Prebiotic properties of jiaogulan in the context of gut microbiome

Abstract Jiaogulan (Gynostemma pentaphyllum) is a traditional Chinese medicinal herb that has been widely used in food and supplemental products. In the last 20 years, extensive research has been conducted to investigate the medicinal prospects of jiaogulan, and in this regard, more than 200 compounds have been isolated with various medicinal properties such as anticancer, anti‐obesity, anti‐inflammation, and antioxidation. In respect of potential benefits, jiaogulan market is likely growing, and various food items comprised of jiaogulan (beverage, sport drinks, cola, beer, tea, bread, and noodles) have been commercialized in the United States of America, China, and other Asian countries. More recently, there has been growing interest in the prebiotic potential of jiaogulan, especially at the interface of the gut microbiota. This review focuses on the prebiotic and therapeutic aspects of saponins and polysaccharides of jiaogulan tea by summarizing the literature on cancer, obesity, antioxidant activity, and immune‐modulatory properties.


| INTRODUC TI ON
Gynostemma pentaphyllum Makino (Cucurbitaceae; Gp) is a perennial creeping plant and has been used for herbal tea (called jiaogulan) in China. For centuries, the herbal tea made from the aerial part (including stems and leaves) of Gp has been consumed in China as a general tonic. Today, it is progressively popularizing around the world for lowering serum lipid and cholesterol levels (Chen et al., 1991;Lin et al., 2000). Like green tea, jiaogulan tea also holds anticarcinogenic and antioxidative activities (Lin et al., 2000;Razmovski-Naumovski et al., 2005). Increasing research interest in Gp is evident from a search of the PubMed database ( Figure 1).
Traditionally, jiaogulan has been broadly applied for the treatment of various illnesses, including hepatitis, diabetes, and cardiovascular disease . However, in the last 20 years, extensive research has been conducted to investigate the medicinal prospects of jiaogulan, which has resulted in the discovery of more than 230 compounds with medicinal properties. These compounds have shown a variety of pharmacological properties, including anti-inflammatory (Cai et al., 2016;Quan & Qian, 2010), antioxidative (Zhao et al., 2014), antiproliferative (Yan, Wang, Niu, et al., 2014), anxiolytic activities (Choi et al., 2013), anti-cancer (Hou et al., 1991), lipid metabolism regulation (Qin et al., 2012), anti-diabetes , and cardiovascular disease treatment (Circosta et al., 2005). Out of 230 compounds, 189 are saponins, also known as gypenosides . Among these gypenosides, 165 have been classified into 12 types based on the nature of aglycone moiety (Lin, 2011). The details of these gypenosides and their pharmacological properties have been discussed elsewhere (Nguyen et al., 2021). Gypenosides possess several therapeutic properties including anticancer and anti-obesity (Lee et al., 2019;Lu et al., 2010).
Like other basic research, Chinese medicine research is entering into a new paradigm from one-gene-one-phenotype model toward a much sophisticated and complex model, known as omics strategies, that is based on data-driven untargeted management, diagnosis, and treatment (Cagan et al., 2013;Yoo et al., 2018). Chinese medicine is holistic in nature and it would be impractical to comprehend it with conventional research tools. Therefore, more recently, researchers have started to evaluate the prebiotic potential of jiaogulan at the interface of the gut microbiota (GM).
The pharmacological properties of jiaogulan at the interface of the GM should be evaluated as commensals that play an important role in human physiology. For instance, the human microbiome constitutes about 47% of our body by cell count and encodes 1000 times more genes than our own body genes (Institute for Genome Sciences, 2017; Knight et al., 2017). It is estimated that the human microbiome encodes 2-20 million genes that surpasses the ~20,000 human genes (Knight et al., 2017). These microbial genes are presented to the host for digestion, metabolism, and immune system maturation (Cani, 2009). Targeted intervention to remodel GM composition has shown encouraging results in disease prevention and treatment.
Mounting lines of evidence indicate that various bioactive natural products, such as dietary fibers, phenolic compounds, and undigested carbohydrates, can upregulate beneficial intestinal microbes, improve gut homeostasis, and alleviate disease symptoms (Makki et al., 2018).
In this review, we highlight some key findings by evaluating jiaogulan extracts and the therapeutic nature of purified compounds (gypenosides, polysaccharides, and flavonoids) at the interface of the GM. The study particularly focuses on the anticancer, antiobesity, and antidiabetic properties of jiaogulan.

