Discovery of anticancer function of Febrifugine: Inhibition of cell proliferation, induction of apoptosis and suppression steroid synthesis in bladder cancer cells.

Bladder cancer is a prevalent malignancy affecting the urinary system, which presents a significant global health concern. Although there are many treatments for bladder cancer, identifying more effective drugs and methods remains an urgent problem. As a pivotal component of contemporary medical practice, traditional Chinese medicine (TCM) assumes a crucial role in the realm of anti-tumor therapy, especially with the identification of active ingredients and successful exploration of pharmacological effects. Febrifugine, identified as a quinazoline-type alkaloid compound extracted from the Cytidiaceae family plant Huangchangshan, exhibits heightened sensitivity to bladder cancer cells in comparison to control cells (non-cancer cells) group. The proliferation growth of bladder cancer cells T24 and SW780 was effectively inhibited by Febrifugine, and the IC50 was 0.02 and 0.018 µM respectively. Febrifugine inhibits cell proliferation by suppressing DNA synthesis and induces cell death by reducing steroidogenesis and promoting apoptosis. Combined with transcriptome analysis, Febrifugine was found to downregulate low density lipoprotein receptor-associated protein, lanosterol synthase, cholesterol biosynthesis second rate-limiting enzyme, 7-dehydrocholesterol reductase, flavin adenine dinucleotide dependent oxidoreductase and other factors to inhibit the production of intracellular steroids in bladder cancer T24 cells. The results of animal experiments showed that Febrifugine could inhibit tumor growth. In summary, the effect of Febrifugine on bladder cancer is mainly through reducing steroid production and apoptosis. Therefore, this study contributes to the elucidation of Febrifugine's potential as an inhibitor of bladder cancer and establishes a solid foundation for the future development of novel therapeutic agents targeting bladder cancer.

Design, Synthesis and Antitumor Activity of FAK/PLK1 Dual Inhibitors with Quinazolinone as the Skeleton.

Febrifugine is a kind of quinazolinone compound with high biological activity from a Chinese herb called Chang Shan (Dichroa febrifuga). Febrifugine and its derivatives possess extensive biological activities, some of which exhibited anti-tumor activities as FAK inhibitors. However, they are not very effective at inhibiting tumor metastasis, perhaps because tumors gain energy through compensatory activation of other signaling pathways that promote cell migration and invasion. Therefore, seventeen novel febrifugine derivatives with quinazolinone skeleton were designed, synthesized and acted as potential FAK/PLK1 dual inhibitors. These compounds were determined by 1 H-NMR, 13 C-NMR and MS. Most of the compounds exhibited good inhibitory activity against cancer cell lines by computer-assisted screening, antitumor activity test and FAK/PLK1 inhibitory activity test, wherein compound 3b was screened as a high-efficiency lead compound.

Febrifugine dihydrochloride as a new oral chemotherapeutic agent against visceral leishmaniasis infection.

Visceral leishmaniasis (VL) is the deadliest form of leishmaniasis without a safer treatment option. This study implies drug repurposing to find a novel antileishmanial compound, namely febrifugine dihydrochloride (FFG) targeting Leishmania antioxidant system. Starting with virtual screening revealed the high binding affinity and lead likeness of FFG against the trypanothione reductase (TR) enzyme of Leishmania donovani, followed by experimental validation. The promastigotes inhibition assay gave the IC50 concentration of FFG and Miltefosine (positive control) as 7.16 ± 1.39 nM and 11.41 ± 0.29 µM, respectively. Their CC50 was found as 451 ± 12.73 nM and 135.9 ± 5.94 µM, respectively. FFG has been shown to increase the reactive oxygen species (ROS), leading to apoptosis-like cell death among L. donovani promastigotes. Spleen touch biopsy resulted in 62% and 55% decreased parasite load with FFG and miltefosine treatment, respectively. Cytokine profiling has shown an increased proinflammatory cytokine response post-FFG treatment. Moreover, FFG is safe on the liver toxicity parameter in mice post-treatment.

