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1. Panzhihua University, Panzhihua 617000, China; 2. Panzhihua Tri-Doc Technology Co., Ltd., Panzhihua 617000, China

Abstract　[Objectives] To optimize the ethyl acetate impurity removal method for extracting and isolating mangiferin from mango leaves, and provide raw materials and technical support for development and use of mangiferin related products. [Methods] Five steps (material crushing → ethyl acetate impurity removing → concentrated extract washing → extracting with methanol → crystallization and precipitation) were used. The single factor experiment and L9(34) orthogonal experiment was carried out to optimize the process parameters including extraction time, ultrasonic power, extraction times, and extraction temperature.[Results] The optimum process of ethyl acetate impurity removal method for extracting and isolating mangiferin from mango leaves was as follows: the mango leaves were crushed and sieved; 3 mL/g of ethyl acetate was added, sealed and soaked for 4 h, ultrasonically shaken for 20 min (50℃, 350 W), filtered at room temperature, filtered with 100 mesh sieve, and extracted three times; added 100% methanol to the residue at 3 mL/g, extract by ultrasonic vibration for 20 min (350 W, 55℃) for four times, filtered with 100 mesh sieve when it was still hot; mixed the extract of each time, condensed by vacuum decompression to get the extract; added 100% methanol at 4 mL/g, mixed and washed for 5 min at room temperature, placed for 10 min, filtered with 100 mesh sieve, washed 3 times repeatedly, and dried the filter residue at 60℃ to obtain the crude mangiferin; added 100% methanol at 4 mL/g, mixed and washed at 50℃ for 5 min, placed at 6℃ for 8 h, dried the filter residue at 60℃, and repeatedly crystallized two times. According to the above process, crude and pure mangiferin products could be obtained, the purity of mangiferin of the crude product was higher than 64.00%, the total recovery rate was 83.90%, and the purity of mangiferin of the pure product was higher than 98.00%, and the total recovery rate was about 66.40%. [Conclusions] The optimized ethyl acetate impurity removal method is easy in operation, low in cost, and high in efficiency for extracting and isolating mangiferin, and can be applied for actual production of mangiferin.

Key words　Mangiferin, Mango leaves, Extraction, Isolation, Ethyl acetate impurity removal method

1　Introduction

Mangiferin is a xanthonoid, with molecular formula of C19H18O11. It has pharmacological effects of anti-oxidation, anti-inflammation, hypoglycemic, uric acid lowering, anti-virus, anti-tumor, removing phlegm and relieving cough[1-3]. Mangiferin is a medicinal component and quality indicator control component of many kinds of traditional Chinese medicines in Chinese Pharmacopoeia (2015), and has extremely high medicinal value. In recent years, the pharmacological mechanism and safety evaluation of mangiferin has become a hot topic in China[4-11], showing excellent development and utilization prospect of mangiferin. By now, mango leaf is the plant resource with the highest content of mangiferin[12-13]. It is of great significance for the efficient extraction of mangiferin from mango leaves in reducing mangiferin production costs, promoting the development of new mangiferin products, and increasing the economic benefits of mango industry.

Domestic studies about the extraction and isolation of mangiferin from mango leaves were concentrated in 2008-2014, while there were few studies about extraction process of mangiferin. Deng Jiagangetal.[14]used high-concentration ethanol for reflux extraction and used D101 and D296 macroporous resins for column chromatography to obtain pure mangiferin products. Li Xuejianetal.[15-16]optimized the technology of Deng Jiagang, after extracting mangiferin with weakly alkaline water, used D101 and D301 macroporous resins, and obtained pure products with purity of 93.96% and yield rate of 65.64%. Using ethanol microwave extraction method, Xie Liyaetal.[17]extracted mangiferin from mango leaves, indicating that microwave extraction has certain advantages over traditional reflux extraction. After ethanol heating reflux, Liu Haoetal. used AB-8 macroporous resins for column chromatography to isolate mangiferin, and the yield rate was 54.6%. Using 50% ethanol as the solvent by ultrasonic extraction method, Deng Xingyun[4]extracted and isolated mangiferin from the leaves ofAquilariasinensis, used AB-8 macroporous resin for column chromatography, and obtained mangiferin with purity of 80%.

