A systematic analysis of anti-diabetic medicinal plants from cells to clinical trials

Effects of medicinal plants and phytochemicals on glucose transport and glucose transporters

Defective insulin-stimulated glucose transport is hallmark of T2DM (Ogurtsova et al., 2017; Nandabalan, Sujatha & Shanmuganathan, 2010; Drissi et al., 2021; Stadlbauer et al., 2016). Glucose transporters (GLUT) of the facilitative diffusion type are a multi-gene family of proteins which function to move glucose across cell membranes (Ogurtsova et al., 2017; World Health Organization, 2019; Singhal, Bangar & Naithani, 2012). Among the facilitative GLUT isoforms, GLUT4 is particularly important as it is expressed predominantly in skeletal and adipose tissues and accounts for post-prandial glucose disposal in these tissues (World Health Organization, 2019; Nandabalan, Sujatha & Shanmuganathan, 2010). Skeletal muscle contribute largely to a greater part of the total body mass in humans and it regulates several physiological processes including up to 85% of insulin-mediated glucose up-take through GLUT4 (Ahmad, Choi & Lee, 2020). Many studies have utilized this for therapeutic management of diabetes and in particular the role of extracellular matrix (Ahmad, Choi & Lee, 2020). Skeletal muscle contraction during exercise improves GLUT4 translocation to the cell membrane for glucose uptake and insulin-sensitivity (Jiang et al., 2013). Also, altered muscle glycogen synthesis play a major role in insulin resistance, and glycogen synthase, hexokinase, and GLUT4 are the major culprit involved in the skeletal muscle pathogenesis of type 2 dibetes (Petersen & Shulman, 2002; Saini, 2010). GLUT2 and GLUT5 are responsible for intestinal glucose and fructose uptake (Schreck & Melzig, 2021), while GLUT1 is present ubiquitously in all the body tissues (Galochkina et al., 2019). The dominant glucose transporters found in the small intestine are sodium-glucose linked transporter 1 (SGLT1) which accumulates glucose into adsorptive epithelial cells against its concentration gradient and GLUT2 which mediates movement of glucose from the epithelial cells into the blood (Schreck & Melzig, 2021); inhibition reduces the amount of glucose absorbed into the body. The hemodynamic activities of the glucose transporters have been extensively researched (Ogurtsova et al., 2017; World Health Organization, 2019; Singhal, Bangar & Naithani, 2012; Nandabalan, Sujatha & Shanmuganathan, 2010). Medicinal plants’ products and phytochemicals that modify the action of the glucose transporters could significantly contribute to the search for effective drugs in the management of diabetes. Table 1 is a collation of studies of medicinal plants known to modulate glucose transport in cell lines. Some notable highlights of this extensive literature are discussed briefly below.

In one of the most comprehensive studies, Schreck & Melzig (2021) used Caco-2 cells exposed to a range of plant extracts to identify potential inhibitors of glucose transport. They reported between 40% to 80% reduction using the methanolic extracts of a range of plants including the fruits of Aronia melanocarpa, Valcheva-Kusmanova et al. (2007) Cornus officinalis, Crataegus pinnatifida, Lycium chinense, and Vaccinium myrtillus; the leaves of Brassica oleracea, Juglans regia, Peumus boldus, and the roots of Adenophora triphylla. The authors also reported 50% to 70% reduction by aqueous extract from the bark of Eucommia ulmoides and fruit skin of Malus domestica. These effects are likely acting via inhibition of GLUT1, the predominant transporter in these cells.

One of the key facets of insulin action is to drive the delivery of GLUT4 molecules from intracellular stores to the surface of fat and muscle cells, a process called ‘translocation’. Stadlbauer et al. (2021) used CHO-K1 cells expressing GLUT4 and total internal reflection microscopy to identify GLUT4 translocation-inducing effects of some thirty plant extracts. Though the taxonomy of some of the plants were not fully defined, they included Hoodia, Sapindus mukorossi, Quillaja saponaria, Papaver, Castanea, Bitter orange (genus and species not specified), Oregon grape (genus and species not specified), Common daisy flowers, Rosebay willowherb leaves and Goldenrod flower as potential compounds that could be exploited as potential anti-hyperglycemic agents in the treatment of T2DM via effects on GLUT4 redistribution.

