Hyperglycemia metabolism and excessive free fatty acids can lead to the production of lots of free radicals, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS). These free radicals can cause oxidative stress, impair the structures and functions of islet β-cells, and cause insulin secretion deficiency. Besides, they can also lead to insulin resistance by affecting multiple insulin signaling pathways. The antioxidant activity of O. stamineus is related to protecting islet cells and reducing insulin resistance. Researchers have always tested antioxidant activity by 1,1-diphenyl-2-picrylhydrazyl radical 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl (DPPH) assay, ferric ion reducing antioxidant power (FRAP) assay, and 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonate) (ABTS) assay. The activity of superoxide dismutase (SOD) and the level of malondialdehyde (MDA) are also used to determine antioxidant activities. SOD can scavenge free radicals and MDA is the end product of lipid oxidation [44,45].

The antioxidant properties of the ethanol extracts of some genotypes ranged up to 15.55 μmol trolox equivalent (TE)/g dry weight (DW) in DPPH assay, and ranged up to 1.60 mmol TE/g DW in FRAP assay [46]. The half maximal inhibitory concentration (IC50) value of the 70% ethanol extract was 58.85 ± 7.11 μg/mL in DPPH assay, a little higher than 15.05 ± 2.03 μg/mL of the positive control, rosmarinic acid [47]. The concentration value for 50% of maximal effect (EC50) of methanol extract was 0.67 mg/mL in DPPH assay [48]. The IC50 values of 50% methanol extract of O. stamineus leaves were 0.145 ± 0.030, 1.143 ± 0.056, 0.192 ± 0.012, and 0.013 ± 0.001 mg/mL in DPPH, ABTS, iron chelating and FRAP assays, respectively, a little higher than the positive control, rutin and caffeic acid [49]. The EC50 values of O. stamineus aqueous extract were 53.51 and 284.9 μg/mL, respectively, in DPPH and ABTS assays, higher than the positive control, ascorbic acid [50]. The antioxidant capacities of aqueous extract were higher than 20 mg ascorbic acid equivalents (VCEAC)/100 mL in ABTS assays, and about 40 mg VCEAC/100 mL in FRAP assays [51]. The DPPH free radical-scavenging activities of aqueous, 50% methanol, methanol, 70% acetone, and chloroform extracts (0.05 mg/mL) were about 85%, 90%, 88%, 83% and 70%, respectively, higher than some positive controls [52].

From these studies, it could be seen that the aqueous extract, ethanol extract, 70% ethanol extract, methanol extract, 50% methanol extract, 70% acetone extract, and chloroform extract all had free radical-scavenging activities in different assays.

O. stamineus ethanol extract (200 and 400 mg/kg) enhanced SOD activity and reduced MDA level in the liver homogenate of the high-fat diet group. Thus, O. stamineus extract might counteract oxidative stress in the liver [40]. The 50% ethanol extracts of O. stamineus roots, stems, and leaves (50 μg/mL) scavenged intracellular ROS and significantly increased cell viability under oxidative stress in IPEC-J2 cells. They could also decrease the MDA level in jejunal homogenates compared to the high-fat group. The extracts of roots and leaves significantly increased the jejunal SOD activity of mice [53].

In the pathogenesis of diabetes, inflammatory factors, such as interleukin (IL)-1β, IL-8, tumor necrosis factor (TNF)-α, and induced nitric oxide synthase (iNOS), are important factors related to insulin sensitivity. They interfere with insulin signal transduction by participating in the insulin signaling pathway, leading to insulin resistance. They also possibly damage islet β-cells. In addition, inflammatory factors also interact with oxidative stress, further aggravating insulin resistance. Therefore, anti-inflammatory activity is essential to attenuate the inflammatory response, protect islet cells, and improve insulin resistance. It is always tested through the levels of inflammatory factors and the inhibition of nitric oxide (NO) production in cells [54,55].

