A novel absorbent, HOF-3@PU: Preparation and application for sustainable and efficient purification of catalpol and ajugol from Rehmannia glutinosa leaves

Abstract This study introduced the preparation of a novel HOF-loaded PU sponge (HOF-3@PU) composite for the sustainable and efficient purification of catalpol and ajugol from Rehmannia glutinosa leaves for the first time. HOF-3 was selected as the best adsorbent from the five synthesised HOFs. HOF-3@PU was prepared by ultrasonication, and the loading conditions were optimised. The results showed that the optimum adsorption conditions are as follows: adsorption liquid volume: 160 mL, flow rate: 3.0 mL/min, pH: 6.0, concentration: 1.62 mg/mL for catalpol and 2.18 mg/mL for ajugol. The optimum desorption conditions are as follows: desorption agent: ethanol, volume fraction: 60%, flow rate: 2.0 mL/min, volume: 300 mL and pH: 6.0. Under the optimal process conditions, the adsorption capacities of catalpol and ajugol were 75.62 and 68.41 mg/g, the desorption rates were 78.5 and 86.4% and the purities were 38.7 and 36.5%, respectively. Graphical Abstract


Introduction
Rehmannia glutinosa Libosch. is a perennial herb belonging to the Rehmannia and Scrophulariaceae families (Zhou et al. 2021).Known as one of four major Huai-yao plants, it has a long cultivation history of more than 1000 years in our country.Traditionally, the medicinal part of R. glutinosa is the rhizome, and its active ingredients mainly include iridoid glycosides, phenylethanol glycosides, triterpenes, flavonoids and polysaccharides (Tian et al. 2021).It has many pharmacological uses which can serve as a blood supplement and anti-depressant, it has hypoglycemic, antiinflammatory and antibacterial, and antitumor properties, and it can provide liver and kidney protection (Vu et al. 2021).R. glutinosa leaves contain the same active ingredients as the rhizomes, though they are often abandoned as waste.Thus, it is necessary to further develop and utilise the resources of R. glutinosa leaves.Ajugol and catalpol are important active components of R. glutinosa leaves, with high contents and extensive pharmacological effects (Liu et al. 2021).Ajugol and catalpol can reduce blood sugar, has antiviral, antibacterial, antioxidant, antidiabetic, anti-inflammatory, anti-asthma, and antitumor properties, provides liver and gallbladder protection, enhances immunity and has other pharmacological effects (Chen et al. 2020).At present, the methods of separating and purifying active pharmaceutical ingredients include column chromatography and the macroporous resin purification et al (Wei et al. 2022).However, these methods are generally limited by cumbersome operation and high cost.Therefore, it is urgent to find a simple and economical separation and purification method.
Hydrogen-bonded organic frameworks (HOFs) are a new type of porous material composed of organic components connected by intermolecular hydrogen bonding interactions (Suzuki et al. 2021;Yin et al. 2022).Compared with metal-organic frameworks (MOFs) and covalent organic frameworks (COFs) that rely on metal coordination and covalent bonds to form porous structures, the hydrogen bonding interactions of HOFs are much weaker and more dynamic, providing unique flexibility and structural adjustment (Wang et al. 2017).In the past decade, many HOFs with different morphologies and topological structures have been constructed (Wu et al. 2018).In addition, they also have high specific surface areas, good solution processing, significant stability, easy regeneration and mild synthesis conditions (Gao et al. 2021).These excellent properties enable them to be widely used in gas storage, catalysis, fluorescence sensing, proton conduction, gas separation and absorption (Wang et al. 2020;Han et al. 2019).A polyurethane (PU) sponge is an adsorptive material with a three-dimensional (3D) porous structure.It has excellent properties, such as high porosity, high adsorption capacity, easy desorption, low price, low density and good mechanical properties.PU sponges are widely used in catalysis, oil-water separation, flame retardancy and sensing (Jamsaz et al. 2021;Pang et al. 2021;Khalilifard and Javadian 2021;Chen et al. 2021).
Combining the advantages of both, we first prepared a new composite HOFs @ PU. PU sponge can be modified by HOFs, and they can not only give HOFs a more stable structure but also reduce the loss of adsorbent in traditional dispersive solid phase extraction and facilitate recycling.Thus, HOF-3 @ PU was prepared and applied in purification of ajugol and catalpol from R. glutinosa leaves for the first time.Furthermore, the adsorption and desorption conditions of the target components were optimised.