| ANTI C AN CER PROPERTIE S
Jiaogulan is known to possess potent anticancer abilities.
So far, several mechanisms of action have been determined regarding the anticancer activities of jiaogulan, including antioxidant , cell cycle arrest, apoptosis, prevention of invasion and metastasis (Yan, Wang, Niu, et al., 2014), and immunomodulating activities. For instance, G. pentaphyllum saponin (GpS) was reported for the anticancer properties by upregulating Prdx1 and Prdx2 expression and suppressing Ras, RAF/MEK/ERK/STAT, PI3K/ AKT/mTOR signaling (Tai et al., 2016). Another in vitro study also showed that GpS revealed the anti-proliferation effect by arresting cell cycle at the G0/G1 phase, and induced apoptosis of HepG2 cells via death receptor and mitochondrial pathway (Hussain et al., 2020).  showed that GpS could significantly upregulate the intracellular ROS level, which induced cell toxicity, apoptosis, and mitochondrial damage in colorectal cancer cells. The anticancer abilities of jiaogulan, both direct and indirect, have been summarized in Figure 2.

| Anticancer properties of jiaogulan's saponin
Unlike the in vitro system, saponins are poorly absorbed and have a long residence time in the intestine when tested in preclinical models (Navarro del Hierro et al., 2018). However, through the recent integration of herbal medicine and GM research, the long residence of saponin in the intestine turned out to improve its efficacy. We and several other studies have demonstrated that GpS improves gut microbial composition by promoting the growth of beneficial bacteria and suppressing potential pathogens (Chen et al., 2015Huang et al., 2017Huang et al., , 2018Khan et al., 2019;Shen, Zhong et al., 2020). While evaluating the anticancer effects of GpS in a Apc Min/+ mouse model, GpS displayed a stimulating effect on the abundance of Lactococcus, Bifidobacterium, Lactobacillus, and short-chain fatty acids (SCFAs) producing bacteria. However, the growth of potential pathogens, for example, Dysgonomonas spp., Helicobacter spp., sulfate-reducing bacteria, were suppressed after GpS introduction to mouse gut (Chen et al., 2015Huang et al., 2017;Khan et al., 2019;Liao et al., 2020) (Figure 3).

F I G U R E 1 Bar chart presentation
of Gynostemma pentaphyllum reported in PubMed database. These data were generated by including clinical trials, research articles, review articles, and abstracts. Numbers at the top of the bar show G. pentaphyllum reported times per year, ranging from 2000 to 2021. The search was conducted on August 4, 2021 After noticing the stimulating effect of GpS in SCFAs producer in the mouse gut, we proved that GpS could increase the growth of Bifidobacterium animalis, Lactobacillus casei, and Lactobacillus reuteri (Liao et al., 2020). By gavaging B. animalis and butyrate (separately) to a cancer preclinical mouse model and noticing anticancer effects, we proved that the anticancer effect of GpS is partly through stimulating the growth of beneficial bacteria in the gut (Liao et al., 2020).