While the above study used a non-classical insulin target tissue (for good experimental reasons), others have focused upon more physiological cell systems. Through bioassay-guided fractionation, Kanaujia et al. (2010) reported two chebulinic acid derivatives from Capparis moonii with significant stimulatory effects on glucose uptake effects concomitant with increased IR-β, Insulin receptor substrate-1 (IRS-1) phosphorylation, and mRNA expression of GLUT4 and PI3K in L6 muscle cells. Carnosol from rosemary extract stimulated AMPK-dependent GLUT4 translocation with no effect on Akt phosphorylation in L6 myotubes (Vlavcheski et al., 2018). Methanolic extract of Gundelia tournefortii potentiated insulin-stimulated GLUT4 translocation to the plasma membrane in skeletal muscle L6 cells (Kadan et al., 2018). An aqueous extract of Cassia abbreviata induced a two-fold increase in GLUT4 translocation in C₂C₁₂ (mouse) skeletal muscle cells probably via activation of the canonical PI3K/Akt pathway (Kamatou, Ssemakalu & Shai, 2021).

Naowaboot and colleagues reported the mechanism of antihyperglycemic effects of Morus alba leaf extract, including increasing glucose uptake via activation of the PI3K pathway and the plasma membrane translocation of GLUT4 in rat adipocytes (Naowaboot et al., 2012). Ethyl acetate extract and 3β-taraxerol isolated from Mangifera indica promoted increased GLUT4 translocation and glycogen synthesis in 3T3-L1 adipocytes (Nandabalan, Sujatha & Shanmuganathan, 2010). The study also noted the effect on glycogen synthesis was due to PI3K-dependent activation of Akt with subsequent inactivation of glycogen synthase kinase 3B (GSK3β) phosphorylation (discussed further below). The fruit of Citrullus colocynthis enhanced insulin-induced GLUT4 translocation and Akt phosphorylation in 3T3-L1 adipocytes (Drissi et al., 2021).

Stadlbauer et al. (2016) screened further natural products as alternatives to insulin through quantitation of GLUT4 translocation in insulin-sensitive CHO-K1 and importantly extended their remarkable study into commercial adipocyte cells. Of the seven medicinal plants tested, Portulaca oleracea and Coccinia grandis were found to induce GLUT4 translocation together with increased glucose concentration uptake likely mediated by PI3K/Akt pathway in adipocytes (Stadlbauer et al., 2016).

Such findings, together with the many others noted in Table 1 and which space preclude detailed discussion of here, suggest natural products can drive re-distribution of GLUT4 to the plasma membrane in an insulin-mimetic manner. However, the effects are not confined to GLUT4. For example, Kang and colleagues reported upregulation of GLUT2 in the liver (and up-regulated GLUT4 in muscle) as the possible mechanism of the antidiabetic effects of Panax ginseng (black ginseng). Manzano & Williamson (2010) investigated the glucose uptake inhibition of polyphenols, phenolic acid, and tannins from strawberry (var. Abion) and apple (var. golden delicious) in Caco-2 intestinal cell monolayers and reported increased inhibition of GLUT2 and SGLT1 and reduced glucose intestinal bilayer transport. Enhancement of glucose uptake by mitragyna speciosa and mitragynine in rat L6 myotubes is associated with increased GLUT1 protein content (Purintrapiban et al., 2011). And the glucose uptake (GLUT4) enhancement activity of G. sylvestre in L6 myotubes alongside the amelioration of insulin resistance in the 3T3-L1 adipocytes cells have been reported (Kumar et al., 2016). One further study of note is the report that the methanol extract of the aerial part of Selaginella tamariscina enhanced glucose uptake in 3T3-L1 adipocytes, possibly by inhibition of PTP1B (El-Zein & Kreydiyyeh, 2011; Giro et al., 2009). Collectively, these studies reveal that medicinal plants have a range of action on glucose transport across multiple tissues of relevance to the treatment of diabetes.