Preparative separation of two isomeric antimalaria alkaloids febrifugine and isofebrifugine from Dichroa febrifuga roots by countercurrent chromatography.

Two antimalaria alkaloids, febrifugine and isofebrifugine, were successfully separated from total alkaloids of Dichroa febrifuga roots by one-step preparative countercurrent chromatography with a selected biphasic solvent system. The selected biphasic solvent system was composed of chloroform: methanol: water (2:1:1, v/v) according to partition performance of the two target components. Selection of biphasic solvent system was conducted by high performance liquid chromatography combined with high performance thin layer chromatography, which greatly assisted the screening procedure for biphasic solvent system. Totally, 50 mg of total alkaloid was separated by one-step preparative countercurrent chromatography, yielding 12 mg of febrifugine and 9 mg of isofebrifugine with more than 98.0% purity, respectively.

Differential Effects of Halofuginone Enantiomers on Muscle Fibrosis and Histopathology in Duchenne Muscular Dystrophy.

Progressive loss of muscle and muscle function is associated with significant fibrosis in Duchenne muscular dystrophy (DMD) patients. Halofuginone, an analog of febrifugine, prevents fibrosis in various animal models, including those of muscular dystrophies. Effects of (+)/(-)-halofuginone enantiomers on motor coordination and diaphragm histopathology in mdx mice, the mouse model for DMD, were examined. Four-week-old male mice were treated with racemic halofuginone, or its separate enantiomers, for 10 weeks. Controls were treated with saline. Racemic halofuginone-treated mice demonstrated better motor coordination and balance than controls. However, (+)-halofuginone surpassed the racemic form's effect. No effect was observed for (-)-halofuginone, which behaved like the control. A significant reduction in collagen content and degenerative areas, and an increase in utrophin levels were observed in diaphragms of mice treated with racemic halofuginone. Again, (+)-halofuginone was more effective than the racemic form, whereas (-)-halofuginone had no effect. Both racemic and (+)-halofuginone increased diaphragm myofiber diameters, with no effect for (-)-halofuginone. No effects were observed for any of the compounds tested in an in-vitro cell viability assay. These results, demonstrating a differential effect of the halofuginone enantiomers and superiority of (+)-halofuginone, are of great importance for future use of (+)-halofuginone as a DMD antifibrotic therapy.

Chemical synthesis of febrifugine and analogues.

The quinazolinone-containing 2,3-disubstituted piperidines febrifugine and isofebrifugine have been the subject of significant research efforts since their occurrence in Dichroa febrifuga and their anti-malarial actions were first described in the late 1940s. Subsequently they have also been shown to be present in other plants belonging to the hydrangea family and various analogues of febrifugine have been prepared in attempts to tune biological properties. The most notable analogue is termed halofuginone and a substantial body of work now demonstrates that this compound possesses potent human disease relevant activities. This review focuses on the literature associated with efforts dedicated towards uncovering the structures of febrifugine and isofebrifugine, the development of practical methods for their synthesis and the syntheses of structural analogues.

[Quantitative determination of febrifugine by proton nuclear magnetic resonance spectroscopy with internal standard method].

A quantitative nuclear magnetic resonance method(qNMR) was established for determination of the absolute content of febrifugine. Proton nuclear magnetic resonance spectroscopy of febrifugine was obtained in DMSO-d6 with hydroquinone as the internal standard substance on a Bruker Ascend 600 MHz superconducting nuclear resonance spectrometer at 298 K. The specific parameters were as follows: the observing frequency was 600 MHz,spectra width was 7 211 Hz, pulse width was 9.70 µs, pulse sequence was zg30,scan times was 32 and relaxation time was 2 s. The proton signal peaked at δ 7.71 for febrifugine and δ 6.55 for hydroquinone were selected as the quantification peaks. Linear regression of quantitative peak area ratio of febrifugine-hydroquinone versus their mass ratio yielded a correlation coefficient of 0.999 6 and a regression equation of Y=0.083 3X+0.008 6．The linear range of febrifugine was 2.17-17.07 g·L⁻¹,the precision RSD was 0.78%(n=6),the repeatability RSD was 1.2%(n=6),and the contents of three batches of febrifugine sample were 94.91%,95.09% and 95.52%,respectively. The content of febrifugine was 96.44% determined by high performance liquid chromatography(HPLC). The relative error of the content of febrigugine determinted by qNMR and HPLC methods was 1.27%. The results showed that the internal standard method of proton nuclear magnetic resonance spectroscopy could be used to determine the absolute content of febrifugine.

