Optimization Of Fermentation Process For Hedyotis Diffusa And Its Effect On Immune Regulation In Mice

Objective and Significance: Microbial fermentation of traditional Chinese medicine is a novel technology in pharmaceutical manufacturing,which employs one or several microorganisms under controlled conditions to interact with Chinese medicine,resulting in specific metabolites.This process reduces toxicity,enhances efficacy,and generates new active substances.Hedyotis Diffusa,a medicine capable of clearing heat,detoxification,and boosting immunity,contains various chemical compounds,with flavonoids being one of its main active immunoregulatory components.However,there is a lack of research on the immunomodulatory effects of total flavonoids and fermented extracts from Hedyotis Diffusa.This study uses Bacillus subtilis and Saccharomyces cerevisiae to ferment Hedyotis Diffusa for total flavonoid extraction.We examined the influence of different fermentation strains and conditions on the total flavonoid content and studied the effects of fermentation on immune indices of immunosuppressed mice.The goal is to provide experimental foundations for the development and utilization of active ingredients in Hedyotis Diffusa.Methods: We added different concentrations of Hedyotis Diffusa to the growth medium of Bacillus subtilis and Saccharomyces cerevisiae,acclimatized the microorganisms,and determined the optimal Hedyotis Diffusa concentration for fermentation.We used the total flavonoid yield in the fermentation broth as the research indicator,and optimized fermentation conditions by controlling variables such as inoculum size,fermentation temperature,initial p H of fermentation medium,shaker speed,and fermentation time.Mice(7-8 weeks old,weighing 18-20g)were divided into 16 groups,with each group having equal numbers of males and females.For each fermentation strain,there were 8 groups.Apart from the blank control group,cyclophosphamide at a dose of 80 mg/kg was administered to mice for three consecutive days in the other 7 groups,to establish an immunosuppressed mouse model.The total flavonoids of Hedyotis Diffusa,pre-and post-fermentation,were extracted,purified,and administered at high,medium,and low doses(300,200,100mg/(kg·d)),to the mice for seven consecutive days.Control groups were treated with physiological saline.Twentyfour hours after the last dose,mice were euthanized,eye blood was collected,serum was separated,and spleen indices were determined by weighing.Serum immunoglobulins(Ig G、Ig M、Ig A)and serum cytokines(IL-2、IL-4、TNF-α、 IFN-γ)were measured by enzyme-linked immunosorbent assay(ELISA).Results: After multiple rounds of acclimatization,Bacillus subtilis could stably grow and reproduce in a 12% Hedyotis Diffusa environment,while Saccharomyces cerevisiae could do the same in a 16%Hedyotis Diffusa environment.Through single-factor control,the optimal conditions for maximum flavonoid production were 3% inoculum size,initial fermentation,24 h fermentation time,28℃ fermentation temperature,and 200r/min shaker speed for Bacillus subtilis,and 4% inoculum size,initial fermentation p H 7.5,32 h fermentation time,30℃fermentation temperature,and 200r/min shaker speed for Saccharomyces cerevisiae.After microbial fermentation,the total flavonoid content in Hedyotis Diffusa was higher than before fermentation,with Bacillussubtilis increasing the total flavonoid yield by 11.14%(P<0.05),and Saccharomyces cerevisiae by 9.27%(P<0.05).From the perspective of animal spleen indices,compared with the blank control group,the organ indices(including thymus and spleen indices)of the experimental control group significantly decreased(P<0.01),as did the serum immunoglobulin(Ig G 、 Ig A 、 Ig M)and serum cytokine content(IL-2、IL-4、IFN-r、IFN-a)(P<0.01),indicating statistical significance.At the same dose,the immune level of immunosuppressed mice significantly improved with fermented Hedyotis Diffusa total flavonoids compared to the non-fermented group.Compared with the experimental control group,low-dose groups of total flavonoids of Hedyotis Diffusa before and after fermentation by the two fermentation strains significantly improved the organ indices,immunoglobulin content,and serum content of immunosuppressed mice(P<0.05).The medium and high-dose groups significantly improved these indicators(P<0.01).Conclusion: The use of acclimatized Bacillus subtilis and Saccharomyces cerevisiae to ferment total flavonoids of Hedyotis Diffusa and determine the optimal fermentation process shows that at the same dose,fermented Hedyotis Diffusa total flavonoids have a higher effect on improving immune indices of immunosuppressed mice than the unfermented group.This indicates that microbial fermentation can significantly enhance the effective components of traditional Chinese medicine,demonstrating that this fermentation method is feasible and reliable.