Optimization of Extraction Process for Polysaccharides ofUrticafissa E. Pritz.

Supported by Project of Natural Science Foundation of Tibet Autonomous Region (2016ZR-15-31).

1 Introduction

Urticafissa E. Pritz., also called nettle, belongs to the family Urticaceae, and is a perennial herb. It has transverse rhizomes. It is monoecious. On its surface, there are maroon wart points. Its florescence is during August and October, and the fruit period is during September and November. U. fissa E. Pritz. is shade-requiring plant, has vigorous life, its requirement for soil is not stringent, the growth is rapid, and it is thermophilic and moisture tolerant. U. fissa E. Pritz. is rich in nutrients, suitable for making cold dish, vegetable dish, and soup, and also can be rapidly frozen or dehydrated for long-term storage[1-3]. U. fissa E. Pritz. is distributed in Eurasia. In China, it is mainly distributed in Yunnan, Guizhou, southeast Sichuan, and Tibet[4]. In foreign countries, as early as 2 000 years ago, there were records of eating U. fissa E. Pritz.[5] and the tradition has been extended to the present day. In several regions such as Tibet of China, eating U. fissa E. Pritz. has a history of few hundred of years. Many regions do not have reasonable use of U. fissa E. Pritz., leading to waste of enormous resources[6].

能够在颜师父的书法课上不打瞌睡的同学，要么是找宇晴师父讨过赫赫有名的“万花谷风油精”，余下都是万花谷一等一的好弟子，星雨算其中的一个。她在家里已由蒙师开过笔，学习楷字，悄悄问师兄们什么时候能将这二十四字咒写到草书，曲风师兄不紧不慢地鬼画着桃符，一边回过头来对她讲：“我还没听说过万花谷有谁能由颜师父这里学到草书的，楷书十年可通神，草书一天乱脚跟，宁要欧公奇，不要张旭狂，小师妹你莫做梦了。”

Polysaccharides are natural macromolecular substances, essential components of all living organisms, and interconnected with the mechanism of maintaining physiological functions of the body. At present, more than 300 kinds of the polysaccharides have been extracted from natural products, of which the most important is water-soluble polysaccharides in Chinese Herbal Medicines. Extensive studies have shown that polysaccharides have anti-tumor, anti-aging, anti-infection, anti-hyperlipidemia, treatment of AIDS biological activities. In recent years, the sugar chain of polysaccharides plays a decisive role in controlling the cell division and differentiation, and regulating the cell growth and aging, and polysaccharides have shown broader and broader application prospects. However, there are few studies about polysaccharides of U. fissa E. Pritz. In this experiment, we extracted polysaccharides from leaves of wild U. fissa E. Pritz. in Nyingchi of Tibet, analyzed factors influencing the extraction rate, determined the optimal extraction conditions, in the hope of providing certain reference for reasonable development and utilization of wild U. fissa E. Pritz. resources in Tibet.

2 Materials and methods

2.1 Source of materials Intact leaves of U. fissa E. Pritz. were collected in Nyingchi of Tibet, dried at 60℃, and used as materials for extracting polysaccharides from U. fissa E. Pritz.

3.1 Single factor experiment

2.3 Instruments and equipment FA2004 Electronic Balance (Mettler-Toledo Instruments Co., Ltd.); Solid Sample Pulverizer (XA-1, Jintan Jingda Instrument Factory); Electric Thermostatic Air Blowing Drying Box (DHG-9240A, Zhejiang Top Instrument Co., Ltd.); Electroheating Thermostatic Water Bath (Changzhou Putian Instrument Manufacturing Co., Ltd.); Digital Magnetic Stirrer (JB-3, Jintan City Kexing Instrument Factory); Freezing Desktop Centrifuge (5804R, Centrifuge); Rotary Evaporator (RE52CS, Shanghai Yarong Bio-chemical Instrument, Co., Ltd.); Circulating Water Vacuum Pump (Hangzhou Mingyuan Instrument Co., Ltd.).

