CN103169750B - Ultrasonic extraction method for extracting flavone from dianthus superbus - Google Patents
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Abstract
The invention provides an ultrasonic extraction method for extracting flavone from dianthus superbus. According to the ultrasonic extraction method, the dianthus superbus is taken as a raw material and ultrasonic wave is adopted for treating crushed dianthus superbus cells to obtain dianthus superbus flavone with high purity. In an experiment, total flavone content is selected as an evaluation index to obtain the optimal ultrasonic extraction process of Mongolian dianthus superbus; an ultraviolet-visible spectrophotometry is adopted for determining the total flavone in the dianthus superbus and the content of the total flavone in the dianthus superbus is 0.6367%; and the ultrasonic extraction method has the advantages of simple operation method, and good stability, precision, repeatability and sample feeding and recycling rates. The ultrasonic extraction method is good for improving the extraction rate of the flavone, shortening the extraction time and saving an extraction solvent; and the method can be used for evaluating the quality of the Mongolian dianthus superbus.
Description
Technical field
The present invention relates to technical field of bioengineering, particularly relate to a kind of ultrasonic extracting method extracting flavone from Herba Dianthi.
Background technology
Herba Dianthi, has another name called wild wheat, stone Flos Osmanthi Fragrantis, huge sentence wheat etc., is the wounded in the battle aerial parts of pinkwort Herba Dianthi (Dianthus superbusL.), is distributed in Korea, Mongolia, Russia, Northeast China, North China, northwest, East China, Sichuan, the ground such as Guizhou.The wounded in the battle overground part of Herba Dianthi is divided into mongolian medicine, and Herba Dianthi bitter in the mouth is cold in nature, blunt, light, rare, removing heat from blood, pain relieving, removing toxic substances.Heat in blood controlled by Herba Dianthi, heat in blood twinge, liver-heat, and " precious day " heat is cold to be held concurrently assorted phase, acute filthy disease, puerperal fever, has inducing diuresis for treating stranguria syndrome, an effect that removing blood stasis stimulates the menstrual flow.Clinical efficacy is extensive, compound recipe or be used alone, and is conventional Chinese medicine.
Herba Dianthi chemical composition is mainly flavonoid and saponins material, and flavone compound is extensively present in each position of plant, especially flower, leaf site, and it mainly contains flavone glycoside and aglycon two kinds of forms.Flavone compound has blood pressure lowering, Adjust-blood lipid, prevents thrombosis, prevents and treats cardiovascular and cerebrovascular disease, strengthens immunity, reduces vascular fragility, improves the effects such as blood circulation.For this reason, extracting and developing, the purification process of studying flavone in Herba Dianthi are significant to its biological activity of follow-up study.
At present, from Herba Dianthi, extract flavone many employings organic extractant solution method, there is the problems such as extraction time length, solvent-oil ratio is large, extraction yield is lower, safety is not high.There are article (Liao Zhixiong etc., the assay of total flavones in Herba Dianthi, medical Leader, 2006,25 (5): 472-473; Wang Y C et al., Cyclopeptides fromdianthus superbus, Physiochemistry, 1998,49 (5): 1453-1456) flavone compound is obtained from the aerial parts separation of Herba Dianthi, and cyclic peptide compound, it adopts organic extractant solution, this method adopts plurality of reagents to extract Herba Dianthi flavone, the method step is many, complex operation, and extraction conditions is complicated, safety is low, the total flavones average content 0.6556mgg of its gained
-1, namely 0.06556%, the productive rate that obtains of this method is not high yet as seen.
From Herba Dianthi, flavone will be obtained fully, Herba Dianthi cellularity should be destroyed as much as possible.Ultrasonic extraction method Flavonoid substances is used in the extraction stage, it is a kind of innovation, its principle is that ultrasound wave can produce " cavitation " in a liquid, and the shock wave that " cavitation " produces and jet can destroy plant cell and membrane structure, thus increase the penetration capacity of cellular content by cell membrane, contribute to release and the stripping of flavone compound, ultrasound wave makes extracting solution constantly shake, contribute to solutes accumulation, hyperacoustic heat effect has water-bath effect to raw material simultaneously.At present, the research adopting ultrasound wave to extract flavone from Herba Dianthi in the extraction stage is not yet reported.