A Novel Synthesis of the Efficient Anti-Coccidial Drug Halofuginone Hydrobromide.

Background: Halofuginone hydrobromide (1) is recognized as an effective drug against several species of Eimeria (E.) in poultry. In this paper, we describe a convenient and low cost preparation method for the compound, as well as primary validation of its activity. Methods: First, 7-bromo-6-chloroquinazolin-4(3H)-one (2) was prepared from m-chlorotoluene by a conventional process, and then chloroacetone was creatively introduced in two steps. Finally, halofuginone hydrobromide (1) was obtained from 7-bromo-6-chloro-3-(3-cholroacetonyl) quinazolin-4(3H)-one (4) by a four-step reaction sequence including condensation, cyclization, deprotection and isomerization. The structures of the relative intermediates and target compound were characterized by melting point, IR, MS and ¹H-NMR. Besides, the protective effect of compound 1-supplemented chicken diet at doses of 6, 3 and 1.5 mg per 1 kg were evaluated on chickens infected with E. tenella, by reduction in mortality, weight loss, fecal oocyst excretion and gut pathology, respectively. Results: Halofuginone hydrobromide (1) was prepared successfully by and improved and innovative method based on traditional research. Moreover, the synthesized halofuginone hydrobromide significantly exhibited an anti-coccidial property. Conclusions: The fruitful work described in this Communication has resulted in halofuginone hydrobromide, which has a good pharmaceutical development prospects, becoming more available for large-scale production.

[Investigation on stability and degradation kinetics of febrifugine].

To investigate the stability and degradation kinetics of febrifugine. The results showed that within 24 hours, febrifugine content was decreased by only 1% in mobile phase solvent, but its content was decreased to be 90% of the initial content in the water, methanol, 50% methanol and 10% acetonitrile solution. When the pH value of the solution was between 3 and 7, the retention rate of febrifugine in 24 hours was over 98%, but its content was decreased by about 12% in alkaline solution (pH 9.0). The higher the temperature, the worse the stability of febrifugine. At 40-80 ℃, the content of febrifugine was decreased to be 60% of its initial content in 10 hours, but the content was decreased by only 5% in 10 h at 20 ℃.However, no matter 40 ℃or 60 ℃, febrifugine was mainly transformed into isofebrifugine, and the total content of febrifugine and isofebrifugine was equal to their initial total content in 10 hours, while incase of 80 ℃, the total content was decreased to be 83.33% in 10 h, which suggested that the structure of febrifugine was absolutely changed, not just isomerized to be isofebrigugine at high temperature. Light had a significant impact on the stability of febrifugine. Under bright light, the content of febrifugine was reduced by about 23% in 108 h, but it only decreased by about 10% in the natural light or darkness. In artificial gastric fluid (pH 1.4) and artificial intestinal fluid (pH 6.8), the content of febrifugine was decreased by less than 5% in 10 h. After storage at high temperature(60 ℃), high humidity [(75±1)%] and strong light (3 000 lx) conditions for 10 d, the content of solid febrifugine was decreased by 0.27%, 7.6% and 5.39%, respectively. The degradation of febrifugine basically complied with the first-order reaction kinetic process in the following conditions: in water, methanol, 50%methanol and 10% acetonitrile solvents, alkaline solution (pH>7), different light intensity and different temperatures (20,40 ℃). Therefore, no matter the isolation and purification of febrifugine or the production of the related preparations, it should be done fast in the acidic solution, low temperature and dark conditions, while the febrifugine solid should be kept in dry and dark conditions.