The swelling in auricle was inhibited by the treatment of ethanol extract, ethyl acetate (EtOAc), and aqueous fractions in acute inflammatory mice induced by xylene. The inhibition ratios were 48.2%, 63.3%, and 46.0% at the dose of 200 mg/kg. Some compounds isolated from EtOAc fractions, orthosiphol M, orthosiphonone A, orthosiphol B, neoorthosiphol A, orthosiphol D, fragransin B₁, sinensetin and 5, 6, 7, 4′-tetramethoxyflavone, also showed marked repression in the observed auricle swelling at the dose of 50 mg/kg. Besides, some of these compounds inhibited pro-inflammatory cytokines production in lipopolysaccharide (LPS)-induced HK-2 cells, such as the levels of TNF-α, IL-1β, and IL-8 [56]. The isolated compounds (clerodens A–D) were studied for anti-inflammatory activities on LPS-induced NO production in RAW264.7 macrophages. The results showed that clerodens A–D had inhibitory activities with IC50 values of 18.9 ± 1.2, 14.7 ± 0.48, 12.4 ± 1.5, and 6.8 ± 0.92 μmol/L, respectively, a little higher than the positive control aminoguanidine [16]. Neoorthosiphonone A, isolated from O. stamineus, showed obvious inhibitory activity on NO production in LPS-activated macrophage-like J774.1 cells with the IC50 value of 7.08 μmol/L, which was more potent than the positive control N^G-monomethyl-_L-arginine (_L-NMMA) [57]. The isolated siphonols A–E also inhibited NO production in LPS-activated macrophage-like J774.1 cells [58].

Diabetic patients often have abnormal lipid metabolism. In the pathogenesis of diabetes, disorders in lipid metabolism increase the levels of free fatty acids and total triglycerides (TG), damaging islet β-cells and leading to insulin resistance in other tissue cells. Because of insulin resistance, the serum levels of TG, total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) increase, while the level of high-density lipoprotein cholesterol (HDL-C) decreases [59]. In addition, leptin and adiponectin, which are secreted from adipocytes, are also associated with insulin resistance. Leptin can antagonize insulin and produce insulin resistance, while adiponectin can improve insulin sensitivity by increasing fatty acid oxidation and glucose uptake in skeletal muscle cells [60,61].

The inhibitory effect of O. stamineus ethanol extract against pancreatic lipase in vitro was determined by using orlistat as the positive control. The IC50 value of the extract was 5.7 mg/mL, compared to the value of orlistat (0.1 mg/mL). In vivo study, the mice were fed on HFD. The ethanol extract reduced the serum levels of TG, TC, LDL-C, and lipase. It also decreased the leptin level and increased the adiponectin level. The extract also attenuated excessive accumulation of fat in liver tissues through histological examination. These results all showed that the extract might regulate lipid metabolisms in adipocytes, downregulate lipid accumulation in the liver [40]. The aqueous extract lowered TC level and increased the ghrelin level in diabetic rats [62]. The aqueous extract also lowered TG level and increased HDL-C level in diabetic rats [43]. 3-Hydroxybutyrate (3-HBT) and acetoacetate were the representative metabolites of fatty acid metabolism, so their levels might be related to the lipid metabolism in the liver. In ¹H-NMR spectroscopic analysis of urine of Azam’ study, aqueous extract showed a remarkable drop in acetoacetate and 3-HBT levels. The reason for that might be that the extract inhibited the abnormal lipid and fatty acid metabolism and re-established energy metabolism [63].

α-Amylase and α-glucosidase are the two key enzymes in the digestion and absorption of carbohydrates in the body. α-Amylase breaks down long-chain carbohydrates, and α-glucosidase hydrolyzes glucoside bonds to release glucose. They are directly involved in the metabolism of starch and glycogen. Therefore, inhibiting the activities of α-amylase and α-glucosidase can reduce the release of glucose from carbohydrate hydrolysis, slow down the absorption of glucose in the small intestine, and effectively lower postprandial blood glucose level [44,64,65]. The inhibitory activities of these enzymes were always tested in vitro.