Summary of the invention
The invention provides a kind of ultrasonic extracting method extracting flavone from Herba Dianthi, be raw material with Herba Dianthi, utilize ultrasonic extraction, the Herba Dianthi flavone of higher degree can be obtained high yield pulp1.
The invention provides a kind of ultrasonic extracting method extracting flavone from Herba Dianthi, comprising:
Take Aerial Parts of Chinese Pink Dianthus, add in round-bottomed flask, add petroleum ether, reflux 3 hours under 70 DEG C of water-baths, filter medicinal residues, volatilize solvent, be placed in conical flask, add alcoholic solution, supersound extraction, sucking filtration after ultrasonic, after discarding medicinal residues, filtrate is transferred in volumetric flask, with 70% ethanol standardize solution, obtains final product.
As a kind of detailed description of the invention, the invention provides a kind of ultrasonic extracting method extracting flavone from Herba Dianthi, comprising:
Take Aerial Parts of Chinese Pink Dianthus 3g, add in round-bottomed flask, add petroleum ether 30ml, reflux 3 hours under 70 DEG C of water-baths, filter medicinal residues, volatilize solvent, add in 100ml conical flask, add alcoholic solution, supersound extraction, sucking filtration after ultrasonic, after discarding medicinal residues, filtrate is transferred in 100ml volumetric flask, with 70% ethanol standardize solution, obtains final product.
Preferably, the concentration adding ethanol before described supersound extraction, between 30%-80%, is more preferably 60%.
Preferably, ultrasonic time is 20-60 minute, is more preferably 50 minutes.
Preferably, solid-liquid ratio is 1:10(g/mL) to 1:30(g/mL) between, be more preferably 1:20(g/mL)." solid-liquid ratio ", refers to the quality of solid-state " material " and the ratio as the volume of " liquid " of lixiviating solution.Unit g, the mg of " material ", unit L, the mL of " liquid ", so there are g/L, mg/L, g/mL, mg/mL etc. in the unit of " solid-liquid ratio ".Unit of the present invention is g/mL.
Ultrasound wave processes Herba Dianthi as a kind of physical energy, more be conducive to the extraction of flavone in Herba Dianthi, on the one hand, produce great pressure by cavitation, cause the even whole organism of the cell wall of the thing that is broken to break and make intracellular organic matter discharge, spread or be dissolved out; On the other hand, the ultrasound wave of suitable intensity can make the conformation generation forward of flavone molecule transform, thus improves the activity of flavone.
Because ultrasonic frequency is high, energy large, can produce significant heat effect in mechanism, in order to reduce the impact of heat effect on Herba Dianthi extraction flavones content, described hyperacoustic power is 100-250W, preferred 125W.
The present invention utilizes and ultrasonicly to extract the flavone in Herba Dianthi, first degreasing decoloring dewaxing is carried out by petroleum ether, flavone in Herba Dianthi cell is carried out ethanol extraction, the Herba Dianthi cell of synergistic supersonic wave process fragmentation, efficiently can obtain the Herba Dianthi content extracting solution containing flavone; Again by sucking filtration process with after removing the medicinal residues in extracting solution, the higher Herba Dianthi flavone of purity can be obtained in high yield pulp1 ground.Compare traditional extraction method, this method has and uses solvent few, simple process, mild condition, is easy to features such as controlling, safe and reliable; This method extraction efficiency is higher, and the operating time is short, extract yield and product purity all higher.
Accompanying drawing explanation
Fig. 1: the rutin standard curve of drafting;
Fig. 2: concentration of alcohol is on the impact of Herba Dianthi total flavones extraction ratio;
Fig. 3: ultrasonic time is on the impact of Herba Dianthi total flavones extraction ratio;
Fig. 4: the impact of different feed liquid comparison Herba Dianthi total flavones extraction ratio;
Fig. 5: ultrasonic power is on the impact of Herba Dianthi total flavones extraction ratio.
Detailed description of the invention
Below embodiments of the invention are described:
Embodiment 1
1, the preparation of reference substance solution
Get control substance of Rutin 10mg, accurately weighed, put in 50ml volumetric flask, with 70% appropriate dissolve with ethanol, and be diluted to scale, shake up, obtain (containing rutin 0.2mg in every 1ml).
