Supported by Project of Sichuan Provincial Health Department (120385); Project of Sichuan Provincial Science and Technology Department (14JC0137); Project of Luzhou City Department of Science and Technology (2015LZCYD-S09(2/8)).

中国汽车制造业的产业地图及影响产业布局的因素....................................................................................................................贺正楚 王 姣 曹文明（1）

1 Introduction

Paliurusramosissimus (Lour.) Poir. is a deciduous multi-branched shrub in the genus Paliurus (family Rhamnaceae). It is mainly growing in the shrubbery of hillsides, or barren hills and grasslands; in villages, it is often planted as a fence. It is mainly located in the middle and lower reaches of the Yangtze River in China, especially in Sichuan and Guangxi. According to ChronicleofTraditionalChineseMedicinesinSichuan, its roots, stems, leaves, flowers, fruits can be used for medicinal purpose, have the functions of detoxifying, eliminating swelling, eliminating stasis and stopping bleeding, and promoting blood flow and relieving pain; in local areas, it is commonly used in treating toothache, fever, fever, bronchitis, cough, bruises, rheumatism, swelling and pus[1]. At present, there are few studies about chemical components of P. ramosissimus. There are reports of flavonoids[2], pentacyclic triterpenoids[3-5]: such as 24-hydroxy-ceanothic acid dimethylester, 27-hydroxyceanothic acid dimethylester, cean-othic acid, ceanothic acid 28-β-glucosylester, and isoceanothic acid 28β-glucosylester in roots of P. ramosissimus. The roots and stems also contain a cyclic peptide alkaloid, such as paliurine B[6]. From the ethanol extract of its fruit, Yu Lei etal[7]. separated 9 compounds: umbelliferone, artemisia coumarin, delta-Gluconolactone, Bergapten, isopimpinellin, byakanrgelicin, Xanthotol, Isosakuranin, and Poncirin. In recent years, studies on the chemical components of P. ramosissimus. have shown that betulinic acid and paliurusene have obvious in vitro antitumor activity[8]. However, there is still no report about chemical components in leaves of P. ramosissimus.

产犊之前需要达到成母牛体重的90%，这是需要关注的一个点。有研究表明，体重在1300～1375磅之间时产犊可使第一泌乳期产奶量最大化。体重在1300磅以下，每降低100磅=损失450磅产奶量。

Flavonoids have anti-inflammatory and antioxidant functions, and are main active components of Medicinal Plants. According to previous studies, both the root and the fruit of P. ramosissimus. contain flavonoids[9], so it can be inferred that the leaves also contain flavonoids. Based on the pre-test results of the hydrochloric acid magnesium powder reaction, it preliminarily determined that leaves of P. ramosissimus. contain flavonoids. This experiment tried to use ultrasonic extraction method, and design orthogonal test to explore the optimum extraction conditions of the total flavonoids in the leaves of P. ramosissimus. The microplate spectrophotometer was used to measure the content of flavonoids in leaves of P. ramosissimus. The results of this study are expected to provide a basis for the development and utilization of the medicinal resources of P. ramosissimus.

本文所指的新生代农民主要是指出生于20世纪80年代与90年代，年龄在16岁以上，在异地以非农就业为主的农业户籍人口。

2 Materials and methods

3.1 UV scanning results Rutin reference substance had absorption peak at the wavelength of 410 nm, and the absorbance was 0.590; the test sample had absorption peak at the wavelength of 410 nm, and the absorbance was 0.358, indicating that P. ramosissimus. contains the flavonoid rutin, thus it is feasible to take rutin as the reference substance. And 410 nm was taken as the measurement wavelength, as shown in Fig.1.

2.2 Reagents and instruments Rutin reference substance (Shanghai Yuanye Bio-Technology Co., Ltd., batch number YM0513SB14, and purity 98%); aluminum trichloride (Al2O3) and ethanol were of analytical reagent (AR); water adopted was distilled water. FC104 electronic balance; UV1700PC Ultraviolet-Visible Spectrophotometer; AS3120A Ultrasonic Cleaner; Microplate Reader Spectra Max M3; volumetric flask; pipette; alcohol meter; conical flask; graduated cylinder; beaker; vacuum suction pump; pipette gun; cuvette; EP tube; colorimetric tube.