| Anticancer properties of jiaogulan's polysaccharides
In addition to the GpS, GP's polysaccharides (GpP) also be reported for anticancer properties. Polysaccharides are potential prebiotic polymers that have been extensively studied. They promote the growth of certain beneficial bacteria (e.g., Bifidobacterium, Lactobacillus) and in the large intestine, metabolize into lactic acid and SCFAs that improve host physiology, particularly gastrointestinal health (Azmi et al., 2012;Devillé et al., 2007;Zaporozhets et al., 2014). Accumulating evidence reports that GpP revealed the anticancer effect in vivo and in vitro. The molecular weight, degree of branching, and solubility of GpP are closely related to its anticancer property. Different molecular weights in the range of 10 3 -10 6 Da have been found in various GpP using different experimental conditions (Ji et al., 2018). A neutral polysaccharide fraction of GP was found to effectively inhibit the solid tumor growth of H22 hepatocarcinoma transplanted in ICR mice . Another study showed that GpP improved the pro-

| Anticancer properties of jiaogulan's flavonoid
Flavonoid is a main polyphenolic compound that is widely found in herbal medicines. Flavonoid is also a major constituent of GP, and re-

F I G U R E 3 Schematic presentation of GpS' anticancer effects through gut microbiota. (a) In colorectal cancer (CRC), the intestinal track
is characterized by polyp formation, imbalanced gut microbiota, reduced mucus layer, suppressed population of goblet and Paneth cells, and inflamed immune milieu. (b) Treated CRC preclinical mouse model with GpS reinstates the inflamed mucosal immunity, promotes goblet and Paneth cell population -that results in mucus layer thickness and higher secretion of lysozyme. Most importantly, the gut microbial composition improves with the prevalence of SCFA producers. (c) At the subcellular level, GpS-associated increase in SCFAs upregulates fatty acid-sensing GPCRs that results in the suppression of histone deacetylases and PPARγ, which downstream inhibits PI3K/AKT oncogenic signaling pathways, as well STAT3 and Src. This graph is based on results published by Hsiao's group Khan et al., 2019;Liao et al., 2020) gypenoside UL4 enriched in GP extract exerted the hepatoprotective effect on diet-induced NAFLD through increasing levels of sirtuin 6 and phase 2 antioxidant enzymes in vivo and in vitro. A recent study showed that GpS could change the GM composition of NAFLD mice to alleviate disease progression. The results showed that GpS reduced the ratio of Firmicutes to Bacteroidetes, elevated GM diversity, and decreased the relative abundance of Fissicatena and Akkermansia, which are enriched in high-fat and high-cholesterolinduced NAFLD mice . Similar research also showed that GpS alleviated NAFLD by maintaining the gut barrier and reversing gut dysbiosis in a high-fat diet-induced NAFLD rat model. Results showed that GpS reduced the ratio of Firmicutes to Bacteroidetes; meanwhile, GpS enriched the abundance of beneficial bacteria (Lactococcus spp.) and inhibited potential pathogens (Shen, Zhong, et al., 2020).

| A NTI -OB E S IT Y EFFEC T
AMPK is an intracellular energy sensor and regulates the wholebody and cellular energy balance in response to energy demand and supply. Nguyen et al. (2011) demonstrated that dammaranetype glucosides from GP were the potential activator of AMPK.
Further study from this research team also showed that GP enriched with saponins could improve obesity in ob/ob mice by activating AMPK (Gauhar et al., 2012). Lee et al.

