Study on Ultrasonic and Microwave Assisted Extraction of Flavonoids From Clemati

Abstract: The paper selects the dry medical materials of Clematis huchouensis Tamura as the material, and researches the extracted technical parameters of flavonoid including liquid material proportion, microwave power, extraction time and extraction temperature. The single-factor experiment indicates that the liquid material proportion is 20:1, the microwave power is 120w, the extraction time is 30s and the temperature is 30℃, which has good extraction effect. The orthogonal design experiment indicates that the best extraction process is liquid-material ratio is 15:1, the microwave power is 160W, the time is 35s, the temperature is 35℃, and the extraction rate reaches 0.706mg/g.

Key words: Clematis huchouensis Tamura, flavonoid, ultrasonic microwave, orthogonal design

Clematis Huchouensis Tamura is also called Gold Scissors and Ranunculaceae Clematis Huchouensis, and it is the plant of Ranunculaceae clematis. It mainly grows in Huzhou of Zhejiang. It has the efficacy of anticancer, expelling wind-damp, detoxification and decreasing swelling and relieving pain. Using external application or decoction of medicinal ingredients to treat rheumatic arthritis, deep abscesses and brain tumor has a long history in folk [1, 2], and the efficacy is evident. But there is no agreement on effective materials especially anti-cancer?activity?components. The early research results indicate that cancer-inhibition active substances relate to flavonoid especially kaempferol.

The methods of extracting flavonoid of Clematis Huchouensis Tamura include Soxhlet extraction [3], ultrasound assisted extraction [4] and ultrasonic-assisted extraction [5]. The paper selects alcohol?as solvent, uses the ultrasound and ultrasonic-assisted collaboration method to extract flavonoid of Clematis Huchouensis Tamura, and observes the influence of liquid-material ratio, power, extraction time and temperature on extraction effect.

1.Materials

1.1Medical materials and reagent

The dry medical materials of Clematis Huchouensis Tamura are provided by Dadongwu Pharmaceutical Limited Company in Zhejiang, and are determined by Zhang Haiying in Huzhou Normal College. The standard substance of kaempferol is produced by Golden Biological Technology Co, Ltd in Shanghai, and the other reagents are analytical pure.

1.2 Instrument

Ultrasonic microwave collaboration extractor(CW-200)is produced by Xintuo Microwave Digestion Test Technology Co. Ltd, and spectrophotometer (UV1100-Ⅱ)shows the ultraviolet?and is produced by Tianmei Scientific Instrument Co. Ltd in Shanghai.

2Methods

2.1 Establishment of standard curve

2 mg of kaempferol standard substance is used, 30 mL of alcohol with 70%(V:V)is added and oscillated about 3 min for 3-4 times. It is placed for 15 min. And the constant volume is 100 mL, which prepares working mother liquor. 0.3, 0.6, 0.9, 1.2, 1.5 and 1.8 mL of mother liquor are taken, and 70% of alcohol is added until the total volume is 3mL.

70% of alcohol is blank control. Standard solution of kaempferol is scanned in the range of 200-580 nm of wavelength. The maximal absorption?peak wavelength is determined. Under the wavelength, the absorbance of six standard solutions with different concentrations is determined. The absorbance is used as the vertical coordinate, and the concentration is used as the horizontal coordinate to draw standard curve.

2.2 Extraction technology of flavonoid

The dry medical material of Clematis Huchouensis Tamura is dried for 12 hours under the temperature 60 ℃ and is smashed. It is screened for 40 times for preparation.

Extraction process is as follows. 1.000 g of Clematis Huchouensis Tamura powder is selected and put into the beaker. 70% of alcohol solution is added and is transferred into the triangular flask matched with ultrasonic microwave extractor. 40Hz ultrasonic wave and microwave with power is extracted collaboratively. Three triplicates of 0.5 mL of filtrate are selected. 2.5mL of 70% alcohol is added. The absorbance is measured, and the average value is computed. The formula, concentration (mg/mL) × dilution?ratio × volume of extracting solution (mL), is used to compute the production.

2.2.1 Single-factor experiment

2.2.1.1 Influence of liquid-material ratio

Under the condition that there are 4 triplicates of Clematis Huchouensis Tamura powder, 10mL, 20mL, 40mL and 60mL of alcohol solution is added, the microwave power is 120W, the microwave extraction time is 20s, and the temperature is 30℃, the extraction is made in ultrasonic-microwave collaboration extractor.