From the point of view of the existing technology, no matter which method is applied for extract mangiferin, it is necessary to go through a complicated column chromatography procedure to obtain pure mangiferin. In addition, due to the extremely high colloidal content in the mango leaves, the concentrated solution after extraction is very viscous, and it is very easy to block the column or filter device, thus it is extremely difficult for subsequent isolation operation. This fundamentally leads to the complex, low efficiency and high cost of existing mangiferin extraction and isolation process. Therefore, it is necessary to improve the existing extraction and isolation process of mangiferin in order to greatly increase the extraction and isolation efficiency and production cost of mangiferin.

On the basis of fully understanding the variation of mango content in mango leaves, after removing most of the colloid from mango leaves with ethyl acetate, we used the solubility difference of mangiferin in hot and cold methanol to extract and isolate mangiferin. This greatly simplified the process steps, increased the product purity and yield, and greatly reduced the production cost of mangiferin.

2　Materials and methods

2.1ExperimentalmaterialsThe experimental mango leaves were collected from a farmer’s mango orchard in Renhe District, Panzhihua City of Sichuan Province, naturally dried, crushed and screened with a 20-mesh sieve. The mangiferin standard substance (batch No.100268-200401, purity 98.2%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products. The mobile phase of high performance liquid chromatograp (HPLC) used the acetonitrile as chromatographically reagent (CR), water was redistilled water, and other reagents were analytical reagents (AR). Main equipments: high rotated speed disintegrator FW100 (Tianjin Taisite Instrument Co., Ltd.), YM-2000CT multi-purpose constant temperature ultrasonic extractor (Shanghai Yumin Instrument Co., Ltd.), R-1005Ex vacuum rotary evaporator (Zhengzhou Great Wall Branch Industry & Trade Co., Ltd.), Agilent 1200 HPLC and ChemStation (Agilent Technologies, Inc.), XS205DU electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd. and electrothermal thermostatic blast dryer (Shanghai Langgan Laboratory Equipment Co., Ltd.).

2.2DeterminationofmangiferininmangoWith reference to the standard curve method of Wei Huipingetal.[18], we precisely determined the content of mangosteen in mango leaves. Eclipse XDB-C18chromatographic column (4.6 mm×150 mm, 5 μm); mobile phase adopted acetonitrile and 0.2% glacial acetic acid solution (15/85,v/v); detection wavelength of 258 nm; column temperature was room temperature; sample injection volume was 20 μL.

2.3Optimizationofmangiferinextractionprocess

2.3.1Selection of extracting solvent. Precisely weighed 100 g of mango leaf powder (six pieces), and added 300 mL of six kinds of extraction solvents[alkaline water (NaOH solution with pH 9), 100% methanol, 50% methanol, anhydrous ethanol, 50% ethanol, and ethyl acetate]. After soaking at room temperature for 8 h, performed the ultrasonic extraction for 30 min (50℃, 350 W), filtered with 100-mesh sieve, repeatedly extracted five times, combined the filtrate together, decompressed and concentrated in vacuum to obtain the concentrated extract, weighed, sampled and measured the purity and extraction rate of mangiferin (the mass percentage of product obtained through extraction to the content of mangiferin in the material before extraction), so as to determine the optimum extraction solvent.

2.3.2Optimization of impurity removal process. According to the experimental results of Section2.3.1, we selected the solvent with lower purity and extraction rate as the impurity removal solvent, to remove the impurities from mango leaves, and optimize the removal process. Optimization of extraction times: weighed 100 g of mango leaves, added 300 mL of impurity removal solvent, sealed and soaked at room temperature for 4 h, performed ultrasonic vibration extraction for 30 min (50℃, 350 W), filtered with 100-mesh sieve, repeatedly extracted five times, combined the filtrate together, decompressed and concentrated in vacuum to obtain the concentrated extract, weighed, measured the mangiferin content (the loss of mangiferin), and calculated the loss rate of mangiferin in each time of extraction (the percentage of loss of mangiferin in each time of extraction to total mangiferin before extraction), to determine the optimum extraction times. Optimization of extraction time: weighed 100 g of mango leaf powder (three pieces), added 300 mL of impurity removal solvent, sealed and soaked at room temperature for 4 h, performed ultrasonic vibration extraction for 30 min (50℃, 350W), filtered with 100-mesh sieve, extracted three times respectively, each with extraction time of 10, 20, and 30 min, combined the filtrate together, decompressed and concentrated in vacuum to obtain the concentrated extract, weighed, measured the mangiferin content (the loss of mangiferin), and calculated the loss rate of mangiferin in each time of extraction, to determine the optimum extraction time.

