Lyciumbarbarum's diabetes secrets: A comprehensive review of cellular, molecular, and epigenetic targets with immune modulation and microbiome influence

Abstract

Diabetes, a metabolic disease stemming from impaired or defective insulin secretion, ranks among the most severe chronic illnesses globally. While several approved drugs exist for its treatment, they often come with multiple side effects. Therefore, there is a pressing need for safe and effective anti-diabetic medications. Traditional Chinese medicine has recognized Lycium barbarum (LB; goji berry) plant, commonly known as “wolfberry fruit” in China, for over 2,000 years. Natural compounds derived from LB show promise in reducing diabetes levels. Although research on the impact of LB on diabetes is still limited, our review aims to explore the potential of LB in reducing the risk of diabetes and examine the underlying mechanisms involved. LB can modulate diabetes through various pathways, such as inhibiting α-amylase and α-glucosidase activities, promoting β-cell proliferation, stimulating insulin secretion, inhibiting glucagon secretion, improving insulin resistance and glucose tolerance, and enhancing antioxidant and anti-inflammatory activities. Additionally, LB improves gut flora and immunomodulation, further aiding diabetes management. These findings highlight the potential clinical utility of LB in managing diabetes and its complications within the framework of evidence-based modern medicine.

Graphical abstract

Highlights

• •LB aids diabetes by inhibiting α-amylase, α-glucosidase, supporting β-cell growth, insulin release, and reducing insulin resistance.
• •LB's natural antioxidants and anti-inflammatory effects show promise in diabetes care and complication prevention.
• •LB supports gut flora and immune health, offering a microbiome-based strategy for diabetes management..
• •Findings support LB 's role in diabetes care, promoting its potential for safe, effective anti-diabetic treatments.

1. Introduction

Diabetes is a chronic and severe medical condition characterized by high levels of glucose in the bloodstream. This condition arises from the malfunctioning of β-cells and disrupts the normal functioning of insulin [1]. According to the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) in 2019, diabetes was the eighth most significant cause of both death and disability on a global scale. In 2019, the disease affected almost 460 million people of all ages and from all nations [2]. The prevalence of diabetes in 2021, as estimated by the International Diabetes Federation, was 537 million, substantially burdening healthcare systems. As a result, worldwide healthcare expenditures reached 966 billion USD, with projections indicating that they will exceed 1,054 billion USD by 2045. The 2016 NCD Risk Factor Collaboration (NCD-RisC) study estimated that the likelihood of achieving worldwide targets to reduce the increasing incidence of diabetes by 2025 is less than 1% for women and significantly lower for men [3].

Furthermore, diabetes is recognized as a significant risk factor for ischemic heart disease and stroke, which have been designated by GBD (2019) as the primary and secondary leading causes, respectively, of the global disease burden [4]. Individuals with diabetes face a higher risk of both macrovascular and microvascular complications. These complications contribute to increased premature deaths, reduced productivity, and a poor quality of life [3]. The prevalence of diabetes is growing at an alarming rate, and the search for effective strategies to prevent and manage this condition has become a priority in medical research. While pharmacological interventions exist with associated side effects, there is a growing interest in natural remedies and complementary therapies to complement conventional treatments.

Among the various natural remedies gaining attention today, one of the most prominent is Lycium barbarum (LB), which is a shrub species (sp.) belonging to the nightshade family, also known as boxthorn. The ellipsoids, or LB berries, are orange-red, have an acidic yet sweet flavor, and are roughly 2 cm long. The fruit of the LB plant is utilized both as a functional food and in Chinese herbal medicine [5].

Research examining the hypo-glycemic and hypo-lipidemic characteristics of LB bark has revealed its ability to decrease blood glucose and lipid levels, improve sugar metabolism, boost insulin production, and stimulate weight gain. These findings suggest that LB barks can potentially be used as a treatment for diabetes [6]. LB contains various bioactive components, including LB polysaccharides (LBPs), LB flavonoids (LBFs), LB carotenoids (LBCs), LB dietary fibers (LBDFs), LB fatty acids (LBFAs), LB vitamins (LBVs), and LB minerals (LBMs). These compounds have shown various biological effects [7], including the ability to improve insulin sensitivity, regulate blood glucose levels, and protect pancreatic β-cells—all critical factors in diabetes treatment. However, scientific studies examining the potential benefits of LB bioactive substances for diabetes treatment have been scarce in recent years. Therefore, there is a significant need to explore the role of LB, particularly its bioactive compounds, in managing diabetes pathways [8].

