-ray crystal diffraction pattern of allantoin.

Analyses of allantoin were performed on a Waters Alliance 2695 High Performance Liquid Chromatography (HPLC) instrument (Waters, USA) consisting of a Waters Quaternary Pump, a Waters 2996 Photodiode Array Detector, and the Empower Pro software. The conditions were as follows: mobile phase CH3OH/H2O (10/90); flow rate 0.4 ml/min; UV detective wavelength 224 nm; and column temperature 30℃. Before injection, the samples were filtered through a 0.22 μm Millipore filter. The standards for allantoin were purchased from the Shanghai Aladdin Bio-Chem Technology Co., LTD, China. The results of HPLC for crystalline compounds were consistent with those of allantoin standard (Figure 2a, 2b).

Fig. 2 HPLC of allantoin crystal(a) and standard product(b)

NMR and LC–TOF-MS/MS methods were used to identify allantoin structure. The results showed short retention time and its peak almost mixed with that of the solvent in the UPLC system (Figure 3). The data was shown as: ¹³C NMR (100 MHz, DMSO-d6) δ 174.0 (C-2), 157.2 (C-4), 62.9 (C-5), 157.8 (C-7)^[16].

Fig. 3 H-NMR(a) and C-NMR(b) of allantoin extracted from Chinese yam

2.2　Allantoin directly excites DRG neurons

The content of allantoin from Chinese yam changed in volatility^[17]. The content of allantoin extracted from yam (origin from Jiaozuo, Henan) was about 3.567 mg/g in our experiments. Allantoin was an important compound in many plants, such as Chinese yam, Arabidopsis. It could increase cadmium tolerance in Arabidopsis via activation of antioxidant mechanisms ^[18]. In clinic, allantoin was used as an independent marker associated with carotid intima-media thickness in subclinical atherosclerosis and a stable marker for in vivo free radical activity^[19,20]. Calcium was a second messenger, playing an important role in excitable cells and signal transduction. Several versions of the original GCaMP sensor have been published^[21,22,23]. We cultured DRG neurons from Pirt-GCaMP3 heterozygotes examined their responses to allantoin using calcium imaging techniques. Allantoin (1 mmol/L) was added into the perfusion system containing DRG neurons. DRG neurons were activated by allantoin (Figure 4a). However, when administered again, the response of DRG neurons was significantly reduced, because of desensitization (Figure 4b). The response curves of two cells were showed in Figure 4c. These results verified the allantoin could induce calcium influx in cultured DRG neurons.

Fig. 4 Allantoin induced calcium ion influx in cultured DRG neurons in vitro

NOTE: (a)Allantoin (1 mmol/L) could active DRG neurons in vitro. Number 1 indicated by white flat end arrow was the responsive neurons. Number 2 indicated by pointed arrow was the negative cell. After washout in normal buffer, the responsive neurons could recover. (b)Ca²⁺ influx caused by the second addition of allantoin. (c)Allantoin induced fluorescence change in the cell-1 and cell-2 in figure(a)and cell desensitization induced by secondary dosage.

2.3　Allantoin induces inward current in DRG neurons and scratching behavior in mice

We also examined the electrophysiological characteristics of the neurons induced by allantoin. Neurons plated on cover slips were transferred into a chamber with the extracellular solution (Figure 5a). Patch pipettes had resistances of 3-4 MΩ. In wild type mouse, inward current was induced upon allantoin treatment (Figure 5b). In whole-cell voltage clamp recordings, inward current measurements were performed with an Axon 700B amplifier and the pCLAMP 10.1 software package (Axon Instruments). Neurons were perfused with 1 mmol/L allantoin for 20 sec. All experiments were performed at room temperature (~25℃). The diameters of allantoin sensitive neurons were about 8-20 μm. The data suggested that allantoin active DRG neurons directly and then play an important biological function

Fig. 5 The electrophysiology response of DRG neurons to allantoin (1 mmol/L)

NOTE: (a)Bright-field image of a neuronal recording(arrow)with an extracellular electrode(outlined with dashed red lines).(b)Inward current induced by allantoin in DRG neurons.(c)Scratch bouts induced by allantoin(7.9 mg/kg) were more than control group (allantoin,(64 ± 11),vs. saline,(21 ± 5),n=10,**P<0.01).

Although we already knew that allantoin could activate DRG neurons, what kind of behavioral responses did these neurons cause after stimulation was still unknown. To detect the role of allantoin in mice behavior, we intradermally injected allantoin (50 mmol/L) in the neck of the mice. The scratch bouts induced by allantoin in mice more than control group (allantoin, (64 ± 11), vs. saline, (21 ± 5), n=10, **P<0.01) (Figure 5c). This result suggests that allantoin could induce itch in mice.

3　Conclusions

Yam has been widely used for the treatment of diabetes, diarrhea, asthma, and other ailments^[24,25]. But in the process of contacting yam, it often causes itching. We chose fresh yam as raw material, and got the allantoin crystal by simple ethanol extraction. As far as we know, this is the first time to obtain allantoin crystal from fresh yam. This work provides an important reference for us to get allantoin from natural product

Allantoin is widely found in many natural plants, but the effect of allantoin on DRG neurons and animal itching behavior was the first time. Our research shows that allantoin not only activated small DRG neurons, but also caused itch in mice. Allantoin could induce the itching behavior of mice, which not only help us understand the cause of yam to induce itch, more importantly, we found a natural itch causing compound. This discovery will have implications for our future research on the mechanism of itch.

李德峰. E-mail: lidefeng@im.ac.cn

唐宗湘. E-mail: zongxiangtang@njucm.edu.cn

References