Medicinal plants and phytochemical effects on PI3K and Akt activity

In the canonical insulin signaling pathway controlling glucose homeostasis, insulin receptor substrates are phosphorylated on tyrosine residues which then act as docking sites for downstream signaling molecules. Of particular note, IRS-1 recruits phosphoinositide 3-kinase (PI3K) (Petersen & Shulman, 2018). PI3K phosphorylates phosphatidylinositol 4,5-biphosphate to form phosphatidyl-inositol 3,4,5-trisphosphate that in turn promotes Akt phosphorylation and activation (World Health Organization, 2019; Luna-Vital & De Mejia, 2018). Akt is an important nexus on the insulin signaling cascade because of its multi-substrate activities (Naowaboot et al., 2012; Nandabalan, Sujatha & Shanmuganathan, 2010; Tonks et al., 2013; Huang et al., 2018). Phosphorylation of Akt initiates a cascade of downstream events through many substrates, including phosphorylation of Akt substrate of 160 kDa (AS160), a RabGAP (GTPase activating protein); this in turn leads to GLUT4 translocation in muscle and adipose cells (Matschinsky, 2005; Sharma et al., 2021). Defects in Akt phosphorylation, as seen in impaired insulin activation, are associated with development of muscle and adipose insulin resistance in obesity and T2DM (Naowaboot et al., 2012; Joshi et al., 2013; Luna-Vital & De Mejia, 2018; Kim et al., 1999). The insulin PI3K/Akt pathway has been widely targeted in T2DM pharmacotherapy (World Health Organization, 2019; Naowaboot et al., 2012; Luna-Vital & De Mejia, 2018) and is important in the pathophysiology and therapy of other diseases (Nurcahyanti et al., 2021). Evidence of medicinal plants and phytochemicals modifying PI3K/Akt actions have been documented and are presented in Table 2. Some examples are briefly outlined below.

An ethyl acetate extract and 3β-taraxerol of Mangifera indica significantly activate GLUT4 translocation via a PI3K dependent pathway in 3T3-L1 adipocytes (Nandabalan, Sujatha & Shanmuganathan, 2010). Polysaccharides from corn silk (Maydis stigma) increased phosphorylation of Akt in a dose-dependent manner in L6 skeletal muscle myotubes (Guo et al., 2019). Similarly, phosphorylation of Akt in response to an extract of Folium sennae was described, together with a significant enhancement of GLUT4 translocation (Zhao et al., 2018a). Antidiabetic effect of alkaloids from the seeds of Nigella glandulifera increased the activity of the PI3K/Akt pathway in L6 myotubes with a concomitant increase in glycogen synthesis and hexokinase activity (Tang et al., 2017). Four C21 steroidal glycosides A-D from G.sylvestre promoted GLUT4 translocation to the plasma membrane in L6 cells via activation of PI3K/AKT (Li et al., 2019a). These and other studies (Table 2) point to a significant number of useful PI3K/Akt modulators within medicinal plants.

Many studies have examined effects of medicinal plants in hepatoma cells, as liver is a key site of post-prandial glucose disposal with a notable emphasis on PI3K/Akt signaling (Table 2). Heteropolysaccharides of Grifola frondosa (edible mushroom) increased IRS1/PI3K mRNA levels and enhanced insulin sensitivity (Chen et al., 2018). Polysaccharides of Dendrobium officinale increased the activity of PI3K and Akt and partially ameliorated symptoms in diabetic mice, pointing to a clear effect in a complex organism rather than simply in cell culture models (Wang et al., 2018). Inhibition of phosphorylated insulin receptor substrate-1(IRS-1), but activation of PI3K/Akt in insulin-resistant HepG2 cells by polyphenol-rich extract of Zhenjiang aromatic vinegar has been documented (Xia et al., 2021). Homogeneous polysaccharides from Sargassum pallidum ameliorate insulin resistance by upregulation of PI3K, Glycogen synthase (GS), and IRS-1 expression in insulin-resistant HepG2 cells (Cao et al., 2019). Monosaccharides from Anemarrhena asphodeloides Bunge exhibited hypoglycemic effects by activating PI3K/Akt, IRS-1signaling pathway, and inhibiting α-glucosidase activities in insulin-resistant-HepG2 cells (Chen et al., 2022). A natural flavonocoumarin (α-Methylartoflavonocoumarin) isolated from Juniperus chinensis was reported to activate PI3K/Akt pathway in insulin-resistant HepG2 cells (Jung et al., 2017). Thus, effects are evident both at the level of gene expression and activity mediated by a range of extracts.