To ensure a comprehensive review of diabetes-related properties of LB, we conducted a systematic search using multiple databases, including PubMed, Scopus, and Web of Science. The literature search covered studies published between January 2004 and April 2024. Keywords such as LB, diabetes, cellular targets, molecular targets, epigenetic targets, immune modulation, and microbiome influence were used to capture relevant research. Both peer-reviewed articles and gray literature were included, allowing us to compile a robust and diverse set of data for our analysis.

This review offers an in-depth investigation into the cellular, molecular, and epigenetic targets influenced by LB in the context of diabetes, thereby uncovering its potential mechanisms of action, a dimension often overlooked in the existing literature. It also explores the immunomodulatory and gut microbiota modulation effects of LB on diabetes and related pathways. This comprehensive approach differentiates the present review from conventional studies on the glycemic effects of LB.

2. Pathophysiology, complications, and therapies of diabetes

Diabetes mellitus (DM) is believed to arise from the combined effects of genetic and epigenetic factors. The complex characteristics of DM, which is categorized as a combination of metabolic disorders with an intricate origin, add to the difficulty of comprehending its pathophysiology [1]. Diabetes encompasses various forms, such as type 1 diabetes (T1DM), type 2 diabetes (T2DM), gestational diabetes, and other less common varieties. T1DM is closely linked to endocrine autoimmunity, primarily defined by the presence of autoantibodies, and has a genetic basis. On the other hand, T2DM, a multifaceted disorder, arises from the combination of genetic susceptibility and external factors, such as obesity, age, lifestyle choices, and hormone imbalances [2]. Gestational diabetes, which develops during pregnancy and creates temporary difficulties with glucose utilization, usually resolves post-baby-birth, as glucose metabolism returns to normal. Increased insulin release occurs due to increased synthesis of insulin-blocking substances [9]. Hormones such as insulin and glucagon have crucial functions in controlling the balance of blood glucose levels. Insulin resistance (IR) in T2DM is caused by multiple causes, such as heightened hepatic glucose production, oxidative stress, reduced glucose tolerance, and changes in genes and proteins related to insulin signaling pathways [1]. Different enzymes, such as glutamate dehydrogenase, hexokinase (HK), and pyruvate kinase (PK), are essential enzymes that regulate the flow of metabolic substances such as insulin and glucagon in pancreatic β-cells. Their activation shows potential as a new method for treating T2DM [3].

In addition, the diminished functionality of insulin in T2DM results in significant alterations in the metabolism of carbohydrates, lipids, and proteins. An essential part of the pathophysiology of diabetes is inflammation [3]. The cause of insufficient insulin production in T1DM is the demise of pancreatic β-cells, which is mediated by several inflammatory mediators, such as T cell effectors that target β-cell autoantigens and associated peptides. Immune β-cells are changed, and β-cells are directly harmed by macrophages that release reactive oxygen species (ROS), causing islet inflammation. This mechanism may also involve natural killer (NK) T cells, dendritic cells, and NK cells. On the other hand, various cellular stresses associated with T2DM result in inflammation and insulin dysregulation. These stressors include gut microbiota, lipotoxicity, glucotoxicity, oxidative stress, endoplasmic reticulum (ER) stress, pancreatic amyloid deposition, and ectopic deposition in muscles, liver, and pancreas [1].

Hyperglycemia in diabetes causes systemic harm, leading to a multitude of consequences over time. Continued elevation of blood glucose levels can lead to severe medical complications, including microvascular, macrovascular, and non-vascular problems. Microvascular problems refer to the development of nephropathy, retinopathy, and vision loss, whereas macrovascular complications encompass cardiovascular disorders, heart attacks, strokes, and neuropathy [1]. On the other hand, non-vascular complications encompass dental ailments, sexual dysfunction, dermatological disorders, and gastrointestinal (GI) complications. Diabetes-related problems can result in infections, ulcers, and non-healing wounds caused by bacterial and fungal infections. There is also a possibility of developing mental health conditions, such as depression and dementia [10].