(i) Effects of the extraction time on the extraction rate of polysaccharides. Under the conditions of the extraction temperature of U. fissa E. Pritz. at 90℃[4], the mass ratio of dry powder of U. fissa E. Pritz. to water at 1∶25[10], and the extraction time at 1, 2, 3, 4, 5, and 6 h, we measured the extraction rate of polysaccharides from U. fissa E. Pritz.

2.4.1 Pretreatment of raw materials. Cut the leaves of U. fissa E. Pritz. into pieces, put them into the drying box for 12 h, pulverized and screened with 60 mesh sieve, obtained the dry powder of U. fissa E. Pritz., and stored in a closed container for use.

68例老年股骨颈骨折患者在经过解剖分型的检验中，对于符合率的检验，患者应用X线检验符合率可达到60.29%（41/68），16排螺旋CT三维成像的检验 符合率可达到95.59%（65/68），16排螺旋CT三维成像明显高于X线，说明在解剖分型中16排螺旋CT三维成像的应用效果更为明显。

改革开放的总设计师邓小平同志曾说过:“中国的改革是从农村开始的，农村的改革是从安徽开始的”。1978年召开的中共十一届三中全会和小岗村的“大包干”创举，开启了农村改革(包产到户农业生产责任制)的新进程，开创了改革发展新局面。[1]随着1982年家庭联产承包责任制在农村的基本普及、1982—1986年中央5个“一号文件”的连续下发、乡镇企业的蓬勃发展和农业剩余劳动力的外出务工，吃饭的问题逐步得以解决，计划经济体制也逐步向商品经济体制、市场经济体制转化，开始了建设中国特色社会主义的新探索。[1]

(iii) Effects of the extraction temperature on the extraction rate of polysaccharides. Under the extraction time at 4 h, the solid-to-liquid ratio at 1∶25, and the extraction temperature at 40, 50, 60, 70, 80, 90, and 95℃, we measured the extraction rate of polysaccharides from U. fissa E. Pritz.

2.4.3 Single factor experiment. Under the same conditions of other factors, we separately studied the effects of extraction temperature, extraction time, and solid-to-liquid ratio (the mass ratio of dry powder of U. fissa E. Pritz. to water) on the extraction rate of polysaccharides from U. fissa E. Pritz.

⑥低 MWR组（MWR≤0.067）患者 146例（56.8%），高MWR组 （MWR＞0.067）患者111例（43.2%）。两组患者的各临床资料之间，差异无统计学意义。

2.4 Methods

综上，“do/does/did+v.”构式核心构式义是强调人的意识，如情感、感知、认知和观点；其次是强调所属，而且是蕴含转折关系的所属；接着是强调转移和变化，目的是凸显作用力的效果；最后较为延伸的意义是强调竞技、接触、创造、消耗、状态等动作，通过凸显动作引起注意力，进而关注动作产生的事件。可见目标构式的构式义丰富多样，实用性和能产性都较高。

2.4.2 Extraction of polysaccharides[8]. Process route: took 10 g dry powder of U. fissa E. Pritz → added the appropriate amount of distilled water (90℃, 4 h, oscillating) → placed in steady state to cool down to room temperature → separated with centrifuge (8 000 r/min, 5 min) → collected the supernatant → depressurized and concentrated (in rotary evaporator) → added 95% ethanol to the supernatant to 80% alcohol content (constantly stirring) → obtained the precipitate → centrifuged and collected the precipitate → washing (anhydrous methanol, acetone and ether) → drying (40℃) → obtained the crude polysaccharides → weighed and calculated the extraction rate of polysaccharides.

From Fig.1, with the extension of the extraction time, the extraction rate of polysaccharides from U. fissa E. Pritz. increased; when the extraction time was in the range of 1-4 h, with the extension of the extraction time, the extraction rate of polysaccharides from U. fissa E. Pritz. significantly increased; when the extraction time was in the range of 3-6 h, with the extension of the extraction time, there was no significant change in the extraction rate of polysaccharides from U. fissa E. Pritz. Because in the beginning of the extraction, the content of polysaccharides was low; with the extension of the extraction time, the transmission of polysaccharides became balanced; then, extending the time would not increase the extraction rate of polysaccharides.