2, the preparation of need testing solution
Precision takes Aerial Parts of Chinese Pink Dianthus 3.0g, adds in round-bottomed flask, adds petroleum ether 30ml, and reflux 3.0h under 70 DEG C of water-baths, filters medicinal residues, volatilizes solvent, add in 100ml conical flask, add alcoholic solution, supersound extraction.Sucking filtration after ultrasonic, after discarding medicinal residues, is transferred in 100ml volumetric flask by filtrate, with 70% ethanol standardize solution, for subsequent use.According to trial test, choose volume fraction of ethanol, ultrasonic time, ultrasonic power, solid-liquid ratio be investigation factor, carrying out 3 levels are determined, design orthogonal experiment in the basis that single factor test gropes, thus determining ultrasonic method optimum extraction process.
3, the determination of wavelength is measured
Use NaNO respectively
2-Al (NO
3)
3-NaOH development process, AlCl
3development process and KOH development process, carry out all-wave long (200 ~ 800nm) scanning to reference substance and test sample, result display NaNO
2-Al (NO
3)
3-NaOH develops the color, and reference substance and test sample all have absorption maximum at 510nm place, and the interference being subject to impurity at 510nm place is little, therefore finally determines with 510nm as mensuration wavelength.Wherein, NaNO
2-Al (NO
3)
3-NaOH development process is as follows: precision measures control substance of Rutin solution 2.0ml, and sample solution 2.0ml is placed in 25ml volumetric flask respectively, respectively adds 5%NaNO
2solution 1.0ml, places 6min, then adds 10%Al (NO
3)
31.0ml, mixing, place 6min, then add 4%NaOH10ml, adding distil water is diluted to 25ml, places 15min, carries out full wavelength scanner.
4, the drafting of standard curve
Precision measures reference substance solution 1.0,2.0,3.0,4.0,5.0,6.0ml and is placed in 25ml volumetric flask, makes series of standards solution.Add 5%NaNO respectively
2solution 1.0ml, mixing, places 6min, then adds 10%Al (NO
3)
31.0ml, mixing, places 6min, then adds 4%NaOH10ml, use distilled water standardize solution, shake up, and places 15min.With corresponding reagent for blank, measure absorbance at 510nm wavelength place, result display is as table 1.Carry out regression analysis with absorbance (A) and solution concentration (C), result display is as Fig. 1, and regression equation is A=0.0119C+0.0048, r=0.9995, the range of linearity: 8.02-48.10 μ gmL
-1.
Table 1 total flavones standard curve test data
Embodiment 2
1, experiment of single factor
Carry out different ethanol concentration, solid-liquid ratio, ultrasonic time by the preparation method of need testing solution, the experiment of single factor of ultrasonic power, draw the relation of total flavones extraction effect and factor level.
1.1 concentration of alcohol are on the impact of Herba Dianthi total flavones extraction ratio
Precision takes Aerial Parts of Chinese Pink Dianthus 3.0g, solid-liquid ratio 1: 20, ultrasonic power 150W, ultrasonic 40min, carries out supersound extraction with the ethanol that concentration is 30%, 40%, 50%, 60%, 70%, 80% respectively, measures absorbance by assay method described in embodiment 1, result is as shown in table 2, calculates general flavone content.
1.2 extraction times are on the impact of Herba Dianthi total flavones extraction ratio
Precision takes Aerial Parts of Chinese Pink Dianthus 3.0g, extractant concentration of alcohol is 70%, solid-liquid ratio is 1: 20, ultrasonic power 150W, select different extraction time 20,30,40,50,60min carries out ultrasonic extraction, measure absorbance by assay method described in embodiment 1, result is as shown in table 3, calculates general flavone content.