2.3.3Optimization of extraction process. Weighed 100 g of mango leaves, removed the impurities in accordance with the impurity removal process in Section2.3.1, took the residue, added the optimum extraction solvent to perform the ultrasonic vibration extraction, added 300 mL of optimum extraction solvent each time of extraction, after extraction, filtered with 100-mesh sieve when it was still hot, combined the filtrate together, decompressed and concentrated in vacuum to obtain the concentrated extract, weighed, and calculated the purity and extraction rate of mangiferin. Under the above conditions, taking the extraction time (A), ultrasonic power (B), extraction times (C), and extraction temperature (D) as factors, and extraction rate as indicator, we designed the L9(34) orthogonal experiment, determined the optimum extraction process conditions, and carried out three times of verification tests (Table 1).

Table 1　Design of orthogonal experiment

2.4Optimizationofconcentratedextractwashingprocess

2.4.1Selection of washing solvent. Weighed 10 g of concentrated extract (four pieces in total) obtained in Section2.3.3, added four kinds of 40 mL washing solvents (100% methanol, anhydrous ethanol, distilled water, and ethyl acetate) separately, mixed and washed for five min at room temperature, placed for 10 min, filtered with 100-mesh sieve, repeatedly washed five times, dried the filtrate residue at 60℃ to obtain the crude mangiferin, weighed, and calculated the purity and recovery rate of mangiferin (the mass percentage of crude mangiferin to mangiferin content in the concentrated extract before washing), to determine the optimum washing solvent.

2.4.2Optimization of washing times. Weighed 10 g of concentrated extract (four pieces in total) obtained in Section2.3.3, added the optimum washing solvent and washed by the method in Section2.4.1for five times, sampled the filtrate of each washing time, measured the mangiferin content, decompressed and concentrated the filtrate of each washing time to obtain the concentrated extract, weighed, and calculated the mass of impurities and loss rate of mangiferin in each washing time (the mass percentage of washing solvent to the mangiferin content in concentrated extract), to determine the optimum washing times.

2.5Optimizationofcrystallizationprocess

2.5.1Selection of crystallization solvent. Weighed 5 g of crude mangiferin (five pieces) obtained by the optimum extraction conditions in Section2.4, added 20 mL of five kinds of crystallization solvents (100% methanol, 50% methanol, anhydrous ethanol, 50% ethanol, and distilled water) respectively, mixed and washed at 50℃ for 5 min, placed at 6℃ for 8 h, took out and filtered when it was still cool, dried the filtered residue at 60℃ to obtain the crystallized product, weighed, and calculated the purity and recovery rate of mangiferin in crystallized product (mass percentage of mangiferin in the product before and after crystallization).

2.5.2Optimization of crystallization times. Weighed 5 g of crude mangiferin obtained by the optimum extraction conditions in Section2.4, added 20 mL of 100% methanol, treated by the method in Section2.5.1, repeatedly crystallized three times, and calculated the purity and recovery rate of the crystallized product in each time of crystallization.

2.6StatisticalanalysisWith the aid of SPSS19.0, by LSD method, we carried out the difference significance analysis of the experimental data.

3　Results and analysis

3.1DeterminationofmangiferininmangoleavesBy the method in Section2.2, we obtained that the mangiferin in mango leaves in this experiment was 2.61%.

3.2EffectsofextractionsolventonthepurityandextractionrateofmangiferinAccording to Table 2, mangiferin can be extracted from mango leaves using alkaline water, 100% methanol, 50% methanol, anhydrous ethanol, and 50% ethanol. Specifically, the extraction rate of alkaline water was as high as 92.3%, which was significantly higher than other extraction solvents (P<0.05, the same as below), but the purity of extracted mangiferin was low, only 7.60%. The purity of mangiferin extracted by 100% methanol and 50% ethanol was significantly higher than that of other extraction solvents, and the extraction rate was significantly higher than other extraction solvents except alkali water. Although there was no significant difference between the extraction rate and mangiferin purity (P>0.05, the same as below), the extract obtained by 50% ethanol was more viscous, and the energy consumption during the concentration process was greater. Therefore, 100% methanol as the extraction solvent was better than 50% ethanol, 100% methanol was determined as the optimum extraction solvent. In addition, the mass of extract obtained from ethyl acetate as the extraction solvent was up to 20.1 g, only second to alkaline water (31.7 g), but the purity and extraction rate of mangiferin were significantly lower than other extraction solvents, this characteristic could be used to remove impurities in mango leaves.

Table 2　Effects of extraction solvent on the purity and extraction rate of mangiferin

Note: Different small letters in the same column of data denote significant differences (P<0.05), the same in Table 5 and Table 6.

3.3EffectsofextractiontimesandextractiontimeonimpurityremovalAccording to the above experimental results, ethyl acetate was used to remove some of the impurities in the mango leaves. According to Table 3, in the process of extracting impurities by ethyl acetate, the mass of the removed impurities decreased sharply with the increase in the number of extraction times, and the total mass of impurities removed in the first three times accounted for more than 90% of the total mass of impurities removed in all five times, the loss of mangiferin in each time of extraction was about 25.0 mg, with the loss rate of about 1%. Through overall consideration, it would be better to set the impurity removal times at three times. From Table 4, it can be seen that compared with the extraction time of 20 min, there was no significant increase in the mass of impurities removed in the extraction time of 30 min, but the loss of mangiferin was significantly increased. Thus, the extraction time of 20 min was optimum.

Table 3　Effects of extraction times on impurity removal

Table 4　Effects of extraction time on impurity removal

Note: Different small letters in the same line of data denote significant differences (P<0.05), the same in Table 8 and Table 9.

3.4ResultsoforthogonalexperimentAs indicated in Table 5, the extraction time, ultrasonic power, extraction times, and extraction temperature had a significant effect on the extraction rate, and the order of influence was extraction temperature > extraction times > ultrasonic power > extraction time. The extraction temperature exerts the greatest effect on the extraction rate, the higher the extraction temperature, the higher the extraction rate, but the methanol boiling point is low and the volatility is large. From the viewpoint of production safety, the extraction temperature should be not higher than 55℃; for other three factors, level 1 was significantly lower than level 2 and level 3, and there was no significant difference between level 2 and level 3. From the factors of time-consuming, cost, and safety, the optimum solution is A2B2C2D3, which means that the extraction time is 20 min, the ultrasonic power is 350 W, the number of extraction times is four and the extraction temperature is 55℃. Results of three verification tests showed that the average extraction rate was 95.1% and the mangiferin purity was as high as 25.50%.

3.5EffectsofwashingsolventonthepurityandextractionrateofmangiferinAccording to Table 6, there was a significant difference in the purity and recovery rate of mangiferin in the crude product obtained from different washing solvents, the purity of mangiferin obtained from 100% methanol washing was significantly higher than that of other solvents, up to 64.50%, and the recovery rate was up to 85.20%. Therefore, 100% methanol was the optimum washing solvent.

3.6EffectsofwashingtimesonthepurityandextractionrateofmangiferinAs shown in Table 7, with the increase in the washing times, the mass of impurities washed away each time sharply decreased, and the total mass of impurities washed away in the first three times accounted for more than 95% of the total mass of impurities washed out in all five times; the loss of mangiferin in each time of washing was about 76.0 mg, and the loss rate was about 3%. Through overall consideration, the optimum washing times was three times, more than 60% impurities of extract could be washed away, the loss rate of mangiferin was about 9%, and the purity of mangiferin was higher than 64.00%.

Table 5　Results of orthogonal experiment

Table 6　Effects of washing solvent on the purity and extraction rate of mangiferin　(%)

3.7EffectsofcrystallizationsolventonthepurityandextractionrateofmangiferinAccording to Table 8, the purity of mangiferin using 100% methanol as the crystallization solvent was significantly higher than that of other solvents, and the recovery rate was significantly higher than other three solvents except distilled water, but the mangiferin purity of the product obtained with distilled water as the crystallization solvent was the lowest. Through overall consideration, 100% methanol was used as the optimum crystallization solvent.