Diabetes, a complex metabolic disorder, requires accurate diagnosis and a thorough approach to treatment. Treatment includes not only the reduction of blood glucose levels but also the management of cardiovascular diseases (CVDs) and related risk factors, such as obesity, smoking, hypertension, and hyperlipidemia. Timely identification and appropriate treatment alleviate symptoms and minimize immediate and long-term consequences [3]. Oral hypoglycemic medicines, such as biguanides and sulfonylureas, are commonly prescribed for the treatment of non-insulin-dependent diabetes mellitus (NIDDM). Some specific medications in this category include glibenclamide, tolbutamide, and metformin. When exercise, dietary modifications, and oral drugs are ineffective in managing NIDDM, insulin administration becomes necessary to regulate high blood sugar levels and achieve treatment goals. Fig. 1 displays statistics on the top 10 countries with high diabetic prevalence from 2021 to 2045, plus information on the most frequently used medications and their probable antidiabetic mechanisms [11].

Fig. 1.

Overview of diabetes types, contributing factors, and commonly used antidiabetic treatments.

The documented adverse effects of these drugs include injection site pain, weight gain, hypoglycemia, poor blood sugar regulation, GI problems, hypersensitivity reactions, and the possibility of organ damage. Therefore, exploring alternative ways, particularly functional foods for diabetes treatment, is critical for expanding treatment possibilities beyond standard methods and mitigating the potential adverse effects of drugs. Adopting these revolutionary dietary options can provide novel, comprehensive techniques for diabetes management and better overall health outcomes.

3. LB bioactive compounds and their effects on diabetes

LB is well-known for its wide range of nutrients and powerful bioactive substances, which show potential in managing diabetes. LBPs extracted using techniques such as enzymatic, ultrasonic, or hot water extraction have been shown to improve insulin sensitivity and regulate blood glucose levels. Regarding diabetes issues, flavonoids obtained via solvent extraction or mass spectrometry help fight oxidative stress. Similarly, carotenoids acquired through solvent extraction or high-performance liquid chromatography (HPLC) can provide cellular protection and ameliorate diabetic conditions. Its fiber content helps to improve lipid profiles and moderate glucose levels.

Rich in minerals such as calcium and potassium and vitamins such as C, LB may provide comprehensive support for a healthy metabolism and balanced blood sugar levels. Table 1 [7,[12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31]] presents the diverse LB extraction methodologies renowned for their proficiency in maximizing the yield of bioactive compounds essential for managing diabetes effectively and conveniently accessible for researchers. We will analyze and identify the distinct functions of each bioactive component in LB for treating diabetes by modulating diabetes-related cellular, molecular, and epigenetic targets, along with immune modulation and microbiome influence pathways.

Table 1.

Potential bioactive compounds in LB associated with diabetes: Extraction yield and solvents utilized.