Table 2 concentration of alcohol affects data to Herba Dianthi total flavones extraction ratio
| Concentration of alcohol | 30% | 40% | 50% | 60% | 70% | 80% |
| A | 0.182 | 0.193 | 0.213 | 0.206 | 0.204 | 0.213 |
| A | 0.206 | 0.217 | 0.215 | 0.220 | 0.206 | 0.219 |
| Average A | 0.194 | 0.205 | 0.214 | 0.213 | 0.205 | 0.216 |
| Quality | 3.0011 | 3.0078 | 3.0336 | 3.0751 | 3.0042 | 3.0356 |
| Content mg/3g | 19.8666 | 20.9748 | 21.7314 | 21.3357 | 21.000 | 21.9247 |
Table 3 extraction time affects data to Herba Dianthi total flavones extraction ratio
| Time | 20 | 30 | 40 | 50 | 60 |
| A | 0.213 | 0.225 | 0.250 | 0.230 | 0.260 |
| A | 0.209 | 0.225 | 0.244 | 0.231 | 0.259 |
| Average A | 0.211 | 0.225 | 0.247 | 0.231 | 0.260 |
| Quality | 3.0541 | 3.0052 | 3.0082 | 3.167 | 3.0546 |
| Content mg/3g | 21.276 | 23.0901 | 25.3719 | 22.5075 | 26.3277 |
1.3 solid-liquid ratios are on the impact of Herba Dianthi total flavones extraction ratio
Accurately take Aerial Parts of Chinese Pink Dianthus 3.0g respectively, extractant concentration of alcohol is 70%, ultrasonic power 150W, select different solid-liquid ratios 1: 10,1: 15,1: 20,1: 25,1: 30, supersound extraction 40min, measure absorbance by assay method described in embodiment 1, result is as shown in table 4, calculates general flavone content.
Table 4 solid-liquid ratio affects data to Herba Dianthi total flavones extraction ratio
| Solid-liquid ratio | 1:10 | 1:15 | 1:20 | 1:25 | 1:30 |
| A | 0.225 | 0.231 | 0.237 | 0.243 | 0.230 |
| A | 0.221 | 0.230 | 0.229 | 0.244 | 0.229 |
| Average A | 0.223 | 0.231 | 0.233 | 0.244 | 0.230 |
| Quality | 3.0078 | 3.0050 | 3.0085 | 3.0028 | 3.0015 |
| Content mg/3g | 22.8606 | 23.721 | 23.9028 | 25.1025 | 23.6436 |
1.4 ultrasonic powers are on the impact of Herba Dianthi total flavones extraction ratio
Accurately take Aerial Parts of Chinese Pink Dianthus 3.0g respectively, extractant concentration of alcohol is 70%, solid-liquid ratio 1: 20, ultrasonic 40min, select different power 100 (40%), 125(50%), 150(60%), 200(80%), 250W(100%) carry out supersound extraction, measure absorbance by assay method described in embodiment 1, calculate general flavone content.
Table 5 ultrasonic power affects data to Herba Dianthi total flavones extraction ratio
| Power | 40 | 50 | 60 | 80 | 100 |
| A | 0.231 | 0.233 | 0.226 | 0.240 | 0.228 |
| A | 0.230 | 0.234 | 0.242 | 0.244 | 0.227 |
| Average A | 0.231 | 0.234 | 0.234 | 0.242 | 0.228 |
| Quality | 3.0043 | 3.0363 | 3.0008 | 3.0351 | 3.0080 |
| Content mg/3g | 23.7264 | 23.7879 | 24.0693 | 24.4827 | 23.3829 |
2, single factor test result and discussion
2.1 different ethanol concentration are on the result display of total flavones extraction effect impact in Herba Dianthi as Fig. 2, and concentration of alcohol is 30% ~ 50% time, and general flavone content increases with the increase of concentration of alcohol, and concentration of alcohol is 50% time, and general flavone content is maximum; When concentration of alcohol is more than 60%, general flavone content reduces with the increase of concentration of alcohol, and when it reaches 80%, flavones content increases suddenly, and may have impurity stripping, when concentration of alcohol is 40%, 50%, 60%, extraction effect is relatively good.Therefore, concentration of alcohol 40%, 50%, 60% is selected to do orthogonal test under ultrasound condition.
2.2 different times show as Fig. 3 the result that total flavones extraction effect affects, general flavone content increases with the increase of extraction time, the extraction initial stage, general flavone content increases very fast, but close to extraction terminal, flavone component diffusion is very slow, then increases suddenly again, impurity stripping may be had, thus select 30,40,50min does orthogonal test.