Table 7　Effects of washing times on the purity and extraction rate of mangiferin

Table 8　Effects of crystallization solvent on the purity and extraction rate of mangiferin　(%)

3.8EffectsofcrystallizationtimesonthepurityandextractionrateofmangiferinAccording to Table 9, with the increase in crystallization times, the purity of mangiferin of the product gradually increased, but after the crystallization times exceeded two times, the increase in the purity of mangiferin of the product was not significant. Considering that with the increase in the crystallization times, the loss of mangiferin was higher. Therefore, two crystallization times was optimum. At this time, the mangiferin purity of the product was 98.23%, and the recovery rate was up to 88.13%.

Table 9　Effects of crystallization times on the purity and extraction rate of mangiferin　(%)

3.9ResultsoftheverificationtestIn summary, the optimum process for extracting and isolating mangiferin from mango leaves was as follows. (i) Crushing of raw material: mango leaves were dried, crushed and screened with 20-mesh sieve. (ii) Ethyl acetate impurity removal: added ethyl acetate in mango leaves at the rate of 3 mL/g, sealed and soaked for 4 h, extract by ultrasonic vibration for 20 min (50℃, 350 W), filtered with 100-mesh sieve, and repeatedly extracted three times to obtain residue of mango leaves. (iii) Methanol extraction: added 100% methanol to the residue at 3 mL/g, extract by ultrasonic vibration for 20 min (350 W, 55℃) for four times, filtered with 100 mesh sieve when it was still hot, then mixed the extract of each time, condensed by vacuum decompression to get the extract. (iv) Washing of concentrated extract: added 100% methanol at 4 mL/g, mixed and washed for 5 min at room temperature, placed for 10 min, filtered with 100 mesh sieve, washed three times repeatedly, and dried the residue at 60℃ to obtain the crude mangiferin. (v) Crystallization and drying: added 100% methanol at 4 mL/g in the crude mangiferin, mixed and washed at 50℃ for 5 min, placed at 6℃ for 8 h, dried the filter residue at 60℃, and repeatedly crystallized two times. According to the above process, crude and pure mangiferin products could be obtained, the purity of mangiferin of the crude product was higher than 64.00%, the total recovery rate was 83.90%, and the purity of mangiferin of the pure product was higher than 98.00%, and the total recovery rate was about 66.40%.

4　Discussions

At present, the process of extracting the mangiferin from mango leaves or other plants requires column chromatography. However, mangiferin is poorly soluble in a single strong polar, weakly polar, and non-polar solvent, and the content of colloidal substance is high in mango leaves. This has high interference on the extraction and isolation of mangiferin, the concentrated solution after extraction is high, and difficult to filter, and easy to block the chromatographic column. Therefore, the entire extraction process takes much time, high costs, and the efficiency is low. Li Xuejianetal. extracted mangiferin with weakly alkaline water. After two times of separation and purification of column chromatography, the mangiferin was obtained with purity of 93.96% and the total recovery rate was 65.64%. In this study, the mangiferin extraction from mango leaves was carried out without the need of column chromatography for isolation and purification. Before the extraction of mangiferin using methanol, some impurities (including most of the colloidal substance) were removed using ethyl acetate, and the lower the viscosity after concentration, the higher the extraction rate. On the principle of the difference in the solubility of mangiferin between hot and cold methanol, by means of crystallization and recrystallization, we realized the high efficient isolation of mangiferin, greatly simplified the isolation and purification process, obtained mangiferin with purity higher than 98.00%, total recovery rate of 66.40%, indicating that the mangiferin extraction and isolation efficiency were higher. In summary, this study not only greatly simplified the extraction and isolation process of mangiferin, reduced the process requirements and production costs, but also significantly increased the efficiency of extraction and isolation, and can be applied to actual production, but the disadvantage is that the overall recovery rate was low and needs further optimization. And it is necessary to recover and repeatedly extract and isolate the impurities and waste in process steps, to increase the extraction efficiency.

5　Conclusions

The optimized ethyl acetate impurity removal method is easy in operation, low in cost, and high in efficiency for extracting and isolating mangiferin, and can be applied for actual production of mangiferin.