Static adsorption and desorption
The structural formulas of catalpol and ajugol show that these two substances have more hydroxyl groups, while the HOF-3 crystal contains more N-H bonds (Chiu et al. 2021;Xia et al. 2020).Due to the action of highly electronegative elements, such as N and O, the hydrogen atom becomes strongly polar, which causes O-H and N-H to have a strong tendency to form intermolecular hydrogen bonds.In addition, the strong van der Waals force strengthens the interactions between catalpol, ajugol and HOF-3.The PU sponge also contains N-H bonds (Jamsaz et al. 2021), which not only helps the adsorption and desorption of catalpol and ajugol but also provides an advantage for the loading of HOF-3.
PFC-11, PFC-12 and PFC-13 are self-assembled by rigid and planar ligands to form a flat hexagonal honeycomb structure.A large number of dense benzene rings produce strong p-p stacking, resulting in preferential adsorption of aromatic compounds, while no benzene ring is found in catalpol and ajugol.In contrast, the two ligands of HOF-3 were small molecular structures, especially sulphate ions added inorganic properties to balance the aromatic ring effect of melamine.Based on the comprehensive consideration of adsorption capacity, desorption capacity and desorption rate shown in Table S6.HOF-3 had the best adsorption and desorption effect.Therefore, HOF-3 was selected for subsequent experiments.
The static adsorption kinetics curves of HOF-3 are shown in Figure S2(a).In the initial stage, the coverage rate of the solid surface was not high and the adsorption capacity was strong.With the extension of adsorption time, the adsorbed amount still increased, but the adsorption rate was reduced due to the increasing coverage of the surface, especially near adsorption equilibrium.At 7 h of adsorption, the adsorption of catalpol by HOF-3 @ PU reached equilibrium, the adsorption amount of catalpol was 70.25 mg/g.The adsorption equilibrium time of ajugol was 8 h and the equilibrium adsorption capacity was 62.63 mg/g.The static desorption kinetics of catalpol and ajugol are shown in Figure S2(b).At the initial stage of desorption, the concentration of the target component in the desorption agent is very low, and the desorption is rapid.With the increase of the elution concentration, the desorption rate tends to be gentle.When the desorption time was 5 h, the equilibrium was reached and the desorption rates of ajugol and catalpol were 87.2 and 83.4%.

Morphology and structure analysis
Figure S3(a) shows the Fourier transform infrared spectrum (FTIR) spectrum of HOF-3.According to the analysis, the peaks at 2800-3375 cm À1 corresponded to the scaling vibration of associated N-H bond.Due to the existence of hydrogen bonds, a strong broad peak was produced.The peak at 2757 cm À1 was the stretching vibration of saturated C-H, and the peaks at 2491 and 2300 cm À1 were the scaling vibration of C ¼ N. The absorption peaks at 1600 and 1580 cm À1 were caused by benzene ring skeleton vibration, while the absorption peaks at 1251 cm À1 were caused by the stretching vibration of the C-N bond.The peak at 1155 cm À1 was the bending vibration absorption peak of sulphate, while the peaks at 980 and 772 cm À1 were caused by the bending vibration of the N-H bond.This evidence was sufficient to prove the successful synthesis of HOF-3 (Wu et al. 2021).
The X-Ray Diffraction (XRD) pattern of HOF-3 is shown in Figure S3(b).Through the analysis of the X-ray single crystal structure, it was found that HOF-3 belongs to the l2/c point group in the monoclinic system.HOF-3 had obvious diffraction peaks at 19.5 , 19.8 and 29.8 .The higher the peak height is, the higher the degree of crystallisation, and the larger the peak area reflects the higher purity of the crystal phase.The half-peak width is inversely proportional to the grain size, indicating that the grain size of the HOF-3 crystal is large.The deviation of diffraction peak (101) at 2h ¼ 19.5 and 2h ¼ 19.8 resulted in two diffraction peaks due to residual stress in the material.There was a weak diffraction peak (020) at 2h ¼ 29.8 .In addition, compared with the spectra in the literature (Gong 2019), it was sufficient to prove that HOF-3 was successfully synthesised.
Figure S4 shows the scanning electron microscope (SEM images of PU sponge (a,b) and HOF-3 @ PU (c-f).As shown in the enlarged SEM image, the PU sponge skeleton was smooth before modification.After the loading of HOF-3, the hexagonal skeleton of the PU sponge was destroyed and became rough, but it maintained the stability of the frame and did not collapse.In addition, the loading of HOF-3 on the sponge was obvious.Figure S4(e) shows that the distribution of HOF-3 particles was uniform enough to illustrate successful loading (Jin et al. 2022;Sui et al. 2021).