Activity of medicinal plants and phytochemicals on glucokinase

Glucokinase (GCK) is important in the regulation of glucose metabolism in liver and pancreas. GCK is essential for pancreatic insulin secretion and hepatic insulin action via phosphorylation of glucose to glucose 6-phosphate (Naowaboot et al., 2012; Balakrishnan, Krishnasamy & Choi, 2018). Low GCK levels have been observed in T2DM (Haeusler et al., 2015) and could serve as a potential drug target for therapeutic intervention (Kim et al., 2013; Yang, Jang & Hwang, 2012). GCK is currently being targeted as therapeutic for T2DM (Matschinsky, 2009; Matschinsky & Wilson, 2019). Glucokinase activators have been advocated as an alternative approach to restoring and improving glycemic control in T2DM (Zhou et al., 2001; Towler & Hardie, 2007; Toulis et al., 2020). Several medicinal plants and phytochemicals with glucokinase activities have been recognized (Sharma et al., 2021). We have focused on the cellular and molecular glucokinase activities of medicinal plants in tissue culture models (Table 3).

Glucokinase activation by the leaf extract of Costus igneus (known in India as the ‘insulin plant’ for its purported anti-diabetic action) was examined in differentiated human hematopoietic stem cell (HSCs) as a model of β-cells. The extract increased GCK and inhibited of glucose-6-phosphatase activity by C. igneus, thereby improving glucose sensing, insulin production, and decreased gluconeogenesis (Kattaru et al., 2021). It was also reported that C. igneus profoundly increased insulin receptor and GLUT2 gene expression (Table 1). Insulin-like proteins (ILP) purified from C. igneus also showed hypoglycemic activity in insulin-responsive cell line RIN 5f cells (Joshi et al., 2013). Activation of free fatty acid-receptor1 (FFAR1) and GCK by anthocyanin-rich extract from the pericarp of purple corn was demonstrated in HepG2 cells (Luna-Vital & De Mejia, 2018). Significant elevations in glucokinase gene expression in response to ethanol, ethyl acetate, and n-hexane fruit extract of Momordica balsamina, were reported in RIN-m5F cells (Kgopa, Shai & Mogale, 2020).

Medicinal plants modifying activity of glycogen synthase kinase-3 (GSK-3)

GSK-3 inhibits glycogen synthase activity. Insulin phosphorylates GSK-3 and prevents glycogen synthase inactivation (Nabben & Neumann, 2016). This role of GSK-3 in the insulin signaling pathway provides a mechanistic approach to the use of GSK-3 inhibitors in the treatment of insulin-resistant diabetes. Two studies are worthy of comment. Ethanolic extract of Shilianhua (Sinocrassula indica Berge) was found to induce GSK-3β phosphorylation similarly to insulin in 3T3-L1 preadipocytes and rat skeletal L6 myoblasts, indicating a possible mechanism of antidiabetic activity (Yin et al., 2009). This extract also enhanced insulin-stimulated glucose consumption in L6 myotubes and H4IIE hepatocytes, and insulin-independent glucose uptake in 3T3-L1 adipocytes. The result also showed increased GLUT1 protein expression in 3T3-L1 and GLUT4 protein expression in L6 myotubes cells (Yin et al., 2009). Hot water reduction from the root of Sarcopoterium spinosum increased glycogen synthesis via induction of GSK-3 β phosphorylation in L6 myotubes (Sahuc, 2016). S. spinosum also enhanced basal insulin secretion in the pancreatic β-cells and inhibited isoproterenol-induced lipolysis in 3T3-L1 adipocytes (Sahuc, 2016).

Peroxisome proliferator-activated receptor-gamma (PPARγ) and medicinal plants

Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of the nuclear receptor super-family which play integral roles in glucose and lipid metabolism (Mirza, Althagafi & Shamshad, 2019). These receptors are targets for diabetes therapy and also for the treatment of cardiovascular disease, cancer, and inflammation (Mirza, Althagafi & Shamshad, 2019). We present the effects of various medicinal plants on PPARγ activity and gene expression (Table 4).