Ingredients	Extraction method	Yield	Solvent	Refs.
LBCs	LC-MS-Q UPLC-PDA	212.94 mg/g	Bht, MgCO3	[7]
ZDP	LC-MS	84.54 mg/g	Methanol/acetone/hexane	[7]
β-Carotene	LC-MS	19.35 mg/g	Methanol/acetone/hexane	[7]
Cryptoxanthin	LC-MS	72.29 mg/g	Methanol/acetone/hexane	[7]
LBPs
LBP-4a	MR, IEC, DEAE, Heating	3.25% ± 0.14%	Ethanol	[12]
d-mannose	Ultrasound-assisted	2.24%–3.94%	Water/ethanol/potassium dihydrogen phosphate	[13]
d-arabinose	HP-GPC	11.19%–17.35%	Water/ethanol/potassium dihydrogen phosphate	[14]
d-xylose	Ultrasound-assisted	9.88%	Water/ethanol/potassium dihydrogen phosphate	[14]
l-rhamnose	Ultrasound-assisted	0.85%–1.20%	Water/ethanol/potassium dihydrogen phosphate	[14]
d-glucose	Ultrasound-assisted	3.37%	Water/ethanol/potassium dihydrogen phosphate	[14]
d-ribose	Ultrasound-assisted	29.38%	Water/ethanol/potassium dihydrogen phosphate	[14]
LBP-s-1	MR, IEC, DEAE, Heating	5.47%.	Water	[15]
LBP-3	MR, IEC, DEAE, Heating	0.69%	Water	[16]
d-galactose	Ultrasound-assisted	6.70%–10.18%	Water/ethanol/potassium dihydrogen phosphate	[17]
Other extraction methods	Heating	7.46%–7.63%	Water	[18]
	Subcritical medium	10.67%± 0.33%	Water	[19]
	Ultrasound-assisted	2.286%–5.701%	Water	[20]
	Enzyme-assisted	6.81%± 0.10%	Water	[21]
	Ultrasound-enhanced subcritical	2.286%–5.957%	Water	[22]
LBFs	Mix-mode macroporous adsorption resins (MAR)	36.88% of p-FLA	Water	[23]
	High-speed shear dispersing emulsifier	7.11 mg/g	DES	[24]
Rutin	UPLC-MS	16.60–12.84 mg/g	Acetone/water/acetic acid	[25]
Quercetin	UPLC-MS	10.23–9.41 mg/g		[26]
Flavan-3-ol	UPLC-MS	10.40 mg/g		[26]
Catechin	UPLC-MS	1.34–1.13 mg/g		[26]
Epicatechin	UPLC-MS	2.18–1.98 mg/g		[26]
LBFAs	Soxhlet extraction and GS	0.39–4.1 g/g	Methyl esters	[27]
Linoleic acid	GC-MS	37%–43.4%	Methanol	[14,27]
Oleic acid	GC-MS	16%–19%		[14,27]
Palmitic acid	GC-MS	19.77%–21.79%		[14,27]
LBVs	Ultra-Turrax	0.236%–0.786%	Citric acid, EDTA	[28]
Ascorbic acid	Ultrasound-assisted	2.39–48.94 mg/g	Oxalic acid/water	[29]
Tocopherol	HPLC	0.33 mg/g	Acetonitrile	[29]
LBMs
Potassium	–	434–1460 mg/g	Sulfuric acid	[27]
Calcium	Spectrophotometry	29–60 mg/g	Sulfuric acid	[27,30]
Sodium	Plasma emission	75–550 mg/g	Sulfuric acid	[27,30]
Iron	Optical emission	5.4 mg/g	Sulfuric acid	[27,30]
Phosphor	Spectrometers	232 mg/g	Sulfuric acid	[30]
Other extraction	Nitric mineralization	–	Ultrapure nitric acid	[30]
LBDFs	Enzymatic TDF assay kit	3.63 g/g	Ethanol solution	[27]
Soluble	EGP	0.90–5.5 g/g	Ethanol solution	[31]
Insoluble	EGP	2.73–11.7 g/g		[31]

ZDP: zeaxanthin dipalmitate; UPLC-MS: ultra performance liquid chromatography mass spectrometry; MR: macroporous resin; IEC: ion-exchanged column; DEAE: diethylaminoethyl cellulose; LC-MS-Q: liquid chromatography/mass spectrometry quadrupole; UPLC-PDA: ultra performance liquid chromatography photodiode array; HPLC: high performance liquid chromatography; GC: gas chromatography; DES: deep eutectic solvents; EGP: enzymatic-gravimetric procedure; TDF: total dietary fiber; HP-GPC: high performance gel permeation chromatography; EDTA: ethylenediaminetetraacetic acid.

3.1. LB polysaccharides

The primary active ingredient of LB, LBP, is mainly made up of d-arabinose, d-galactose, d-mannose, d-xylose, l-rhamnose, d-glucose, d-ribose, and other monosaccharides, as well as various uronic acids [32]. LBP is a water-soluble poly-glycoprotein complex with a molecular weight range of 10 to 2,300 kDa. LBP has an amino or imino group containing a chemical structure primarily consisting of d-pyranose with O-links connecting sugar and peptide chains. The primary ways of LBP extraction include hot water extraction, alkali extraction, zymolytic extraction, microwave-assisted extraction, ultrasonic extraction, and other techniques (Table 1).

