*4.4. Gut Microbiota Modulation*

Both iron deficiency and excess iron can lead to dysbiosis, characterised by an imbalance in the gut microbial community, and are associated with diseases [128]. Recent research has found ginger to exact prebiotic effects that improve gut microbiota composition. In a study that stimulated digestion and fermentation in vitro, 85% of the polyphenols in a dry ginger powder were still detectable in the digestive fluids after simulated digestion [146]. These polyphenol constituents include 6-, 8-, 10-gingerols and 6-shogaol. The undigested ginger extract significantly modulated faecal microbiota structure following mixed-culture fermentation with faecal inoculation compared with the control group. After 12 h of fermentation, the abundances of the beneficial bacterial groups of *Bifidobacterium*

(*p* < 0.05) and *Enterococcus* (*p* < 0.01) were significantly higher in the ginger group than in the control group. The study also found elevated levels of short-chain fatty acids (SCFA) accompanied by decreased pH value after fermentation with ginger extract compared to control. The results demonstrated that ginger and its polyphenol compounds could improve human health through gut microbiota modulation [146].

Ginger's effects on gut microbiota modulation were also confirmed in a mice model in an in vivo study [147]. Five-week-old C57BL/6J male mice were fed a high-fat diet with or without ginger supplementation for 16 weeks. With ginger treatment, mice on a high-fat diet showed lower body weight and amelioration of liver steatosis, low-grade inflammation, and insulin resistance compared to controls. Analysis of the gut microbiome showed an increase in *Bifidobacterium* genus and SCFA-producing bacteria (*Alloprevotella* and *Allobaculum*) and increases in faecal SCFA concentrations. As a high-fat diet promotes oxidative stress and chronic low-grade inflammation associated with metabolic dysfunction, this study demonstrated that ginger supplementation could mitigate the detrimental impact of a high-fat diet on gut microbiota composition to promote health.

Another in vivo study also found ginger to have therapeutic effects in relieving diarrhoea after antibiotic use through gut microbiota recovery [148]. The study used 5-week-old Sprague-Dawley rats treated with antibiotics by gavage for seven days before administering ginger extract for another seven days. The study found ginger treatment significantly reduced diarrhoea symptoms (*p* < 0.05) compared to the control group that did not receive ginger treatment. Furthermore, the ginger treatment also considerably increased microbiota diversity in the gut, showing accelerated recovery. Specifically, the abundance of Proteobacteria phyla was increased by antibiotics treatment but restored significantly after ginger administration (*p* < 0.0001). In contrast, antibiotic treatment depressed the abundance of Bacteroidetes phyla and saw significant improvement with ginger treatment (*p* < 0.001). The study found *Escherichia Shigella* decreased at the genus levels, whereas Bacteroides increased the most in relative abundance. Histopathological observation of the colon also revealed evidence of intestinal barrier integrity improvement with ginger treatment associated with restoring tight junction protein Zonula occludens-1.

Changes in gut microbiota compositions in humans were also observed after consumption of ginger in a recent RCT [149]. The study recruited 138 healthy adults. All participants were advised to consume their usual diet but avoid ginger-rich products, probiotics, and prebiotics for one week before starting the study. During the intervention period, the participants were randomly assigned to take either fresh ginger juice (ginger group, n = 68) or sterile 0.9% sodium chloride (control group, n = 66) daily for seven days. Blood serum and faecal samples were collected at baseline and after seven days. A total of 4 participants in the control group and 7 in the ginger group were lost to follow-up, with only 123 participants completing the study. The study found increased counts of intestinal bacterial species when comparing the taxonomic composition between the ginger and control groups. The ginger juice intervention decreased the relative abundance of the Prevotella-to-Bacteroides ratio and the pro-inflammatory *Ruminococcus\_1* and *Ruminococcus\_2* genus and increased the Firmicutes-to-Bacteroidetes ratio, *Proteobacteria* and anti-inflammatory *Faecalibacterium*. Hence, the study concluded that consuming ginger juice for a short period had substantial effects on the composition and function of gut microbiota in healthy people.

There is currently no known pre-clinical and clinical study on the effect of ginger on the gut microbiota in the case of anaemia or iron deficiency. Nevertheless, prebiotics, such as inulin, are known to affect gut microbiota to improve iron absorption in IDA [130]. Thus, it is logical to assume a similar positive impact of ginger on IDA through inferences. This is an area of further research.

#### *4.5. Erythropoiesis Stimulation*

Erythropoiesis, the process of producing RBCs, is impeded in IDA due to insufficient dietary iron intake, impairment in iron absorption as affected by inflammation, or an imbalance between surging iron requirements and iron available resulting from rapid growth or heavy blood loss [159]. In fact, the use of ginger for haematopoiesis has an ethnomedicinal origin. Dry ginger is used in traditional Chinese medicine as a warm herb to promote blood flow, remove blood stasis, and alleviate weakness and fatigue. As such, ginger is used in many blood tonic herbal formulas for treating blood circulation, anaemia, and haemorheological conditions [160,161]. Ginger is also a tonic food recommended for postpartum women during the one month confinement period immediately after delivery for recovery from blood loss. Specifically, in Southern China, traditional postpartum dietary practices include the 'ginger vinegar soup' made from sweet vinegar, ginger, egg, and pig's trotters [162]. This is an example of iron-rich food leveraging ginger as a functional ingredient to improve iron bioavailability and promote erythropoiesis. Thus, the ability of ginger to stimulate erythropoiesis can be an added benefit to the effectiveness of oral iron therapy for IDA treatment.

In a study that utilised zebrafish embryos to investigate the effect of ginger extract on haematopoiesis in vivo, Ferri-Lagneau et al. [150] found ginger, with its bioactive compounds of 8-gingerol, 10-gingerol, 8-shogaol, and 10-shogaol, promoted the expression of GATA-binding factor 1 (Gata1) in erythroid cells. Gata1 is an early marker and key regulator of erythropoiesis. Moreover, increases in the expression of haematopoietic progenitor markers cmyb and scl were also observed. The study also identified 10-gingerol as the most potent stimulator in promoting the primitive wave of erythropoiesis in early developing zebrafish embryos. The study further confirmed that the haematopoiesis effect of ginger was mediated through the bone morphogenetic protein (Bmp) signalling pathway.

In a subsequent study, the same group of researchers further demonstrated that ginger/10-gingerol can rescue the expression of haematopoietic stem/progenitor cells (HSPC) in zebrafish embryos with genetic defects [151]. Ginger was found to induce scl/runx1 expression through Bmp and Notch signalling pathways that led to arteriogenesis and HSPC formation. Bmp and Notch are known to regulate nitric oxide (NO) production, which plays an active role in the modulation of haematopoietic cell growth and differentiation. The study also showed that ginger produced a robust up-regulation of NO in the rescued mutant zebrafish embryos. Therefore, the combined effect of ginger on Bmp, Notch and NO production can be beneficial for the regulation of erythropoiesis for regeneration/recovery.