2.2.1.2 Influence of ultrasonic power

Under the condition that there are 4 triplicates of Clematis Huchouensis Tamura powder, 20mL of alcohol solution is added, the extraction time is 20s, the temperature is 30℃, and the microwave powers are 40W, 120W, 200W and 280W, the extraction is made in ultrasonic-microwave collaboration extractor.

2.2.1.3 Influence of extraction time

Under the condition that there are 4 triplicates of Clematis Huchouensis Tamura powder, 20mL of alcohol solution is added, the microwave power is 120W, the temperature is 30℃, and the extraction time is 10s, 35s, 60s and 85, the extraction is made in ultrasonic-microwave collaboration extractor.

2.2.1.4 Influence of extraction temperature

Under the condition that there are 4 triplicates of Clematis Huchouensis Tamura powder, 20mL of alcohol solution is added, the microwave power is 120W,the microwave extraction time is 20s and the temperature is 20℃,30℃,40℃ and 50℃, the extraction is made in ultrasonic-microwave collaboration extractor.

2.2.2 Orthogonal design experiment

Liquid-material ratio, microwave power, extraction time and extraction temperature are selected for orthogonal experiment. Each factor is divided into three levels. Orthogonal design factors and level table is shown in Table 1, and the orthogonal design experiment table is shown in Table 2.

2.2.3 Verification of extraction technology

The best optimization scheme which is achieved according to orthogonal design analysis receives three parallel tests.

3 Results and Analysis

3.1 Establishment of standard curve

After the spectrophotometer scans, in the range of 300-560nm of wavelength, the maximum absorption wavelength of kaempferol standard solution is 370.5nm. And the standard curve is shown in Figure 1. The regression analysis indicates that linear equation is y=119.1429x-0.015667(R2 =0.999535).

Figure 1 Standard curve of kaempferol standard solution

3.2 Extraction technology

3.2.1 Single-factor experiment

3.2.1.1 Influence of liquid-material ratio

Under the condition that the liquid-material ratio is 10:1, 20:1, 40:1 and 60:1, the extraction volume of flavonoid for each gram of medical material is 0.3604mg/g, 0.6654mg/g, 0.672mg/g and 0.6800mg/g. In view of the cost factor, the liquid-material ratio 20:1 is suitable.

3.2.1.2 Influence of microwave power

Under the condition that the microwave power is 40W, 120W, 200W and 280W, the extraction volume of flavonoid for each gram of medical material is 0.4640mg/g, 0.5556mg/g, 0.3834mg/ and 0.3300mg/g. When the power is 120W, the extraction effect is better.

3.2.1.3 Influence of extraction time

Under the condition that the extraction time is 10s, 35s, 60s and 85s, the extraction volume of flavonoid for each gram of medical material is 0.4730mg/g, 0.5959mg/g, 0.5153mg/g and 0.4690mg/g. When the extraction time is 35s, the effect is better.

3.2.1.4 Influence of temperature

Under the condition that the temperature is 20℃, 30℃, 40℃ and 50℃, the extraction volume of flavonoid for each gram of medical material is 0.4146mg/g, 0.6654mg/g, 0.4871mg/g and 0.4136mg/g. When the temperature is 30℃, the effect is the best.

3.2.2 Orthogonal design experiment results

Orthogonal design experiment results are shown in Table 3. From range analysis, we can see that the order of factor influence is microwave power (B)> temperature(D) > liquid-material ratio(A)> time(C). The best extraction technology is A1B3C2D3, which means that the liquid-material ratio is 15:1, the microwave power is 160W, the time is 35s and the temperature is 35℃.

3.2.3 Verification results of orthogonal design

Under the condition of the best extraction technology A1B3C2D3 which means that the liquid-material ratio is 15:1, the microwave power is 160W, the time is 35s and the temperature is 35℃, the extraction volume of three parallel experiments is 0.701mg, 0.711mg and 0.706mg, and the average extraction volume is 0.706mg.

The spectrophotometric method is used to quantify the total volume of flavonoids. The establishment of standard curve can use quercetion and rutin. The objective of the research is to provide basis for extraction technology of kaempferol, so kaempferol is used as quantitative reference substance. Single-factor experiment results indicate that when liquid-material ratio is greater than 20:1, the increase of extraction rate is not evident. The extraction with too low microwave power is not evident. But too large microwave power, too high extraction temperature and too long extraction time may increase the degradation of flavonoids, and the extraction effect is not good. Orthogonal design experiment results indicate that the best parameter of influencing factors including microwave power, extraction time and extraction temperature is consistent with single-factor experiment results. But the best extraction liquid-material ration is 15:1, which is lower than 20:1 of single factor. And the reasons need to be researched.

Reference

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