Rosmarinic acid and 2-caffeoyl-L-tartaric acid were two constituents isolated from O. stamineus. In a recent study, their inhibition ratios on α-glucosidase (0.5 U/mL) were 71.06 ± 1.82% and 69.85 ± 1.27%, respectively, both higher than that of positive control, acarbose, at concentration of 5 mg/mL. Molecular docking results showed that the binding energy of 2-caffeoyl-L-tartaric acid and α-glucosidase was −7.7 kcal/mol, and there were 3 hydrogen bonds between them. The binding energy of rosmarinic acid and α-glucosidase was −8.6 kcal/mol. In the conformation of α-glucosidase-rosmarinic acid complex, there were 6 hydrogen bonds [66]. The 95% EEF showed higher α-glucosidase (86 μg/mL) inhibitory activity (IC50 = 40 ± 0.73 μg/mL) than acarbose (IC50 = 250 ± 1.05 μg/mL) [39]. The ethanol extract (1000 μg/mL) of some genotypes of O. stamineus inhibited α-glucosidase up to 62.84% [46]. The ethanol extract at concentration of 50 μg/mL inhibited α-glucosidase (0.57 U/mL) at 40.74%, α-amylase (1.6 U/mL) at 81.48%, higher than acarbose[67]. The 50% ethanol extract and the isolated compound sinensetin both showed inhibitory activity on α-glucosidase and α-amylase. The IC50 values on α-glucosidase (1.0 U/mL) were 4.63 ± 0.413 and 0.66 ± 0.025 mg/mL, and on α-amylase (0.5 mg/mL) were 36.70 ± 0.546 and 1.13 ± 0.026 mg/mL, respectively. The IC50 values of acarbose on α-glucosidase and α-amylase were 1.93 ± 0.281 mg/mL and 4.89 ± 0.397 mg/mL, respectively [68].

Insulin is a hormone secreted by islet β-cells. It can control blood glucose level and regulate glucose and lipid metabolism. Insulin promotes glucose uptake and utilization in the liver, muscle, and adipose cells to reduce postprandial blood glucose level. However, these functions can be achieved only by combining with insulin receptors (IR). IRs are widely distributed in the body. Muscle, fat, and liver are all insulin target organs or tissues. Insulin resistance occurs when insulin receptors become less sensitive to insulin due to various factors [69]. Normally, glucose is transported and utilized mainly under the stimulation of insulin through a variety of insulin signaling pathways, such as the phosphoinositide 3-kinase/protein kinase B (PI3k/Akt) pathway. Insulin binds to IRs on the cell membrane, causing tyrosine phosphorylation of insulin receptor substrates (IRS), activating the PI3k/Akt signaling pathway and increasing glucose uptake. Any abnormality in insulin signaling pathway may lead to insulin resistance [70,71]. In addition, protein tyrosine phosphatase 1B (PTP1B) is also associated with insulin resistance. High PTP1B activity can lead to the dephosphorylation of IR and IRS tyrosine and weaken insulin signal transduction, leading to insulin resistance [72,73]. In some investigations, it has been proved that the extract of O. stamineus and its active components promoted insulin secretion, improved insulin resistance, and enhanced insulin sensitivity.