The result display of 2.3 different feed liquid comparison total flavones extraction effect impacts is as Fig. 4, and the content of total flavones increases with the increase of solid-liquid ratio and reduces again subsequently, 1: 25 time, reach maximum.Therefore solid-liquid ratio is selected to be 1: 15,1: 20,1: 25 do orthogonal test.
2.4 different capacities show as Fig. 5 the result that total flavones extraction effect affects, and with the increase of power, total flavones extraction effect increases, and when 200W, extraction effect is best.Afterwards, increase with power, extraction effect reduces on the contrary.So select power 125W, 150W, 200W to do orthogonal test.
3, orthogonal
According to above experiment of single factor result, adopt L9 (3
4) orthogonal table is with concentration of alcohol (A), ultrasonic time (B), solid-liquid ratio (C), ultrasonic power (D) 4 factors, to choose in each factor preferably 3 levels, concentration of alcohol (40,50,60) %, ultrasonic time (30,40,50) min, solid-liquid ratio (1: 15,1: 20,1: 25), ultrasonic power (125,150,200) W does orthogonal experiment, and each horizontal parallel does 3 parts, average, in table 6.
The factor level table of total flavones ultrasonic extracting process in table 6 Herba Dianthi
Orthogonal experiments and analysis result are in Table 7-8:
Total flavones ultrasonic extraction process Orthogonal experiment results in table 7 Herba Dianthi
Table 8 variance analysis
The R value size shown by table 7 can find out concentration of alcohol (A) having the greatest impact to total flavones extraction effect, ultrasonic power (D) takes second place, the impact of ultrasonic time (B) and solid-liquid ratio (C) is minimum, i.e. A > D > B > C.Optimum extraction process is combined as A
3b
3c
2d
1, namely extract the optimal extract process of total flavones in Herba Dianthi: concentration of alcohol 60%, ultrasonic time is 50min, and solid-liquid ratio is 1: 20, and ultrasonic power is 125W.
Embodiment 3
Series of process confirmatory experiment is carried out to the above-mentioned Herba Dianthi extracting flavonoids technique of employing:
1, precision is investigated
Precision measures same test liquid solution 6 parts, and every part of 2.0ml is placed in 25ml volumetric flask, develops the color by aforesaid coloration method, take corresponding reagent as blank, and measure absorbance in 510nm place, by regression equation calculation general flavone content, result is as shown in table 9.RSD is 0.29%, shows that precision is good.
Table 9 precision test data
2, study on the stability
Precision measures same need testing solution, develop the color by aforesaid coloration method, at 510nm place, measure absorbance 1 time every 5min, measure 60min, the absorbance of result display need testing solution in 30min is stablized, result is as shown in table 10, RSD is 3.52%, and after 30min, its absorbance declines obviously, shows need testing solution having good stability in 30min.
3, repeated experiment
Precision takes Aerial Parts of Chinese Pink Dianthus 3.0g, 6 parts, and by preparation and the coloration method of test liquid solution, measure absorbance at 510nm place, calculate its content, result is as shown in table 11, RSD%=1.21%, and result shows that the method repeatability is good.
4, average recovery experiment
Precision takes Aerial Parts of Chinese Pink Dianthus 1.5g, 6 parts, adds the control substance of Rutin suitable with test sample content respectively, and by preparation and the coloration method of test liquid solution, measure absorbance at 510nm place, calculate average recovery, result is as shown in table 12.
Table 10 stability experiment data
Table 11 repeated experiment data
Table 12 response rate experimental result
5, sample size measures
Precision takes Aerial Parts of Chinese Pink Dianthus 3.0g, 6 parts, to reflux 3h, under optimal extract process (concentration of alcohol 60%, ultrasonic time is 50min, and solid-liquid ratio is 1: 20, and ultrasonic power is 125W), prepare need testing solution with petroleum ether.Accurate absorption 2.0ml, is placed in 25ml volumetric flask, respectively adds 5%NaNO
2solution 1.0ml, mixing, places 6min, then adds 10%Al (NO
3)
31.0ml, mixing, places 6min, then adds 4%NaOH10ml, mixing, and adding distil water is diluted to 25ml, places 15min, measure absorbance, and by regression equation calculation general flavone content, result is as shown in table 13 in 510nm wavelength place.Result total flavones average content 0.6367%, RSD=1.21%.