Table 1 Level of orthogonal experiment factors for extraction of polysaccharides fromUrticafissa E. Pritz.

LevelFactorA(Time)∥hB(Temperature)∥℃C(Solid⁃to⁃liquidratio)12701∶2023801∶2534901∶30

2.4.5 Calculation of the extraction rate of polysaccharides. The extraction rate of polysaccharides (W/W, %) =[Dry weight of polysaccharides (g)/Weight of raw materials (g)][9].

(ii) Effects of the solid-to-liquid ratio on the extraction rate of polysaccharides. Under the conditions of the extraction temperature of U. fissa E. Pritz. at 90℃ and the extraction time at 4 h[4], and the solid-to-liquid ratio at 1∶5, 1∶10, 1∶15, 1∶20, 1∶25, 1∶30, and 1∶35, we measured the effects of the solid-to-liquid ratio on the extraction rate of polysaccharides.

3 Results and analyses

2.2 Main reagents In this experiment, 95% ethanol, ethanol, ether, anhydrous methanol, and acetone were used, and all these were analytical reagents (AR)[7].

本井采用SF-1.32/22-170型撬装式空气压缩机及美国寿力增压机，先将油管下至井底循环抽洗，然后分别于1300 m和900 m进行压风机气举洗井。利用高压空气压缩机气举洗井、潜水电泵抽水洗井，共历时51 h，直到地热水水清砂净。

3.1.1 Effects of the extraction time on the extraction rate of polysaccharides. Under the conditions of the extraction temperature of U. fissa E. Pritz. at 90℃[4], the mass ratio of dry powder of U. fissa E. Pritz. to water at 1∶25[10], and the extraction time at 1, 2, 3, 4, 5, and 6 h, we measured the extraction rate of polysaccharides from U. fissa E. Pritz. The results were shown in Fig.1.

Fig.1 Effects of the extraction time on the extraction rate of polysaccharides fromUrticafissa E. Pritz.

2.4.4 Orthogonal experiment. First, through the single factor experiment, we determined the factors influencing the extraction rate of water-soluble polysaccharides and influencing levels. Using the extraction rate of polysaccharides as the indicator, we designed the orthogonal experiment of three factors, namely, the extraction time, extraction temperature, and solid-to-liquid ratio. The experiment scheme was listed in Table 1.

3.1.2 Effects of the solid-to-liquid ratio on the extraction rate of polysaccharides. Under the conditions of the extraction temperature of U. fissa E. Pritz. at 90℃ and the extraction time at 4 h[4], and the solid-to-liquid ratio at 1∶5, 1∶10, 1∶15, 1∶20, 1∶25, 1∶30, and 1∶35, we measured the effects of the solid-to-liquid ratio on the extraction rate of polysaccharides. The results were shown in Fig.2.

The polysaccharides were extracted from leaves of U. fissa E. Pritz. According to Fig.2, with the increase in the water volume, the extraction rate increased; at the solid-to-liquid ratio of 1∶5-1∶25, the extraction rate increased with the increase of water volume, and there was a significant difference between the values; at the solid-to-liquid ratio of 1∶25-1∶35, there was no significant change in the extraction rate with the increase of water volume. When the water volume was relatively small, polysaccharides were not completely dissolved in water, the extraction rate of polysaccharides increased with the increase of water volume; when the water exceeded certain volume, nearly all polysaccharides were dissolved in the water, so the extraction rate of polysaccharides would not increase.

Fig.2 Effects of the solid-to-liquid ratio (mass ratio of dry power ofUrticafissa E. Pritz. to water) on the extraction rate of polysaccharides fromUrticafissa E. Pritz.