LBP yields range from 7.6% to 14% (w/w), with protein and phenolic content varying depending on the extraction method [33]. Various extraction techniques result in slightly different monosaccharide compositions of LBP, which have varying implications for the quality of the substance. For instance, acid-extracted LBPs exhibit higher homogalacturonan region proportions. Size exclusion chromatography coupled with multi-angle light scattering and atomic force microscopy confirmed large-size polymers with branched morphologies in alkali-extracted polysaccharides [34,35].

Neutral LBPs, such as LBP-1 and LBP3b, have potent bioactivity. LBP-1, which contains arabinose (37.53%), galactose (28.08%), glucose (14.72%), xylose (7.83%), and mannose (4.50%), possesses anti-inflammatory effects, suggesting its potential relevance in managing diabetes and its related complications [36]. LBP3b, which contains mannose (5.52%), rhamnose (5.11%), glucose (28.06%), galactose (1.00%), and xylose (1.70%), has hypoglycemic effects because it inhibits glucose uptake [33]. In an anti-diabetic investigation, water-soluble polysaccharides were segregated into LBP-1, LBP-2, LBP-3, and LBP-4 peaks using diethyl aminoethyl cellulose chromatography (producing 10.0%, 8.0%, 14.0%, and 16.0%, respectively). LBP-4, with a yield of 16.0%, has the most potent hypoglycemic effects. Further purification yielded LBP-4a (1.07%) and LBP-4b (0.57%), with LBP-4a having 92.2% ± 1.3% sugar, 3.8% protein, and a molecular weight of 33,867 Da [12].

The molecular weight of LBP is closely related to digestion, with maximum digestion yielding the most bioavailable molecules necessary for diabetic treatment. As LBP passes through the GI tract, its molecular weight decreases. For instance, glucose falls from 39.42% to 36.09%, glucuronic acid (GlcA) grows from 1.18% to 1.94%, and galacturonic acid (GalA) climbs from 1.69% to 4.25%. Furthermore, total protein content increases from 9.04% to 10.87%, while phenolic content rises from 3.46% to 3.89%. LBPs exposed to in vitro digestion exhibit improved antioxidant activity [13].

Despite breakthroughs in protein identification, quality control criteria for LBPs remain limited, with glucose levels being the primary indicator. Different LBPs induce anti-diabetic actions that are just as effective as or even more effective than synthetic diabetic drugs and hypoglycemic effects that reduce cell dysfunction (Fig. 2).

Fig. 2.

Potential in vivo pathways for the antidiabetic effects of polysaccharides found in Lycium barbarum (LB). LBPs: Lycium barbarum polysaccharides; GLUT4: glucose transporter type 4; IL-6: interleukin-6; TNF-α : tumor necrosis factor-alpha.

3.2. LB flavonoids

A study identified 37 LBFs, including quercetin, kaempferol, and isorhamnetin derivatives [37]. However, a separate study discovered only three flavonoids in LB fruit extract: myricetin, quercetin, and kaempferol [38]. Another study identified seven phenolic acids and flavonoids in LB: protocatechuic acid, chlorogenic acid, rutin, hyperoside, hesperidin, morin, and quercetin [39]. Among these, hyperoside was found to be the main flavonol in LB [28], while rutin, quercetin, and kaempferol were among the notable flavonoids identified in LB, along with others [40].

LB cultivars contain an average of 75.3% LBFs [7]. Different extraction methods showed various types and yields of LBFs. Ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) screening revealed a diverse flavonoid profile in dried LB, with slight differences in abundance observed: cyanidin (0.22 mg/g), catechin (0.86 mg/g), and luteolin (0.11 mg/g) [41]. A novel method utilizing mixed-mode microporous adsorption resins enhanced flavonoid purification from LB by simulating the molecular size of rutin and myricetin. The optimized process increased flavonoid content from 0.97% to 36.88% [23].

Ultrasonic extraction revealed quercetin-3-O-hexoside as the primary component (169.1–1107.7 mg/kg), followed by quercetin-3-O-rutinoside (7.1–232.7 mg/kg) [7]. Preliminary pH-differential assays revealed LB anthocyanin (LBA) content ranging from 0.02 to 18.29 mg/100 g [42]. The authors suggested that differences in LBF content may be attributed to variations among Lycium sp. growth conditions, as well as extraction and purification methods.