Table 13 sample size determination data
In the present invention during sample ultrasonic extraction with petroleum ether process be in order to degreasing decoloring dewaxing, the extraction ratio of total flavones can be improved.General flavone content is selected to be evaluation index in experiment, draw the best ultrasonic extraction process of mongolian medicine Herba Dianthi, and adopt the total flavones in ultraviolet visible spectrophotometry mensuration Herba Dianthi, the general flavone content recorded in Herba Dianthi is 0.6367%, compare the extracting method in background technology, content almost exceeds 10 times, and its operational approach is simple, and stability, precision, repeatability, average recovery are all better.Ultrasonic extraction contributes to the extraction rate improving flavone, shortens extraction time, and save Extraction solvent, the method can be used as the method for mongolian medicine Herba Dianthi quality evaluation.
Claims (5)
1. from Herba Dianthi, extract a ultrasonic extracting method for flavone, comprising:
Take Aerial Parts of Chinese Pink Dianthus, add in round-bottomed flask, add petroleum ether, reflux 3 hours under 70 DEG C of water-baths, filter medicinal residues, volatilize solvent, be placed in conical flask, add 30-80% alcoholic solution, supersound extraction 20-60 minute, hyperacoustic power is 100-250W, sucking filtration after ultrasonic, after discarding medicinal residues, is transferred to filtrate in volumetric flask, with 70% ethanol standardize solution, adopt NaNO
2-Al (NO
3)
3-NaOH development process carries out full wavelength scanner, drawing standard curve, thus calculates general flavone content.
2. method according to claim 1, is characterized in that: the concentration adding ethanol before supersound extraction is 60%.
3. method according to claim 1, is characterized in that: ultrasonic time is 50 minutes.
4. method according to claim 1, is characterized in that: hyperacoustic power is 125W.
5. from Herba Dianthi, extract a ultrasonic extracting method for flavone, comprising:
Take Aerial Parts of Chinese Pink Dianthus 3g, add in round-bottomed flask, add petroleum ether 30ml, reflux 3 hours under 70 DEG C of water-baths, filter medicinal residues, volatilize solvent, add in 100ml conical flask, add alcoholic solution, supersound extraction, sucking filtration after ultrasonic, after discarding medicinal residues, filtrate is transferred in 100ml volumetric flask, with 70% ethanol standardize solution, adopts NaNO
2-Al (NO
3)
3-NaOH development process carries out full wavelength scanner, drawing standard curve, thus calculates general flavone content.
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| CN105287662A (en) * | 2015-09-15 | 2016-02-03 | 济宁医学院 | Ultrasonic-microwave extraction process for total flavonoids in leaves of Ilex cornuta Lindl.ex Paxt. |
| CN105412197B (en) * | 2015-12-28 | 2019-08-13 | 内蒙古医科大学 | Total alkaloid ultrasonic extracting method in anaesthetic dianthus superbus |
| CN108379327A (en) * | 2018-05-20 | 2018-08-10 | 内蒙古民族大学附属医院 | A kind of anaesthetic and its preparation process for treating nose-nasosinusitis |
| CN112263601A (en) * | 2020-11-25 | 2021-01-26 | 内蒙古自治区农牧业科学院 | Ultrasonic extraction process of artemisia rupestris total flavonoids |
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Inventor after: Chen Jianping Inventor after: Guo Huiqing Inventor after: Du Yanqing Inventor after: Wang Laibing Inventor after: Cheng Riqing Inventor after: Zhu Xiaowei Inventor after: Bao Baoquan Inventor before: Chen Jianping Inventor before: Buren Inventor before: Guo Huiqing Inventor before: Du Yanqing Inventor before: Tang Huaqi |
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Free format text: CORRECT: INVENTOR; FROM: CHEN JIANPING BU REN GUO HUIQING DU YANQING TANG HUAQI TO: CHEN JIANPING GUO HUIQING DU YANQING WANG LAIBING CHENG RIQING ZHU XIAOWEI BAO BAOQUAN |
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Granted publication date: 20150422 |
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| CF01 | Termination of patent right due to non-payment of annual fee |