3.1.3 Effects of the extraction temperature on the extraction rate of polysaccharides. Under the extraction time at 4 h, the solid-to-liquid ratio at 1∶25, and the extraction temperature at 40, 50, 60, 70, 80, 90, and 95℃, we measured the extraction rate of polysaccharides from U. fissa E. Pritz. The results were shown in Fig.3.

From Fig.3, when the extraction temperature at 40℃-90℃, the extraction rate of polysaccharides increased with the rise of extraction temperature, and there was significant difference in values; when the extraction temperature at 90℃-95℃, the extraction rate of polysaccharides dropped with the rise of extraction temperature. The solubility of polysaccharides increased with the rise of the temperature. In the range of certain temperature, the extraction rate of polysaccharides increased with the rise of the temperature; however, after the temperature rose to a certain range, polysaccharides were decomposed due to thermal instability, thus the extraction rate of polysaccharides dropped.

Fig.3 Effects of the extraction temperature on the extraction rate of polysaccharides fromUrticafissa E. Pritz.

3.2 Results of orthogonal experiment From the extreme difference of the orthogonal experiment (Table 2), the order of factors influencing the extraction rate of polysaccharides from U. fissa E. Pritz. was A (extraction temperature)> B (extraction time)> C (solid-to-liquid ratio). From the results of orthogonal experiment and the extreme difference analysis, the optimal process conditions for the extraction of polysaccharides from U. fissa E. Pritz. were A3B3C2, namely, the extraction time of 4 h, the extraction temperature of 90℃, and the solid-to-liquid ratio of 1∶25. Under such conditions, the extraction rate of polysaccharides from U. fissa E. Pritz. was 11.548%. After two times of repeated experiment, the extraction rate of polysaccharides was 11.615% and 11.671% respectively, and the average extraction rate was 11.611%.

Table 2 Orthogonal experiment and extreme difference analysis of extraction of polysaccharides fromUrticafissa E. Pritz.

ExperimentNo．ABCEmptycolumnExtractionrate∥%111117．980212228．238313338．685422338．9975231110．4256212210．7687332210．9788313311．5759321111．767K1j24．90327．95530．32330．172-K2j30．19030．23830．77329．984-K3j34．32031．22030．08829．257-K18．3019．31810．107--K210．06310．07910．258--K311．44010．40710．029--Rj3．1391．0890．229--

According to the variance analysis (Table 3), FA>F0.01(2，4)=18.000, P<0.05; FC>F0.01(2，4)=18.000, P<0.05, showing that the extraction temperature (A) and the solid-to-liquid ratio (C) were extremely significant for the experimental results. F0.05(2，4)=6.944<FB<F0.01(2，4)=18.000, 0.01<P<0.05, showing that the extraction time (B) was significant for the experimental results.

③对于小规模的供水工程，普遍存在设计欠合理的问题，选用的输、配水管道管径偏大。一般情况下，工程规模越小，这一问题越多。

Table 3 Orthogonal experiment and variance analysis of extraction of polysaccharides fromUrticafissa E. Pritz.

VariationsourceSquaresumDegreeoffreedomMeansquareFFαSignificanceTemperature(A)14．85427．42795．205F0．05(2，4)=6．944∗∗Time(B)1．87120．93612．000F0．01(2，4)=18．000∗Solid⁃to⁃liquidratio(C)35．618217．809228．308∗∗Error(e)0．15620．0780．039Total52．499

Note: ** denotes extremely significant difference, and * denotes significant difference.

4 Conclusions

Taking the extraction time, extraction temperature, and solid-to-liquid ratio as factors, using the water extraction method, we carried out three-factor and three-level orthogonal experiment and determined the optimal process parameters for extraction of polysaccharides from U. fissa E. Pritz., namely, the hot water extraction time was 4 h, the extraction temperature was 90℃, and the mass ratio of water and U. fissa E. Pritz. was 1∶25. Under such extraction conditions, the extraction rate of polysaccharides from U. fissa E. Pritz. was up to 11.611%.

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