Enrichment Of Total Flavonoids From Cistanche Deserticola By MOF Materials

Abstract Cistanche deserticola Y. C. Ma, as a traditional "food and medicine" medicinal herb, is characterized by its flavonoids, which have immunomodulatory and antioxidant effects. In this study, we investigated the performance of different MOFs in the enrichment and separation of total flavonoids from Cistanche deserticola Y. C. Ma and the optimal adsorption and resolution conditions. Five MOFs, [Zn(nicotinate)2]n, MIL-101(Cr), MIL-101(Fe), MIL-53(Fe) and MOF-5, were synthesized by hydrothermal synthesis, and characterized by PXRD, FT-IR, SEM and TG to ensure the accurate synthesis, and the adsorption and desorption quantities under different conditions were taken as the indexes for evaluating adsorption and desorption performance. After the screening, the zinc nicotinate coordination polymer [Zn(nicotinate) 2]n ([Zn(NA)2]) was found to have the best overall performance and the optimal adsorption and desorption conditions were determined. The results showed that the optimal adsorption conditions were adsorption time of 6 h, pH value of sample stock solution of 5.0, and concentration of sample stock solution of 2.20 mg∙mL-1, and the adsorption amount of total flavonoids of Cistanchis sinensisby [Zn(NA)2] was 62.91 mg∙mL-1. The optimal desorption condition was a 30% ethanol solution, and the desorption rate was 45.52%. The purity of total flavonoids in Cistanche was increased from 9.33% to 48.23% after adsorption and desorption. The crystalline form of [Zn(NA)2] before and after adsorption and desorption was characterized by PXRD as stable. The experiments showed that [Zn(NA)2] exhibited high adsorption performance with efficient enrichment and purification of flavonoids from Cistanche. Therefore, MOFs possess a wide range of application prospects as emerging materials for the enrichment and separation of active ingredients in traditional Chinese medicine. 

Keywords:Metal Organic Frameworks(MOFs); Cistanche deserticola; total flavonoids

HERBAL CISTANCHE WITH HIGH-LEVEL FLAVONOIDS

Cistanche deserticola Y. C. Ma[1] is a plant listed in the "food and medicine homology" catalog of the National Health Commission[2]. It is known as the "desert ginseng"[3] and has many functions, such as enhancing immunity and neuroprotection[4-9]. Its main active ingredients are flavonoids, which have multiple biological activities such as anti-inflammatory and immune system regulation[10-13]. Traditional separation methods have many disadvantages, such as cumbersome operation and solvent consumption[14-17]. Metal organic framework (MOFs) materials have been widely studied due to their huge specific surface area and strong adsorption capacity, showing their potential in the field of adsorption and separation of traditional Chinese medicine[18-26]. In this study, zinc nicotinate coordination polymer ([Zn(NA)2]) was selected as the adsorbent to explore its adsorption effect on total flavonoids from Cistanche deserticola and the optimal adsorption and analysis conditions. The aim was to provide theoretical support for the separation and enrichment of total flavonoids from Cistanche deserticola, expand the application of MOFs materials in the enrichment and separation of traditional Chinese medicine, and develop an economical, environmentally friendly and efficient separation and purification method.

1 Experimental part

1.1 Instruments and reagents

Cistanche deserticola, purchased from Beijing Tongrentang Pharmacy; rutin, standard substance, purchased from China Food and Drug Administration; niacin, analytical grade, purchased from Tianjin Bodi Chemical Co., Ltd.; sodium hydroxide; anhydrous ethanol; sodium nitrite; zinc sulfate heptahydrate; aluminum nitrate; chromium nitrate nonahydrate; ferric chloride hexahydrate; terephthalic acid; hydrofluoric acid; zinc nitrate hexahydrate; DMF were all domestic analytical grade. TU-1901 UV-visible spectrophotometer, Beijing Puxi General Instrument Co., Ltd.; XRD-6000X-ray diffractometer, Shimadzu Corporation, Japan; Hitachi S4800 cold field emission scanning electron microscope, HITACHI Corporation, Japan; FTIR-650 Fourier transform infrared spectrometer, Beijing Puxi General Instrument Co., Ltd.; SDT Q600 thermogravimetric analyzer, TA Corporation, USA;

1.2 Synthesis of MOF materials

1.2.1 Synthesis of nicotinate zinc coordination polymer [Zn(nicotinate)2]n([Zn(NA)2])

Reference method [28], weigh 2.336 g (8.14 mmol) ZnSO4∙7H2O, add 15 mL deionized water, and dissolve by ultrasonic. Take 1.00 g (8.13 mmol) of nicotinic acid, add 5 mL of DMF and 10 mL of anhydrous ethanol, mix for 30 min, transfer the mixed solution into a reactor, and react at 120 °C for 10 h. Filter the product, wash with deionized water and hot ethanol solution, and dry at 150 °C for 12 h to obtain [Zn(NA)2] as a white solid.