[Simultaneous determination of febrifugine and isofebrifugine in Dichroa febrifuga root by HPLC method].

To develop the HPLC method for simultaneous determination of febrifugine and isofebrifugine in Dichroa febrifuga root, and on the basis of this, the feasibility of quantitative analysis of multi-component by a single-marker (QAMS) model for the determination of the two alkaloids was investigated. The chromatographic separation was performed on an octadecyl bonded silica gel column with mixed solvent consisting of acetonitrile-water-glacial acetic acid-triethylamine (9∶91∶0.36∶0.745) as mobile phase at a flow rate of 1.0 mL•min⁻¹. The detection wavelength was set at 225 nm, and the column temperature was set at 30 ℃. The linear range of febrifugine and isofebrifugine were 10.7-426 ng and 10.6-424 ng, respectively. Their average recovery were 98.33% (RSD 2.7%) and 100.4% (RSD 1.8%), respectively. On the basis of this established method, febrifugine was used as the internal reference substance to calculate the relative correction factors (RCF) and the relative retention values (RRV) of isofebrifugine to febrifugine. Through a series of methodology evaluations, the two alkaloids were simultaneously assayed only by quantitative determination of febrifugine. This result played the part of demonstration role for the application of QAMS model in the determination of isomers.

Prospects of halofuginone as an antiprotozoal drug scaffold.

Halofuginone is a clinically active derivative of febrifugine that was first isolated from the Chinese herb Dichroa febrifuga. The beneficial biological effects of halofuginone on various diseases including parasitic diseases, cancer, fibrosis, and autoimmune disorders have been investigated. Halofuginone has reduced toxic side effects when compared to febrifugine, an advantage that has led to the commercial availability of halofuginone-based antiparasitic drugs for animal use, and to human clinical trials for the treatment of tumors and fibrosis. This review summarizes advances in determining the mechanism of action of halofuginone, focusing on its antiparasitic role in malaria, cryptosporidiosis, coccidiosis, toxoplasmosis, and leishmaniasis. We discuss mechanistic insights into halofuginone's primary mode of action which involves inhibition of the prolyl-tRNA synthetase enzyme, which is crucial in protein synthesis. Halofuginone exemplifies the untapped wealth of plant-derived compounds in disease therapeutics.

Synthesis and in vitro antileishmanial efficacy of novel quinazolinone derivatives.

Currently available drugs being used to treat leishmaniasis have several shortcomings, including high toxicity, drug administration that requires hospitalization, and the emergence of parasite resistance against clinically used drugs. As a result, there is a dire need for the development of new antileishmanial drugs that are safe, affordable, and efficient. In this study, two new series of synthesized quinazolinone derivatives were investigated as potential future antileishmanial agents, by assessing their activities against the Leishmania (L.) donovani and L. major species. The cytotoxicity profiles of these derivatives were assessed in vitro on Vero cells. The compounds were found to be safer and without any toxic activities against mammalian cells, compared to the reference drug, halofuginone, a clinical derivative of febrifugine. However, they had demonstrated poor antileishmanial growth inhibition efficacies. The two compounds that had been found the most active were the mono quinazolinone 2d and the bisquinazolinone 5b with growth inhibitory efficacies of 35% and 29% for the L. major and L. donovani 9515 promastigotes, respectively. These outcomes had suggested structural redesign, inter alia the inclusion of polar groups on the quinazolinone ring, to potentially generate novel quinazolinone derivatives, endowed with effective antileishmanial potential.

Research Note: Study on the residue depletion of febrifugine and isofebrifugine in broiler chicken.

