Study on the Extraction Techniques of Impatiens balsamina (Tougucao)

Lida CHEN, Yuanzhe ZHANG, Yuling LUO, Xianmei XU, Hai ZHAO, Yan WU

Abstract [Objectives] This study was conducted to investigate the extraction method of Impatiens balsamina L. （Tougucao）， and to optimize the extraction process conditions and parameters.

[Methods]Different ethanol concentrations， ethanol amounts and extraction time were investigated by an orthogonal test L9（34） and high performance liquid chromatography （HPLC）， and the extraction process parameters were evaluated by the yield of extract and the content of quercetin.

[Results] Under the three optimal extraction conditions of impregnation， heating reflux and ultrasound， the extract yield and quercetin contents of I. balsamina were 8.27%， 9.50%， 7.09% and 1.60， 3.05， 3.03 μg/g， respectively. Heating reflux extraction and ultrasonic extraction showed higher extract yields and quercetin contents of I. balsamina， while the values were lower under the impregnation extraction conditions. The primary and secondary factors were： ethanol amount （C）>ethanol concentration （A）>extraction time （B）. The optimal extraction process of I. balsamina was as follows： adding 10 times of 60% ethanol and ultrasonically extracting for 30 min.

[Conclusions]The method is simple， convenient， accurate and reliable， and provides a scientific basis for the research on the extraction process of I. balsamina and its preparations.

Key words Impatiens balsamina; Impregnation; Heating reflux; Ultrasonic extraction; Orthogonal test; HPLC; Quercetin

Received： March 2， 2022  Accepted： May 20， 2022

Supported by Scientific Research Project of Guizhou University of Traditional Chinese Medicine （[2019]64）.

Lida CHEN （1982-）， male， P. R. China， pharmacist-in-charge， devoted to research about hospital preparation processes and quality.

*Corresponding author. E-mail： 357984356@qq.com.

Impatiens balsamina L. of Balsaminaceae is harvested in summer and autumn， and the roots， leaves， flowers and fruit are removed to obtain dry stems， which is called Tougucao. The medicinal material is pungent， warm， and slightly poisonous， and attributive to the liver and kidney meridians. It has the functions of dispelling wind-damp， promoting blood circulation， and relieving pain， and is used for treating rheumatic arthralgia， contracture of muscles and bones， cold and dampness of limbs， sore， ringworm， swelling and pyogenic infections[1]. Due to the unique curative effect of Tougucao， it is widely used in clinical practice. Tougucao has the main pharmacological activity of anti-allergic， anti-inflammatory and anti-fungal[2-7]. Since it contains complex active components， it is not included in the Chinese Pharmacopoeia at the moment， and it is only recorded in the local standards of Hubei， Liaoning， Yunnan， Guangxi， and other provinces. According to the relevant literature， most of the extraction methods of Tougucao use the extract as the investigation index[8-9]. In this study， ethanol extract and the content of quercetin in I. balsamina determined by high performance liquid chromatography were used as indexes for extraction. The method is simple， accurate and reliable， and provides a scientific basis for the research on the extraction process of I. balsamina and its preparations.

Experimental materials and instruments

Experimental materials

I. balsamina （Guiyang Jirentang Chinese Herbal Pieces Factory， batch number： 20220301）; anhydrous ethanol （Chongqing Wansheng Chuandong Chemical Co.， Ltd.， batch number： 20200801）; methanol （Chongqing Wansheng Chuandong Chemical Co.， Ltd.， batch number： 20211101）; phosphoric acid （Sinopharm Chemical Reagent Co.， Ltd.， batch number： 20210425）; hydrochloric acid （Chemical Reagent Factory of Chongqing Chuandong Chemical （Group） Co.， Ltd.， batch number： 20210601）; chromatographic methanol （Anhui Tiandi High Purity Solvents Co.， Ltd.， batch number： 2112530）.