1.2.2 Synthesis of MIL-101(Fe)

Refer to [29], accurately weigh 1.350 g (0.024 mol) of ferric chloride hexahydrate and 0.412 g (2.45 mmol) of terephthalic acid, add 30 m DMF, stir for 30 min to dissolve, react in a hydrothermal reactor at 110°C for 20 h, cool naturally, wash the product with DMF, purify it twice with 70°C hot ethanol solution, each time for 2 h, and dry the purified product at 150°C for 8 h to obtain a reddish brown solid powder.

1.2.3 Synthesis of MIL-101 (Cr)

Accurately weigh 2.699 g (16.06 mmol) of terephthalic acid, 1.101 g (2.75 mmol) of cadmium nitrate nonahydrate and 100 µL of hydrofluoric acid, add them into 30 mL of deionized water and stir for 30 min to obtain a uniform solution. Transfer it to a reactor and keep it at 220 °C for 8 h, cool it naturally, filter and wash the product, and dry it in an oven at 150 °C overnight [30] to obtain a gray-green solid powder.

1.2.4 Synthesis of MOF-5

Accurately weigh 0.356 g (2.12 mmol) of terephthalic acid and 1.662 g (5.60 mmol) of zinc nitrate hexahydrate, add 40 mL of DMF and stir the solution until it is clear, react in an autoclave at 130°C for 4 h, cool naturally, wash with DMF several times, replace with dichloromethane for 12 h to obtain a white powder solid, dry and activate the sample at 160°C for later use [31-32].

1.2.5 Synthesis of MIL-53 (Fe)

Weigh 0.473 g (1.75 mmol) of ferric chloride hexahydrate and 0.291 g (1.73 mmol) of terephthalic acid and dissolve them in 38 mL of DMF, react in an autoclave at 170°C for 15 h, cool naturally, and wash with DMF solvent several times to obtain a light orange powder solid. Dry at 150℃ for 12 h[33].

1.3 Determination of total flavonoid content in samples

1.3.1 Preparation of rutin standard curve

Accurately weigh 10.0 mg of rutin standard sample, dissolve it in 70% ethanol and dilute to 100 mL to prepare a 0.100 mg∙mL-1 standard solution. Take 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 mL of standard solution respectively, add 0.4 mL of 5% NaNO2 solution, react for 6 min; then add 0.4 mL of 10% Al(NO3)3 solution, react again for 6 min; then add 4 mL of 4% NaOH solution, dilute to 10 ml volumetric flask and let stand for 15 minutes. Measure the absorbance at 510 nm. The fitting relationship between absorbance A and rutin mass concentration C was established: C=0.08637A + 0.000701 (Figure 1), and the correlation coefficient was 0.9992, indicating that rutin has a good linear correlation in the range of 0~0.045 mg∙mL-1.

1.3.2 Sample Content Determination

Take an appropriate amount of Cistanche deserticola powder sample and extract it with 70% ethanol solution at 25℃ for 2h according to a certain solid-liquid ratio. After filtration, remove ethanol by rotary evaporation and adjust the volume to 250 mL. Take 1 mL of sample solution for color development and determine its absorbance. The content of total flavonoids in Cistanche deserticola was calculated by formula (1) [27]:

W=C×V×N/m (1) Where: V represents the dilution volume of the sample solution, mL; W represents the total flavonoid content in the sample, mg∙g-1; C represents the concentration of flavonoids in the sample solution, mg∙mL-1; m represents the sample mass, g; N represents the dilution multiple.

Fig. 1 Standard curve of Rutin

1.4 Static adsorption and desorption test of total flavonoids in Cistanche deserticola by MOFs

1.4.1 Preparation of crude extract of total flavonoids in Cistanche deserticola

Weigh 5 g of Cistanche deserticola powder sample, add 150 mL of 70% ethanol by volume, extract at 60℃ for 2 h, and obtain the crude extract of total flavonoids in Cistanche deserticola. In this experimental scheme, the content of total flavonoids in Cistanche deserticola was 51.67 mg∙g-1, and the purity was 9.33%.

1.4.2 MOFs static adsorption and desorption test

Accurately weigh 100.0 mg of each of the five MOFs materials and add them to a 50 mL centrifuge tube, then add 35 mL of crude extract and perform ultrasonic dispersion. Adsorb for 24 h at 150 r/min on a shaker at 25°C. Centrifuge after adsorption and extract 1 mL of the supernatant to determine the total flavonoid content, thereby calculating the total flavonoid adsorption amount of each MOF material.

The MOF material after adsorption equilibrium was washed with deionized water, and 20 mL of desorption solution was added. The material was placed on a constant temperature shaker at 25°C and desorbed at 150 r/min for 24 h. Centrifuge after desorption and extract 1 mL of the supernatant to determine the total flavonoid content in the desorption solution. Based on the above steps, the equilibrium adsorption amount, desorption amount and desorption rate of the five MOFs materials for total flavonoids were calculated[34]. Qe=(C0− Ce)×Vi/m (2)

Qd=Cd×Vd/m (3)

Rd(%)=Qd/Qe×100% (4)

Where: m represents the weight of MOFs, g; Qd represents the desorption amount, mg∙g-1; Qe represents the equilibrium absorption amount, mg∙g-1; Cd represents the concentration of the desorption solution, mg∙mL-1; Rd represents the desorption rate, %; C0 represents the initial concentration of the adsorption solution, mg∙mL-1; Ce represents the equilibrium concentration of the adsorption solution, mg∙mL-1; Vd represents the volume of the desorption solution, mL; Vi represents the volume of the sample solution, mL.