| ANTID IAB E TI C EFFEC T
Gynostemma pentaphyllum saponin has been reported for hypoglycemic properties by enhancing the Nrf2 signaling pathway in streptozotocin-induced diabetic rats . GP containing standardized gypenosides significantly elevated the plasma insulin concentration and profoundly affected the intraperitoneal insulin tolerance test compared with the control group (Wang, Ha, et al., 2018). In the streptozotocin-induced diabetic rat model, GpS showed the hypoglycemic effect through enhancing the Nrf2 signaling pathway. Results also showed that GpS increased the level of insulin in the blood, as well as increased SOD and GSH-px activities . Norberg et al. (2004) isolated a novel gypenoside from GpS, which was named phanoside, and the results showed that phanoside and its stereoisomers could significantly stimulate insulin secretion. Wang, Ha, et al. (2018) found that two compounds from gypenosides could significantly enhance 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose  (Ding et al., 2010). Aktan et al. (2003) found that GpS could suppress NO synthesis in murine macrophages by inhibiting iNOS enzymatic activity and attenuating NF-κB-mediated iNOS protein expression. Tsang et al. (2019) reported that GpS induced melanogenesis and activated cAMP/PKA and Wnt/β-catenin signaling pathways in both B16 and B16F10 cells. Yang et al. (2013) found that two new saponins from Gp could inhibit lipopolysaccharide (LPS)-induced IL-1β, IL-6, and COX-2 mRNA expression in RAW 264.7 which showed a prominently anti-inflammatory effect. Li et al. (2015) isolated three acid polysaccharides from GP-GPA1 (19.6 kDa), GPA2 (10.6 kDa), and GPA3 (6.7 kDa)that displayed the antioxidant effect through scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and hydroxyl radical, chelating ferrous ion, and reducing ferric ion. GpP increased the scavenging activity of DPPH, hydroxyl radical, superoxide anion, and ABTS radical in vitro . Furthermore, an animal experiment showed that GpP could enhance SOD, CAT, GSH-Px activities, and decrease MDA activity

| IMMUNOMODUL ATION EFFEC T
Mounting evidence indicated that polysaccharides from Chinese herbal medicines usually act as an immunomodulator that provides benefits for the host Gan et al., 2004;Khan et al., 2019;Xu et al., 2011;Zhao et al., 2010). GpP activated macrophage phagocytosis and NK cells, and exhibited activity on none or Con A/LPS-stimulated splenocytes in C57BL/6 mice. GpP also increased CD4 + lymphocyte quantitation and the ratio of CD4 + /CD8 + , and increased IL-2 secretion in serum and spleen in immunosuppressed mice (Shang et al., 2016). Ren et al. reported that the acid polysaccharide fraction from Gp could markedly promote the secretion of NO, TNFα, IL-1β, and IL-6 in murine macrophage RAW264.7. The authors claimed that MAPK, PI3K/Akt, and NF-κB signaling pathways were involved in these GpP-induced macrophage activations (Ren et al., 2019). The neutral polysaccharide fraction from GP could modulate the activity of NK cells and cytotoxic T lymphocytes besides increasing the secretion of IL-2, TNFα, and IFNγ in tumor-bearing mice .
The hepatoprotective effect of GpP was proved by decreasing serum ALT and AST levels, as well as the hepatocyte MDA content and hepatocyte necrosis in the liver-injured animal model (Song et al., 2013;Zhang, 2013). GpP possessed hypoglycemic and hypolipidemic effects in a streptozotocin-induced type 2 diabetes rat model (Du et al., 2011). Jia et al. (2015) investigated the neuroprotective effect of GpP and found that GpP could be effective against Aβ (25-35)-induced neurotoxicity in PC12 cells by inhibiting oxidative stress and suppressing the mitochondrial apoptotic pathway (Jia et al., 2015). Moreover, an associated research also showed that treatment with GpP could markedly increase the exhaustive exercise time of mice through scavenging excessive ROS produced during the exercise regimen (Chi et al., 2012).

| CON CLUS ION
This study summarized the therapeutic and prebiotic properties of various saponins and polysaccharides from jiaogulan. The study also highlighted GM-modulating properties of various compounds from jiaogulan. This review further highlighted the therapeutic effect of jiaogulan on the diversity and composition of the GM.

ACK N OWLED EG M ENT
We thanks all the authors' contribution to this work. Thanks for the suggestion from Prof. Li Qingnan from Shantou central hospital.
Thanks for the foundation of The Hospital Incubation Project from Shantou Central Hospital (2020-2022).

CO N FLI C T O F I NTE R E S T
The authors declare no competing financial interest.

E TH I C A L A PPROVA L
Not applicable.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article because no new data were created in this study.