Experimental instruments

Brava high performance liquid chromatograph （Panna （Changzhou） Instruments Co.， Ltd.）; BHS-6 digital constant temperature water bath （Joan Lab Equipment Co.， Ltd.）; AUW220D analytical balance （Shimadzu）; SK5210HP ultrasonic cleaner （Shanghai Kudos Ultrasound Instrument Co.， Ltd.）; 101-2AB type electric heating blast drying oven （Tianjin Taisite Instrument Co.， Ltd.）.

Methods and Results

Impregnation extraction

First， 20 g of I. balsamina coarse powder was accurately weighed， and put in a 250 ml conical flask. An L9（34） orthogonal test was designed to investigate the influence factors the extraction process of I. balsamina， including of ethanol concentration （60%， 70% and 80%）， impregnation time （12， 24 and 48 h） and ethanol amount （6， 8 and 10 times）， with the extract yield and quercetin content as index components.

Extract yield

Each filtrate was placed in an evaporating dish with constant weight， evaporated to dryness in a water bath， and dried at 80 ℃. The yield of extract was determined according to General Examples： Accuracy， Constant Weight in volume 1 of Chinese Pharmacopoeia （2020 edition）.

Content determination

Chromatographic conditions and system suitability test

The chromatographic conditions[10-12] were as follows： packed column： octadecyl silane chemically bonded silica， mobile phase： methanol-0.4% phosphoric acid solution （50∶50）， column temperature： 30 ℃， flow rate： 1.0 ml/min， and injection volume： 20 μl. The detection wavelength was 360 nm. The number of theoretical plates should not be less than 2 000 according to the quercetin peak.

Solution preparation

An appropriate amount of quercetin reference substance was accurately weighed， and added with 80% methanol to make a 20 μg/ml solution， that is， the reference substance solution.

The above solid was accurately weighed and added with 15 ml of 80% methanol for dissolution. The obtained liquid was transferred to a 25 ml volumetric flask， added with 7 ml of 25% hydrochloric acid solution， and hydrolyzed in a water bath at 85 ℃ for 30 min. It was cooled rapidly after being taken out， and then added with 80% methanol to the mark and shaken uniformly. The filtrate obtained after filtration served as the test solution.

Results and Analysis

Impregnation extraction

According to "Impregnation extraction"， impregnation investigation was carried out on I. balsamina. The results are shown in Table 1-Table 4.

Specificity test

From each of the reference solution， sample solution and blank solvent， 20 μl was precisely drawn and injected for determination according to the proposed chromatographic conditions. In the chromatogram of the test solution， there was a corresponding chromatographic peak at the corresponding retention time with the reference solution chromatogram， but the blank solvent had no corresponding chromatographic peak， indicating that the blank solvent did not interfere with the sample determination （Fig.1）. The resolution between the target peak and adjacent chromatographic peaks was >1.5.

Orthogonal test results

Based on the orthogonal test results of the extraction process L9（34）， the range analysis of the extract yield results reflected the primary and secondary relationship of the influencing factors： C>A>B， and the range analysis of the quercetin content determination results reflected the primary and secondary relationship of the influencing factors： A>C>B. The results of variance analysis of extract yield showed that there were no significant differences between the influencing factors of ethanol concentration A （60%， 70% and 80%） and extraction time B （12， 24 and 48 h）， while the amount of ethanol C （6， 8 and 10 times） showed significant differences. The analysis of variance for the determination of quercetin content showed that the influencing factors of ethanol concentration A （60%， 70% and 80%）， impregnation time B （12， 24 and 48 h） and ethanol amount （6， 8 and 10 times） resulted in no significant differences. In this experiment， under the condition of C3B3A1， the extract yield and quercetin content were the highest.