Optimisation of adsorption and desorption conditions
In order to determine the experimental range of each factor and establish a control method, the influencing factors of adsorption and desorption must be explored.Except for the effects of volume, flow rate, concentration and pH of the adsorption solution on the adsorption, the effects of the type, flow rate, volume fraction, volume and pH of the desorption agent on the desorption were tested.The results are shown in Figure S5 and S6.The effects of these factors may come from the following reasons.When the volume was small, the adsorption rate was large due to the low coverage of the surface.When the surface was close to saturation, the adsorption rate decreased.At the lower flow rate, due to the full contact between the adsorbent and the sample solution, the adsorption speed was faster, but the time required to reach adsorption equilibrium was longer.A fast flow rate led to insufficient contact so adsorption decreased.Because more hydroxyl groups are present in catalpol and ajugol, these compounds are more stable under relatively acidic conditions.When the acidity was too strong, the structure of catalpol and ajugol was destroyed, so the adsorption capacity decreased.The low concentration is beneficial for adsorption.
However, when equilibrium is reached, the continuous increase in concentration does not significantly change the adsorbed amount.
Catalpol and ajugol are polar molecules, ethanol and methanol have obvious advantages because of their strong polar.Methanol has a slight advantage over ethanol, but methanol is toxic.Thus, ethanol is more suitable than methanol as desorption agent.And the polarity of the desorption agent was adjusted by changing the volume fraction of ethanol so that the desorption was different.It can be seen that when the flow rate was low, the desorption solution made full contact with the HOF-3 @ PU so that the concentration of target components in the effluent decreased rapidly, but the time required was longer, which is similar to the effect of desorption agent dosage.If the acidity or basicity was too strong, the structure of catalpol and ajugol were damaged to some extent.The weak acidic conditions stabilised the target components which improved the desorption rate.Considering the energy consumption and production efficiency, the optimised adsorbent concentration was diluted four times as the original solution, the flow rate was 3.0 mL/min, the pH was 6.0, and the volume was 160 mL.Using 300 mL ethanol with pH 6 and volume fraction of 60% as desorption agent, the optimal desorption effect was obtained by controlling the flow rate of 2.0 mL/min.

Conclusion
In summary, we successfully synthesised HOF-3 @ PU and applied it to the purification of catalpol and ajugol in R. glutinosa leaves.The single-factor experiment allowed us to determine the best adsorption and desorption conditions for catalpol and ajugol by HOF-3 @ PU, and the desorption rate of the two target compounds reached more than 75%.Before this study, there had neither been an attempt to apply them to the enrichment and purification of medicinal active ingredients, nor combine them with PU sponges.This study fills the gap in this field and opens up a new direction for application of HOF-3 @ PU.The optimum conditions were explored to make the adsorption amount of catalpol and ajugol reached 75.62 and 68.41 mg/g, and the desorption rates were 78.5 and 86.4%.After purification by HOF-3 @ PU, the purities of the catalpol and ajugol reached 38.7 and 36.5%,respectively.