In this study, 105 broiler chickens were fed with dietary feeds containing different contents of Dichroae Radix extract for 10 consecutive days. Then the residue depletions of its main alkaloids (febrifugine and isofebrifugine) in muscle, kidney and liver samples at different withdrawal times were determined by an ultra-performance liquid chromatography method. Results showed that the 2 alkaloids were mainly at tissue-bound formation. At withdrawal period of 0 d, their concentrations in all samples were high but decreased rapidly after 1 day of cessation (35-91%). After 5 to 7 days of cessation, their residues in muscle and kidney were not detectable, and after at least 10 days of cessation they were not detectable in liver. These results indicated that an appropriate withdrawal time for Dichroae Radix preparation was required if it is licensed as a new drug, and the best target tissue for monitoring its residue was liver.

In vitro effects of febrifugine on Schistosoma mansoni adult worms.

BACKGROUND: Reports on the antischistosomal effect of several antimalarial drugs such as artesunate, mefloquine, and amodiaquine suggest that febrifugine, which exerts an antimalarial effect, can also be expected to possess antischistosomal potential. The present study investigates the antischistosomal effects of febrifugine. METHODS: In experiment 1, Schistosoma mansoni adult worm pairs were incubated in a medium alone as a control or supplemented with febrifugine at 0.05, 0.1, 0.2, and 0.5 µg/ml for 14 days. The morphology of the worms and the egg production of the female worms were observed simultaneously. In experiment 2, the incubation was conducted as in experiment 1, except that the febrifugine concentrations were reduced to 0.005, 0.01, and 0.02 µg/ml. In addition, S. mansoni adult worms were incubated with either 0.5 µg/ml febrifugine or none as a control for 5 days and stained with neutral red dye. RESULTS: Febrifugine significantly reduced the survival of S. mansoni male and female worms at concentrations of 0.02-0.5 µg/ml following incubation for 14 days and remarkably inhibited the daily egg output of the female worms. The non-treated male and female worms remained morphologically normal within the period of 14 days, whereas male and female worms treated with febrifugine at different concentrations gradually twisted and subsequently died. In addition, S. mansoni adult worms were incubated with either 0.5 µg/ml febrifugine or none as a control for 5 days and stained with neutral red dye. Non-treated male worms were morphologically normal and stained dark red with neutral red, while febrifugine-treated male worms appeared similar to those in the control group and were stained at a slightly lower level of dark red than the non-treated male worms. Non-treated female worms were morphologically normal, and their intestinal tract and vitellaria were stained deep red and dark red, respectively. In contrast, febrifugine-treated female worms were morphologically damaged, and their intestinal tract and vitellaria remained mostly unstained and stained dark red, respectively. CONCLUSION: Febrifugine exerts potent antischistosomal effects and can be expected to contribute to the development of a novel antischistosomal drug.

Exploring dual inhibitory role of febrifugine analogues against Plasmodium utilizing structure-based virtual screening and molecular dynamic simulation.

Malaria is an endemic disease caused by the protozoan parasite Plasomodium falciparum. Febrifugine analogues are natural compound obtained from the traditional Chinese herbs have shown significant antimalarial and anticancerous efficacy in experimental model. Development of resistance against the existing antimalarial drug has alarmed the scientific innovators to find a potential antimalarial molecule which can be further used by endemic countries for the elimination of this disease. In this study, structure-based virtual screening and molecular dynamics (MD) base approaches were used to generate potential antimalarial compound against plasmepsin II and prolyl-tRNA synthetase of Plasmodium. Here, we have docked series of febrifugine analogues (n = 11,395) against plasmepsin II in three different docking modes and then it was compared with previously reported target prolyl-tRNA synthetase. Extra precision docking resulted into 235 ligands having better docking score were subject for QikProp analysis. Better ligands (n = 39) obtained from QikProp analysis were subject for ADMET prediction and docking protocol validation through the estimation of receiver operator characteristics. In the later stage, 24 ligands obtained from ADMET study were subject for the estimation of binding energy through MM-GBSA and same were also docked against prolyl-tRNA synthetase to get compounds with dual inhibitor role. Finally, MD simulation and 2D fingerprint MACCS study of two best ligands have shown significant interaction with plasmepsin II and homology against known active ligand with noteworthy MACCS index, respectively. This study concludes that FA12 could be potential drug candidate to fight against Plasmodium falciparum parasites.