2.3 Methods

3.2 Methodological study

Table 1 Design of factor and level for orthogonal experiment

LevelFactorA(Ethanolconcentration)∥%B(Ultrasonictime)∥hC(Solid⁃to⁃liquidratio)∥g/mLD(Extractingtimes)1651．01∶1522801．51∶2033952．01∶254

2.3.2 Preparation of the test solution. Precisely weighed 1.000 0 g powder of leaves of P. ramosissimus, placed in 100 mL capped conical flask, added ethanol with the optimal extraction conditions for ethanol concentration and solid-to-liquid ratio to soak 24 h, and carried out ultrasonic extraction, sucked and filtered with vacuum suction pump, the filtrate was extracted with ethanol to fix the concentration to 100 mL volumetric flask, obtain the test solution.

[25]博登海默：《法理学：法律哲学与法律方法》，邓正来译，北京：中国政法大学出版社，2004年，第279页。

2.3.3 Determination of UV absorption wavelength. Precisely absorbed 1 mL reference substance and sample to a 10 mL colorimetric tube, added 4 mL of 1% aluminum trichloride solution, and then fixed the volume with ethanol to 10 mL, placed 15 min, and used 0 as the blank to conduct baseline correction, and used UV spectrophotometer to scan at the wavelength of 200-800 nm.

(iii) Determination of total flavonoids in samples. Precisely absorbed 0.2 mL test solution, placed in 1 mL EP tube, added 0.4 mL of 1% aluminum trichloride solution for color development, fixed the volume with 65% ethanol to 1 mL, placed 15 min, measured the absorbance (A) at the wavelength of 410 nm, and took the blank reagent as a reference, and calculated the sample extraction rate according to the standard curve regression equation. Calculation formula for the extraction rate of total flavonoids:

Extraction rate (%) =(C×V1×V)/(V2×m)

① 印出的产品图形完整，线划印色饱满、注记、网线（点）光洁实在，无双影、花糊、虚断和脏污。大面积的平网颜色均匀一致。

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2.3.4 Determination of total flavonoids. (i) Preparation of reference solution. Precisely weighed 2.0 mg rutin reference substance with the electronic balance and placed in a 10 mL volumetric flask, ultrasonic dissolved with 65% ethanol, cooled down, and fixed the volume to the desired value, to prepare 0.2 mg/mL standard solution for use.

(ii) Methodological study. (a) Linear relationship test. Precisely absorbed 0, 0.05, 0.10, 0.20, 0.30, 0.40, 0.50 mL rutin reference solution, and placed in 1 mL EP tube, added 0.4 mL of 1% aluminum trichloride solution, and then fixed the volume with 65% ethanol to 1 mL, placed 15 min, and used 0 as the blank to measure the absorbance (A) with the microplate reader at the wavelength of 410 nm, and took the absorbance as the ordinate and the concentration as abscissa, to plot the standard curve and obtain the regression equation. (b) Precision test. Precisely absorbed 0.2 mL test solution, placed in 1 mL EP tube, added 0.4 mL of 1% aluminum trichloride solution for color development, fixed the volume with 65% ethanol to the desired value, placed 15 min, measured the absorbance, repeated measurement 6 times, and calculated the RSD. (c) Stability test. Precisely absorbed 0.2 mL test solution, placed in 1 mL EP tube, added 0.4 mL of 1% aluminum trichloride solution for color development, fixed the volume with 65% ethanol to the desired value, placed 15 min, measured the absorbance every 20 min within 2 h for 6 times, analyzed and calculated the total flavonoids, and calculated the RSD. (d) Reproducibility test. Took 6 samples in the same batch, precisely weighed 1.000 0 g, prepared in parallel 6 pieces of the test solution, precisely absorbed 0.2 mL test solution, placed in 1 mL EP tube, added 0.4 mL of 1% aluminum trichloride solution for color development, fixed the volume with 65% ethanol to 1 mL, placed 15 min, measured the absorbance, substituted into the regression equation and calculated the content of total flavonoids and RSD. (e) Sample recovery rate test. Precisely weighed 0.2 mL test solution with known content of total flavonoids, placed in 1 mL EP tube, added 0.1, 0.14, 0.18, 0.22, 0.28, and 0.32 mL of 0.2 mg/mL rutin reference solution, then added 0.4 mL of 1% aluminum trichloride solution for color development, fixed the volume with 65% ethanol to 1 mL, placed 15 min, measured the absorbance and calculated the sample recovery rate and RSD.

