The Study Of Separation And Purification Of The Active Ingredient In Yam Caused Itch And Relations With Mrgprs Family

Chinese yam is the tuber of Dioscorea opposite Thunb., species of Dioscoea L genus of Dioscoreaceae. Itch can be induced by Chinese yam when contacting or possessing it directly. However, no studies have been published that what ingredients of Chinese yam cause itch.Itch is unpleasant and it can cause scratching desire or scratching reflex. Itching is occurred in skin diseases and some systemic diseases. Itching seriously affects daily life. Except conventional antihistamine therapy, there is no better methods in treatment of itching.Research shows that the itch-causing substances are divided into two categories: one is the class of histamine including histamine and substances produced by stimulating mast cells; the other is the class of non-histamine itch-causing substances such as chloroquine, small peptide BAM8-22, SLIGRL peptide, bromelain, black Mucuna protein, which the receptor is Mrgprs protein family and PAR-2. Itch induced by contacting with fresh yam juice directly can not cause red and inflamed scar as induced by histamine, thus it is probably induced by non histamine substances.Mrgprs can be activated by non histamine itch-causing substances such as chloroquine, small peptides BAM8-22, SLIGRL, beta alanine. It is also related with some itching disorders such as chronic itching, contact dermatitis. Therefore, Mrgprs may play an important role in non histamine itch-causing.The study is expected to separate and purify the active components of itch-causing from yam, investigate the relation between its active component and the Mrgprs family, clarify the mechanism of causing, provide related information for simplifying the application and processing of Chinese yam, and provide laboratory evidence for the function of Mrgprs family, and provide a new way for the treatment of pruritus as well as theory research and development of new drugs.Part1Theory researchThrough summarizing related literatures in the purification and separation of various components in pharmacological research of yam, We systematically reviewed itch research situation of traditional Chinese medicine and Western medicine and mainly introduced Mrgprs family and its role relevant with itch.Part2Research on extraction of itch-causing active components from Chinese yam and its relation with Mrgprs familyResearch on animal behavior shows that fresh yam juice via subcutaneous injection can cause the scratching response in wild-type mice, while subcutaneous injection of saline, potato homogenate, mouse scratching time were reduced significantly (P<0.05), but the boiled yam juice doesn’t affect the activity. The results showed that scratching reaction induced by Chinese yam has nothing to do with the solvent, high molecular starch granules, and large molecular proteins. Compared with WT mice, in Mrgpr-cluster Δ-/-mice, the scratching response induced by fresh yam juice significantly decreased(P<0.05), which showed that itch-causing by yam is relevant to Mrgprs family.The active component from yam can be extracted by the ethanol. Research on animal behavior shows that ethanol extractum of Chinese yam induces scratching response of mouse. After extracted by chloroform, ethyl acetate, n-butanol extraction step by step, ethanol extraction precipitation can not cause animal itching. These polysaccharide substances presumably not play a role in itch-causing. The chloroform and ethyl acetate extract phase showed no better itch activity either. With the solvent polarity increased further, more polar phase-n-butanol showed activity of mouse scratching itch. Extraction of residual water showed the highest scratching responses.The results showed that a kind of soluble and high polar compound substances may plays a role in itch-causing by Chinese yam.Part3Separation and purification of active component from Chinese yam and its structure identificationYam ethanol extract was separated and purified using Sephadex-G50gel column by The AKTA purifier automatic liquid chromatography system. Fraction-1and fraction-2were obatain. Fraction-1was separated using Mono Q5/50anion exchange column by The AKTA purifier automatic liquid chromatography system, gain peak of Ⅰ (fraction-Ⅰ), the peak of Ⅱ (fraction-Ⅱ) and peak Ⅲ (fraction-Ⅲ). Using Waters-2695-2996automatic high-performance liquid chromatography system separated fraction-Ⅰ, obtain fraction-Ⅰ-1, fraction-Ⅰ-2and fraction-Ⅰ-3. The further separation and purification of fraction-Ⅰ-2hanging drop method to obtain high purity, crystal compounds.The crystal structure was refined by dual wavelength X-ray diffraction. According to the literature, it was identified as NJUTCM-01.The results showed that itch-causing active ingredients from Chinese yam with high purity can be separated and purified by Sephadex gel chromatography, ion exchange chromatography, HPLC chromatography and crystallization. It’s crystallographic parameters can be obtained by X-ray diffraction and was identified as NJUTCM-01according to relevant literature.Part4Identification of itch-causing active components of Chinese yam and research on its molecule mechanism.Animal behavior results showed that compared with the solvent control group, fraction-1and fraction-2can cause animal scratching reaction, fraction-1can cause scratching reaction significantly, which were significantly higher than those of the same dose of fraction-2.Fraction-1and fraction-Ⅰ,-Ⅱ,-Ⅲ separated and purified by ion exchange were made into5mg/ml, each with20μl. WT mouse DRG neurons were obtained by the primary cells. After cultured cell in Fura-2incubated for40min, cell activity was measured by calcium imaging. The results showed that fraction-I had the highest activity, followed by fraction-Ⅲ and fraction-Ⅱ. The activity of Fraction-Ⅰ-1, fraction-Ⅰ-2and fraction-Ⅰ-3separated by C18reversed-phase were test by calcium imaging.The statistic results show that fraction-I-2activity is the highest activities, and fraction-Ⅰ-1and fraction-Ⅰ-3activity is very weak or inactive. Fraction-Ⅰ-2separated and purified by crystallization were test by calcium imaging. The results showed that adding the crystal can cause calcium influx in DRG neurons reached peak reaction in a short time.Animal behavior results showed that compared with the solvent control group, NJUTCM-01can cause animal scratching reaction; NJUTCM-01injected in WT mice and Mrgpr-cluster Δ-/-mice, animal behavior results showed that compared with the solvent control group, NJUTCM-01induced animal scratching were reduced significantly in WT mice.NJUTCM-01activity was measured by calcium imaging. The results showed that NJUTCM-01perfusion can induce DRG neuron intracellular calcium influx to peak reaction in a short time; and8%of DRG neurons respond to the NJUTCM-01.NJUTCM-01was also used for electrophysiological experiments. The results show that, cells in response to NJUTCM-01stimulation belongs to small sense neurons.Part of the cells in Mrgpr A3and Histamine receptor agonized by chloroquine and Hiatamine can induced calcium influx, after given NJUTCM-01again. In addition,24.5%of cells responded to menthol also were responded to NJUTCM-01; while92%of cells responded to NJUTCM-01were responded to capsaicin.Part5Conclusion1. Animal behavior studies showed that fresh yam juice can cause scratch response and it has nothing to do with the solvent, high molecular starch granules, and large molecular proteins.2. Itch-causing by Chinese yam is associated with the Mrgprs family.3. The active component from yam can be extracted by the ethanol. The results showed that a kind of soluble and high polar compound substances may plays a role in itch-causing by Chinese yam.4. Fraction-1and Fraction-2can be got by Sephadex gel chromatography. Fraction-I-1, fraction-I-2and fraction-I-3can be got by HPLC. Crystallographic parameters can be obtained by X-ray diffraction and was identified as NJUTCM-01according to relevant literature. 5. The results showed that itch-causing active ingredients from Chinese yam with high purity can be separated and purified by Sephadex gel chromatography, ion exchange chromatography, HPLC chromatography and crystallization. These compounds showed a good itch-causing activities.6. Behavior studies showed that NJUTCM-01can induce itch and associated with Mrgprs. Calcium imaging and patch clamp also proved that NJUTCM-01is the main factor of Chinese yam itch-causing.7. The results showed that the mechanism of itch induced by NJUTCM-01may by activating Mrgpr A3and Histamine receptor, and is associated with TRPM8and TRPV1.