Compound antimalarial ethosomal cataplasm: preparation, evaluation, and mechanism of penetration enhancement.

Malaria is still a serious public health problem in some parts of the world. The problems of recurrence and drug resistance are increasingly more serious. Thus, it is necessary to develop a novel antimalarial agent. The objectives of this study were to construct a novel compound antimalarial transdermal nanosystem-ethosomal cataplasm, to investigate its characteristics and efficiency, and to systematically explore the penetration-enhancing mechanisms of ethosomal cataplasm. Artesunate-loaded ethosomes and febrifugine-loaded ethosomes were prepared, and their characteristics were evaluated. Drug-loaded ethosomes were incorporated in the matrix of cataplasm to form the compound antimalarial ethosomal cataplasm. With the help of ethosomal technology, the accumulated permeation quantity of artesunate significantly increased at 8 hours after administration, which was 1.57 times as much as that of conventional cataplasm. Soon after administration, the ethosomal cataplasm could make a large quantity of antimalarial drug quickly penetrate through skin, then the remaining drug in the ethosomal cataplasm could be steadily released. These characteristics of ethosomal cataplasm are favorable for antimalarial drugs to kill Plasmodium spp. quickly and prevent the resurgence of Plasmodium spp. As expected, the ethosomal cataplasm showed good antimalarial efficiency in this experiment. The negative conversion rates were 100% and the recurrence rates were 0% at all dosages. The mechanism of penetration enhancement of the ethosomal cataplasm was systematically explored using an optics microscope, polarization microscope, and transmission electron microscopy. The microstructure, ultrastructure, and birefringent structure in skin were observed. Data obtained in this study showed that the application of ethosomal technology to antimalarial cataplasm could improve the transdermal delivery of drug, enhance the efficacy, and facilitate practical application in clinic.

Pharmacophore modeling and 3D quantitative structure-activity relationship analysis of febrifugine analogues as potent antimalarial agent.

Febrifugine and its derivatives are effective against Plasmodium falciparum. Using PHASE algorithm, a five-point pharmacophore model with two hydrogen bond acceptor (A), one positively ionizable (P) and two aromatic rings (R), was developed to derive a predictive ligand-based statistically significant 3D-quantitative structure-activity relationship (QSAR) model (r(2) = 0.972, SD = 0.3, F = 173.4, Q(2) = 0.712, RMSE = 0.3, Person-R = 0.94, and r(2) pred = 0.8) to explicate the structural attributes crucial for antimalarial activity. The developed pharmacophore model and 3D QSAR model can be a substantial tool for virtual screening and related antimalarial drug discovery research.

Quantitative determination of febrifugine by proton nuclear magnetic resonance spectroscopy with internal standard method / 中国中药杂志

A quantitative nuclear magnetic resonance method(qNMR) was established for determination of the absolute content of febrifugine. Proton nuclear magnetic resonance spectroscopy of febrifugine was obtained in DMSO-d₆ with hydroquinone as the internal standard substance on a Bruker Ascend 600 MHz superconducting nuclear resonance spectrometer at 298 K. The specific parameters were as follows: the observing frequency was 600 MHz,spectra width was 7 211 Hz, pulse width was 9.70 μs, pulse sequence was zg30,scan times was 32 and relaxation time was 2 s. The proton signal peaked at δ 7.71 for febrifugine and δ 6.55 for hydroquinone were selected as the quantification peaks. Linear regression of quantitative peak area ratio of febrifugine-hydroquinone versus their mass ratio yielded a correlation coefficient of 0.999 6 and a regression equation of +0.008 6．The linear range of febrifugine was 2.17-17.07 g·L⁻¹,the precision RSD was 0.78%(=6),the repeatability RSD was 1.2%(=6),and the contents of three batches of febrifugine sample were 94.91%,95.09% and 95.52%,respectively. The content of febrifugine was 96.44% determined by high performance liquid chromatography(HPLC). The relative error of the content of febrigugine determinted by qNMR and HPLC methods was 1.27%. The results showed that the internal standard method of proton nuclear magnetic resonance spectroscopy could be used to determine the absolute content of febrifugine.