where C denotes the concentration calculated according to the regression equation, V denotes the total volume of samples, V1 denotes measured total volume of samples, V2 denotes the sample size during measurement, and m is the mass of the medicine.

3 Results and analyses

2.1 Source of materials The whole herb of P. ramosissimus. was collected from Zigong of Sichuan Province. The collected P. ramosissimus. was separated from each other by roots, stems, leaves, flowers, and fruit, and dried separately.

Note: A. scanning results of rutin reference substance; B. scanning results of test sample.

Fig.1 UV scanning results of rutin reference substance and test samples

2.3.1 Ultrasonic extraction and orthogonal experiment. The dried leaves of P. ramosissimus. were ground into fine powder, screened with No. 6 sieve, accurately weighed with FC104 electronic balance for 9 pieces of 1.000 g powder of leaves of P. ramosissimus, separately placed in 9 capped conical flasks, and added ethanol in accordance with design requirements of Table 1, soaked for 24 h, for ultrasonic extraction. Sucked and filtered with vacuum suction pump, the filtrate was extracted with ethanol to fix the concentration to 100 mL volumetric flask, obtain the test solution. Prepared 1% aluminum trichloride (AlCl3) solution, took 0.2 mL test solution using the pipette gun with a range of 20-200 μL to 1 mL EP tube, added 0.4 mL of 1% aluminum trichloride solution, used the ethanol to fix the volume to 1 mL, placed 15 min, and measured the absorbance (A) with the microplate reader in the wavelength of 410 nm. Took the ethanol concentration group with the highest absorbance as the solvent to prepare rutin reference substance and determine the rutin standard curve, and calculate the extraction rate using the obtained regression equation.

3.2.1 Linear relationship test. Took the concentration C as the X-axis and absorbance A as Y-axis to plot the chart according to the measurement results with the aid of Excel, as shown in Fig.2. The regression equation was A = 8.380 2C + 0.009 6, R = 0.999 5. The results indicated that the rutin reference substance took on an excellent linear relationship.

Fig.2 Standard curve for rutin reference solution

3.2.3 Stability test. The absorbance values of 6 samples measured at 0, 20, 40, 60, 80, 100 and 120 min were 0.405, 0.410, 0.408, 0.409, 0.400, 0.399 and 0.397, respectively, and the RSD of absorbance was 2.3%, showing that the absorbance was basically stable within 120 min.

3.2.2 Precision test. The absorbance values of 6 samples were 0.407, 0.405, 0.405, 0.416, 0.419, 0.410, and 1.61. The RSD of absorbance was 1.61% (n=6), showing excellent precision.

3.2.4 Reproducibility test. The content of total flavonoids of samples 1-6 was 1.584%, 1.602%, 1.587%, 1.566%, 1.574% and 1.586%, respectively. The RSD of content of total flavonoids in samples was 0.78%, indicating good reproducibility.

3.2.5 Sample recovery rate test. The results obtained in Table 2 showed that the average recovery rate was 99.81% and the RSD was 1.17% (n=6), indicating that the method was accurate and reliable and could be used to determine the content of total flavonoids in the leaves of P. ramosissimus.

Table 2 Sample recovery rate of total flavonoids in the leaves ofPaliurusramosissimus

No．MedicineweightgWeightoftotalflavonoidsmgSampleinjectedmgTotalweightmeasuredmgRecoveryrate%Averagerecoveryrate%RSD%10．49822．65122．12104．762199．5298．021．0720．49902．62152．09724．653696．8930．50142．73032．18424．887698．7740．50062．66082．66085．273598．1950．49862．63092．63095．178696．8460．50102．66032．66035．278698．4270．49942．64983．17985．768998．0980．49882．64563．17475．726397．0490．50022．65873．19045．798998．43

3.3 Results of orthogonal experiment To screen the optimum extraction conditions for total flavonoids from P. ramosissimus, the three-level four-factor orthogonal experiment was designed. The influence factors included ethanol concentration (A), ultrasonic time (B), the solid-to-liquid ratio (C), and extraction times (D). L9(34) orthogonal experiment was carried out, and the results were listed in Table 3.