Inhibition of PTP1B activity might improve IR and IRS, leading to the improvement of insulin resistance and enhancement of insulin sensitivity. Hence, five diterpenes isolated from O. stamineus were tested for PTP1B inhibitory activity. The IC50 values of siphonol B, orthosiphols B, G, I, and N were 8.18 ± 0.41, 9.84 ± 0.33, 3.82 ± 0.20, 0.33 ± 0.07, and 1.60 ± 0.17 μmol/L, respectively, compared to the positive control, ursolic acid (3.42 ± 0.26 μmol/L). The inhibition types of these five diterpenes on PTP1B were mixed-competitive, non-competitive, non-competitive, competitive, and uncompetitive, respectively [74]. The hexane fraction of 70% ethanol extract slightly increased insulin secretion in both basal and glucose-stimulated states, and also elevated the mRNA expression of insulin and pancreatic duodenal homeobox-1 (PDX-1) in INS-1 cells under normal and high-glucose conditions. PDX-1 is an essential transcription factor for insulin gene expression. Its main functions are to promote the proliferation of islet β-cells, inhibit the apoptosis of islet β-cells, and regulate the transcription of insulin genes. The fraction also increased p-PI3K levels and Akt phosphorylation in INS-1 cells [75]. The ethanol extract reduced the levels of homeostasis model assessment of insulin resistance (HOMA-IR) index in HFD-induced rats [40].

Hyperglycemia is a typical characteristic of diabetes. Carbohydrates are absorbed by intestinal epithelial cells in the form of glucose after digestion by enzymes. The uptake and utilization of glucose mainly exist in peripheral tissues or cells, such as liver, muscle, and adipose cells. Therefore, reducing the absorption of intestinal glucose and promoting glucose uptake by peripheral cells are very important to reduce blood glucose [76].

The sub-fraction 2 of chloroform extract significantly inhibited the glucose absorption from the small intestine at concentrations of 0.5, 1.0, and 2.0 mg/mL. Sub-fraction 2 (2.0 mg/mL) significantly increased the glucose uptake of hemi-diaphragms during the 90-min incubation period [34]. Some diterpenes in O. stamineus had 2-deoxy-2-((7-nitro-2,1,3-benzoxadiazol-4-yl)amino)-D-glucose (2-NBDG) uptake effect in 3T3-L1 adipocytes. 2-NBDG was always used as a substrate to evaluate the action of compounds as insulin mimickers. Siphonol B, orthosiphols B, G, I, and N stimulated glucose uptake at the concentration of 5 and 10 μmol/L [74]. The aqueous extract of O. stamineus significantly enhanced glucose uptake and glucose consumption in 3T3-L1 adipocytes [77]. The O. stamineus aqueous extract could increase the glucose uptake in cells by measuring the traces of radiolabelled glucose in 3T3-L1 adipocytes model [78].

Gluconeogenesis and glycolysis are two metabolic mechanisms to ensure glucose homeostasis. Glycolysis is the process of breaking down glucose to produce pyruvate, which is one of the most important pathways of glucose metabolism in the body. Increasing the expression of glucokinase and pyruvate kinase can promote glycolysis and reduce blood glucose. Gluconeogenesis is the process of converting non-sugar substances into glucose. Liver is the main organ for gluconeogenesis. Both insulin and glucagon can regulate liver gluconeogenesis through different signaling pathways [79,80].

In ¹H-NMR spectroscopic analysis of urine of diabetic rats, aqueous extract increased the levels of pyruvate, succinate, and citrate compared to the model group. Pyruvate is an end product of glycolysis, and it can enter tricarboxylic acid (TCA) cycle. High glucose level inhibits glycolytic enzymes and decreases the generation of pyruvate, thereby reducing the TCA cycle activity, and thus may contribute to mitochondrial dysfunction. Mitochondrial dysfunction may induce diabetes by affecting insulin secretion of islet β-cells and aggravating insulin resistance. Citrate and succinate are the TCA cycle intermediates. Thus, the increased levels of pyruvate, citrate, and succinate showed that the aqueous extract might reduce blood glucose level by increasing glycolysis and decreasing gluconeogenesis, and it might also modulate TCA cycle and improve mitochondrial dysfunction [63].

GLP-1 is released from intestinal cells and maintains blood glucose homeostasis by increasing insulin secretion and inhibiting glucagon secretion [81]. The aqueous extract of O. stamineus (0.1 g/100 g of body weight) increased GLP-1 level in diabetic rats—non-pregnant or pregnant [62].