Heating reflux extraction

Sample preparation

Referring to "Impregnation extraction"， 20 g of coarse I. balsamina powder was accurately weighed， and according to the L9（34） orthogonal test on the influence factors ofthe extraction process of I. balsamina， i.e.， the ethanol concentration （60%， 70%， 80%）， heating and refluxing time （30， 60 and 90 min） and the amount of ethanol （6， 8 and 10 times）， extraction was performed under heating and refluxing， with the extract yield and quercetin content as index components. The results are shown in Table 5-Table 8.

Results of reflux extraction

Based on the orthogonal test results of the reflux extraction process L9（34）， the range analysis of extract yield results reflected the primary and secondary relationship of the influencing factors： C>A>B， and the range analysis of the quercetin content determination results reflected the primary and secondary influencing factors： A>B>C. The results of variance analysis of extract yield showed that there were no significant differences between the influencing factors of ethanol concentration A （60%， 70% and 80%） and extraction time B （30， 60 and 90 min）， while the amount of ethanol C （6， 8 and 10 times） showed significant differences. The analysis of variance for the determination of quercetin content showed that the influencing factors of ethanol concentration A （60%， 70% and 80%）， impregnation time B （30， 60 and 90 min） and ethanol content （6， 8 and 10 times） all did not caused a significant difference. In this experiment， under the condition of C3B3A1， the yield of extract was the highest， and under the condition of C3B2A2， the content of quercetin was the highest.

Ultrasonic extraction

Sample preparation

Referring to "Impregnation extraction"， 20 g of coarse I. balsamina powder was accurately weighed， and according to the L9（34） orthogonal test on the influence factors of the extraction process of I. balsamina， i.e.， ethanol concentration， heating reflux time and the amount of ethanol， ultrasonic extraction was performed， with the extract yield and quercetin content as index components. The results are shown in Table 9-Table 12.

Ultrasonic extraction results

Based on the orthogonal test results of the ultrasonic extraction process L9（34）， the range analysisof extract yield results reflected the primary and secondary relationship of the influencing factors： C>A>B， and the range analysis of the quercetin content determination results reflected the primary and secondary influencing factors： A>B>C. The analysis of variance for the extract yield and quercetin content determination results showed that the influencing factors， ethanol concentration A （60%， 70%， 80%）， ultrasonic time B （30， 60 and 90 min） and ethanol amount C （6， 8 and 10 times）， all did not exhibited significant differences. In this experiment， under the condition of C3B3A1， the extract yield and quercetin content were the highest.

Conclusions and Discussion

According to the local standards， there are differences in the genera and species of Phrymaceae included in the local standards. In Guizhou Province， Tougucao mainly refers to the dried stems and branches of I. balsamina in Balsaminaceae， which is widely used in rheumatic diseases， and the extraction methods are impregnation mostly. Therefore， three extraction methods of impregnation， heating and refluxing and ultrasonic extraction were set up in this study for comparison and different levels of influencing factors， ethanol concentration （60%， 70% and 80%）， impregnation （ultrasonic/heating reflux） time （30， 60 and 90 min/12， 24 and 48 h） and ethanol amount （6， 8 and 10 times） were screened， with the combination of extract yield and quercetin content determination as index components. The results showed that among the three extraction methods， heating reflux extraction and ultrasonic extraction had higher contents of extract and quercetin， while impregnation resulted in lower extract and quercetin contents. Meanwhile， among the three extraction processes， the amount of solvent added had the greatest effects on the extract yield and quercetin content， followed by ethanol concentration， and extraction time had the least effect. Therefore， it was finally determined that the extraction process of I. balsamina was as follows： adding 10 times of 60% ethanol， and ultrasonically extracting for 30 min. This method is simple and convenient to operate.

For the determination of quercetin content， it is difficult for general sample solution preparation methods to reach the detection limit of high performance liquid chromatography （PHLC） due to the low content of quercetin. Therefore， the sampling volume of I. balsamina was changed to 20 g， and the filtrate obtained after extraction was directly prepared into a dry extract， which was then prepared into the test solution. Meanwhile， the injection volume was increased from 10 to 20 μl.

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