Investigation on stability and degradation kinetics of febrifugine / 中国中药杂志

To investigate the stability and degradation kinetics of febrifugine. The results showed that within 24 hours, febrifugine content was decreased by only 1% in mobile phase solvent, but its content was decreased to be 90% of the initial content in the water, methanol, 50% methanol and 10% acetonitrile solution. When the pH value of the solution was between 3 and 7, the retention rate of febrifugine in 24 hours was over 98%, but its content was decreased by about 12% in alkaline solution (pH 9.0). The higher the temperature, the worse the stability of febrifugine. At 40-80 ℃, the content of febrifugine was decreased to be 60% of its initial content in 10 hours, but the content was decreased by only 5% in 10 h at 20 ℃.However, no matter 40 ℃or 60 ℃, febrifugine was mainly transformed into isofebrifugine, and the total content of febrifugine and isofebrifugine was equal to their initial total content in 10 hours, while incase of 80 ℃, the total content was decreased to be 83.33% in 10 h, which suggested that the structure of febrifugine was absolutely changed, not just isomerized to be isofebrigugine at high temperature. Light had a significant impact on the stability of febrifugine. Under bright light, the content of febrifugine was reduced by about 23% in 108 h, but it only decreased by about 10% in the natural light or darkness. In artificial gastric fluid (pH 1.4) and artificial intestinal fluid (pH 6.8), the content of febrifugine was decreased by less than 5% in 10 h. After storage at high temperature(60 ℃), high humidity [(75±1)%] and strong light (3 000 lx) conditions for 10 d, the content of solid febrifugine was decreased by 0.27%, 7.6% and 5.39%, respectively. The degradation of febrifugine basically complied with the first-order reaction kinetic process in the following conditions: in water, methanol, 50%methanol and 10% acetonitrile solvents, alkaline solution (pH>7), different light intensity and different temperatures (20,40 ℃). Therefore, no matter the isolation and purification of febrifugine or the production of the related preparations, it should be done fast in the acidic solution, low temperature and dark conditions, while the febrifugine solid should be kept in dry and dark conditions.

Simultaneous determination of febrifugine and isofebrifugine in Dichroa febrifuga root by HPLC method / 中国中药杂志

To develop the HPLC method for simultaneous determination of febrifugine and isofebrifugine in Dichroa febrifuga root, and on the basis of this, the feasibility of quantitative analysis of multi-component by a single-marker (QAMS) model for the determination of the two alkaloids was investigated. The chromatographic separation was performed on an octadecyl bonded silica gel column with mixed solvent consisting of acetonitrile-water-glacial acetic acid-triethylamine (9∶91∶0.36∶0.745) as mobile phase at a flow rate of 1.0 mL•min⁻¹. The detection wavelength was set at 225 nm, and the column temperature was set at 30 ℃. The linear range of febrifugine and isofebrifugine were 10.7-426 ng and 10.6-424 ng, respectively. Their average recovery were 98.33% (RSD 2.7%) and 100.4% (RSD 1.8%), respectively. On the basis of this established method, febrifugine was used as the internal reference substance to calculate the relative correction factors (RCF) and the relative retention values (RRV) of isofebrifugine to febrifugine. Through a series of methodology evaluations, the two alkaloids were simultaneously assayed only by quantitative determination of febrifugine. This result played the part of demonstration role for the application of QAMS model in the determination of isomers.