1.5 Determination of the optimal adsorption conditions of [Zn(NA)2] on Cistanche deserticola

1.5.1 Static adsorption kinetic curve

Accurately weigh 1.0 g of [Zn(NA)2] sample, add 200 mL of Cistanche deserticola crude extract, and adsorb on a shaker at 25℃ and 150 r/min. Take out 1 mL of supernatant for analysis at a certain time interval to determine the content of total flavonoids. At the same time, continue to add fresh solution until adsorption equilibrium.

1.5.2 Effect of adsorbent dosage on the adsorption of total flavonoids in Cistanche deserticola

Accurately weigh 20, 60, 100, 140, 180, and 200 mg [Zn(NA)2], add 35 mL of extract solution, and after adsorption equilibrium, centrifuge and take 1 mL of supernatant for analysis and determination of total flavonoid content.

1.5.3 Relationship between the adsorption of total flavonoids in Cistanche deserticola and the initial concentration of the sample. Prepare the sample solution of crude flavonoid extract of Cistanche deserticola with concentrations of 0.64, 1.28, 1.76, 2.20, 2.84, and 3.6 mg∙mL-1, add 100 mg [Zn(NA)2], adsorb for 6 h, centrifuge and take 1 mL of supernatant for analysis and determination, and calculate the adsorption of [Zn(NA)2] for total flavonoids.

1.5.4 Relationship between the adsorption of total flavonoids of Cistanche deserticola and the pH value of the sample solution

The experiment selected a sample solution with a concentration of 2.20 mg∙mL-1, and adjusted the pH value of the sample solution to 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0. After 6 hours of adsorption, the adsorption reached a complete state, and 1 mL of the supernatant was centrifuged for analysis. Then the adsorption of [Zn(NA)2] on total flavonoids was calculated.

1.6 Determination of the optimal desorption conditions of [Zn(NA)2] on Cistanche deserticola

1.6.1 Effect of desorption performance and different desorption solutions

For the oscillation desorption of [Zn(NA)2] at adsorption equilibrium, the effect of selecting ethanol, n-butanol, methanol, and ethyl acetate as desorption solutions on the desorption performance of [Zn(NA)2] was investigated.

1.6.2 Effect of desorption effect on ethanol concentration

[Zn(NA)2] at adsorption equilibrium was desorbed, and the effect of ethanol aqueous solution with desorption liquid volume fraction of 10%, 30%, 50%, 70%, and 90% on the desorption effect of [Zn(NA)2] was studied. The optimal analytical conditions were determined.

1.5.2 Effect of adsorbent dosage on the adsorption of total flavonoids in Cistanche deserticola

Accurately weigh 20, 60, 100, 140, 180, and 200 mg [Zn(NA)2], add 35 mL of extract solution, and after adsorption equilibrium, centrifuge and take 1 mL of supernatant for analysis and determination of total flavonoid content.

1.5.3 Relationship between the adsorption of total flavonoids in Cistanche deserticola and the initial concentration of the sample. Prepare the sample solution of crude flavonoid extract of Cistanche deserticola with concentrations of 0.64, 1.28, 1.76, 2.20, 2.84, and 3.6 mg∙mL-1, add 100 mg [Zn(NA)2], adsorb for 6 h, centrifuge and take 1 mL of supernatant for analysis and determination, and calculate the adsorption of [Zn(NA)2] for total flavonoids.

1.5.4 Relationship between the adsorption of total flavonoids of Cistanche deserticola and the pH value of the sample solution

The experiment selected a sample solution with a concentration of 2.20 mg∙mL-1, and adjusted the pH value of the sample solution to 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0. After 6 hours of adsorption, the adsorption reached a complete state, and 1 mL of the supernatant was centrifuged for analysis. Then the adsorption of [Zn(NA)2] on total flavonoids was calculated.

1.6 Determination of the optimal desorption conditions of [Zn(NA)2] on Cistanche deserticola

1.6.1 Effect of desorption performance and different desorption solutions

For the oscillation desorption of [Zn(NA)2] at adsorption equilibrium, the effect of selecting ethanol, n-butanol, methanol, and ethyl acetate as desorption solutions on the desorption performance of [Zn(NA)2] was investigated.

1.6.2 Effect of desorption effect on ethanol concentration

[Zn(NA)2] at adsorption equilibrium was desorbed, and the effect of ethanol aqueous solution with desorption liquid volume fraction of 10%, 30%, 50%, 70%, and 90% on the desorption effect of [Zn(NA)2] was studied. The optimal analytical conditions were determined.