Chloroform extract of the fruit of Momordica charantia has been reported to significantly increase PPARγ gene expression 2.8-fold, comparable to the insulin sensitizer rosiglitazone (2.4-fold) in L6 myotube skeletal muscle cells (Kumar et al., 2009). Huang et al. (2005) demonstrated that Punica granatum flower extract and ethyl acetate fractions enhanced PPARγ gene expression and protein levels in a macrophage cell line. Increased mRNA of PPARγ (and GLUT4) by Nymphaea nouchali seed extract in 3T3-L1 adipocytes as the possible mechanism of its anti-hyperglycemic effect was reported (Parimala et al., 2015). Exposure of 3T3-L1 adipocytes to an ethanol extract of Miconia increased mRNA of PPARγ by 1.4-fold and inhibited α-amylase and α-glucosidase. The extract also increased lipid accumulation by around 30% as a possible anti-diabetic mechanism of action (Ortíz-Martinez et al., 2016). Upregulation of PPARγ together with GLUT4, SREBP and FAS expression was observed in 3T3-L1 adipocytes treated with the leaf extract of Moringa concanensis (Balakrishnan, Krishnasamy & Choi, 2018). Effects in muscle models have also been reported: Kim et al. (2013) reported increased transcription activity and mRNA levels of PPARγ in C₂C₁₂ myotubes by Boehmeria nivea ethanol leaf extract and Korean red peppers (Yeongyang korea) increased glucose uptake in C₂C₁₂ via increased transcriptional activity of PPARγ (Yang, Jang & Hwang, 2012).

AMP-activated protein kinase (AMPK)

AMP-activated protein kinase (AMPK) is a known energy sensor for metabolic homeostasis (Steinberg & Carling, 2019) which plays a central role in regulating lipid and protein metabolism together with fatty acid oxidation and muscle glucose uptake (Sathishsekar & Subramanian, 2005). AMPK plays a crucial role in insulin sensitivity, which explains its place as a potential drug candidate for T2DM therapy (Tasic et al., 2021; Hawkins et al., 2021). AMPK systems have been said to be partly responsible for the health benefits of exercise and AMPK is an important downstream effector of metformin. It has also been proposed as a possible target for novel drugs in managing obesity, type 2 diabetes, and metabolic syndrome (Hu, Zeng & Tomlinson, 2014; Hosseini et al., 2014; Kim et al., 2016). Ethnopharmacological investigators have reported that several medicinal plants modulate the activity of AMPK in cell models, and we have summarized these reports in Table 5, and highlight a few notable studies below.

Ethanolic extract and phytochemical compounds from Cimicifuga racemosa mediate increased AMPK activity in fully differentiated HepaRG cells and is a possible mechanism of antidiabetic activity (Moser et al., 2014). Yuan & Piao (2011) reported activation of AMPK by the petroleum ether fraction of Artemisia sacrorum in HepG2 cells. They showed increased phosphorylation of AMPK (on T172), acetyl-CoA carboxylase (ACC; reside S79), and GSK-3β and reported concomitant downregulation of phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6Pase). Similarly, Zheng et al. (2016) exploring the anti T2DM activity of Entada phaseoloides in primary mouse hepatocytes and HepG2 cells, reported suppression of hepatic gluconeogenesis via activation of the AMPK signaling pathway and Akt/GSK-3β. Extract of Rosmarinus officinalis significantly increased glucose consumption in HepG2 cells via increased phosphorylation of AMPK and ACC and potentially increased liver glycolysis and fatty acid oxidation (Tu et al., 2013). Thus, numerous examples of medicinal plants exerting effects via AMPK in hepatoma cell lines have been described (Table 5).

Effects mediated via AMPK have been reported in other cell types. These include an alcoholic extract of Artemisia dracunculus enhanced insulin release from β-cells isolated from mouse and human islets via activation of AMPK and suppressed LPS/IFNγ-induced inflammation. Effects on glucose transport in muscle lines include an extract of Crocus sativus (saffron) which increased glucose uptake and insulin sensitivity in C₂C₁₂ myotubes by increased phosphorylation of AMPK in a dose and time-dependent manner (Kang et al., 2012) and compounds isolated from the seed of Iris sanguinea was reported to be via AMPK and ACC phosphorylation in the same cell type (Yang et al., 2017).

Ethanolic extract and isolated compound (β-sitosterol) from Malva verticillata seed significantly increased activation of AMPK as the molecular mechanism for glucose uptake in L6 myotubes (Jeong & Song, 2011). Triterpenoids from the stem of Momordica charantia have been reported to overcome insulin resistance in FL83B and C2C12 via AMPK activation (Cheng et al., 2008). Hence, the effects of such compounds on AMPK is an active and vigorous area of research.