Table 3 Results of L9(34) orthogonal experiment for determination of content of total flavonoids in the leaves ofPaliurusramosissimus

No．ABCDMedicineweight∥gExtractionrateoftotalflavonoids∥%111111．00063．49212221．00005．02313331．00036．50421230．99995．54522311．00074．04623120．99904．68731320．99903．55832131．00054．94933211．00013．12K15．004．164．373．55K24．754．674．564．42K33．874．774．705．66R1．130．610．332．11

From Table 3, the extreme difference value R analysis result of orthogonal experiment indicated that the degree of influence of four factors on the extraction rate of flavonoids in P. ramosissimus. was D > A > B > C, i.e. the extraction times > ethanol concentration > ultrasonic time > solid-to-liquid ratio. The effects of solid-to-liquid ratio on the extraction rate of total flavonoids in leaves of P. ramosissimus, thus it was taken as error to make the variance analysis. Further variance analysis showed no significant difference between the levels (P>0.05). The screened optimal extraction process was A1B3C3D3, namely, ethanol concentration of 65%, the solid-to-liquid ratio of 1∶25, ultrasonic time of 2 h and the extraction times of 4.

3.4 Measurement of content of total flavonoids in samples The optimum extraction conditions were used to prepare the test solution. The absorbance (A) of the test solution was 0.456, substituted to the formula, obtain the extraction rate of total flavonoids in leaves of P. ramosissimus. was 2.56%.

4 Discussions

Flavonoids are the main active components in traditional Chinese medicine and have many biological activities such as anti-inflammatory, anti-bacterial and anti-tumor activity[10-11]. Previous reports on the study of P. ramosissimus. showed that there are flavonoids in the root and fruit of P. ramosissimus. The extract fraction of n-butanol from leaves of P. ramosissimus. has anti-inflammatory activity, and its n-butanol and ethyl acetate extracts have antibacterial activity[12-13]. Therefore, the active components of extract of leaves of P. ramosissimus. possibly come from the flavonoids. However, there is no report about whether the leaves contain flavonoids and the content of flavonoids. In the past, the determination of flavonoids in plants was mainly carried out with UV spectrophotometer[14]. However, this method needs operators have skilled technique for measuring the standard curve and samples, and constantly change the cuvette during measurement. For samples with instable color development, slow measurement may lead to failure to plot standard curve and fail to obtain measurement results. The determination of substance content with microplate reader and UV spectrophotometer has the same theoretical basis, both are subject to Beer-Lambert Law. At present, the microplate reader has been successfully used for determination of polysaccharide content[15]. Yet, the use of microplate reader for determination of flavonoids has not been reported. In this study, the reference solution and test solution were placed in a 96-well plate for simultaneous determination, which accelerated the test speed of the samples and ensured performance of experiment for samples and reference substance under the same conditions, excluding the interference of system error with the determination, making the experimental results more rapid and accurate, and realizing high-throughput determination of the samples.

In this experiment, ultrasonic extraction was used to extract the flavonoids in leaves of P. ramosissimus, so it could minimize the damage to the structure of flavonoids such as rutin[16]. The process equipment was simple, easy to operate and the extraction time was short. Through orthogonal experiment, the optimal extraction process for extraction of flavonoids in leaves of P. ramosissimus. was the ethanol concentration of 65%, the solid-to-liquid ratio of 1∶25, ultrasonic time of 2 h and the extraction times of 4, under which condition, the content of total flavonoids in the leaves of P. ramosissimus. was 2.56%. This experiment shows that the leaves of P. ramosissimus. contain the flavonoids. This study is expected to provide experimental basis for the development and utilization of medicinal resources of P. ramosissimus.

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