In vitro, the digestion of LBFs has revealed altered antioxidant capacity. Oral/intestinal digestion decreased by 4.39%–21.22%, whereas gastric digestion increased by 1.26%–16.14%. A strong association was also found between flavonoids and antioxidant capability in digested groups (r > 0.899, P < 0.01), with rutin, a flavonoid glycoside, and p-coumaric acid derivatives demonstrating improved stability and suggesting a functional potential [43].

Flavonoids present in LB fruits have attracted considerable attention in diabetes studies owing to their potent antioxidant, hypolipidemic, and hypoglycemic properties (Fig. 3) [44]. Additionally, they can prevent cardiovascular illnesses and enhance immunity.

Fig. 3.

Potential in vivo pathways for the antidiabetic effects of Lycium barbarum flavonoids (LBFs). Reproduced from Ref. [44] with permission. IL-1β: interleukin-1 beta; IL-6: interleukin-6; TNF-α: tumor necrosis factor-alpha; CRP: C-reactive protein; GLUT4: glucose transporter type 4; IGF-1: insulin-like growth factor 1; IRS-1: insulin receptor substrate 1.

3.3. LB carotenoids

LBCs ranging from 0.03% to 0.5% dry weight (DW) are rich in zeaxanthin, β-carotene, lutein, and astaxanthin, with zeaxanthin being the predominant carotenoid, constituting 31%–56% of the total carotenoid content. Additionally, these LBCs contain reduced levels of neoxanthin, cryptoxanthin, and β-carotene natural pigments that give LB berries their yellow, orange, and red hues [45]. Studies have unveiled variations in carotenoid profiles of LB. One study quantified 10 carotenoids via HPLC without mass spectrometry, while another detected only one zeaxanthin dipalmitate in LB fruits using liquid chromatography-mass spectrometry (LC-MS) [46,47]. Additionally, 11 free carotenoids and seven carotenoid esters were identified in LB extracts, with ester concentrations declining gradually with saponification time [48].

Due to the fat solubility and low polarity of LBCs, extraction techniques have evolved from solvent to supercritical and subcritical processes. This advancement has led to a broader range and higher quantities of LBCs. Researchers developed a sugar reduction process in response to sugar residue extraction challenges. This process decreased total sugar levels from 41%–45% to 25%–29% while achieving Lycium ZDP purity of over 80% [24,49].

Identification of carotenoid components in LB, conducted via ultraviolet–visible (UV–Vis) and positive MS spectra for molecular weight and structural determination, revealed 14 distinct carotenoids, including β-carotene, zeaxanthin, and β-cryptoxanthin, alongside their fatty acid esters. Notably, neoxanthin (601.40 m/z), zeaxanthin (569.40 m/z), β-cryptoxanthin (553.40 m/z), and β-carotene (537.30 m/z) served as critical markers for identification. Carotenoid esters contain a single palmitic acid molecule characterized by UV–Vis absorption maxima and palmitic acid fragments [7].

A total of 17 primary LBCs were identified through analysis using ultra-performance convergence chromatography coupled with a photodiode array detector and quadrupole time-of-flight mass spectrometry. Lycium zeaxanthin exhibited the highest concentration (1315.46 μg/g), followed by β-cryptoxanthin monopalmitate (55.34 μg/g) and zeaxanthin monopalmitate (44.99 μg/g). Notably, free carotenoids, all-trans-β-carotene (9.98 μg/g), and all trans-zeaxanthin (5.98 μg/g) had lower concentrations than their esters [50]. Meanwhile, the conventional extraction method showed a low Lycium zeaxanthin content of 0.3%–0.4% [51].

Despite the ability of the human digestive system to hydrolyze zeaxanthin esters, efficient cleavage of carotenoid esters remains challenging in vitro. LBCs have more substantial positive effects on diabetes and metabolic syndrome, which aligns with their natural antioxidant properties. It has been demonstrated that insulin resistance and fasting plasma glucose concentrations are inversely correlated with LBCs and plasma β-carotene concentrations, respectively [7].

3.4. LB dietary fibers

Previous research has found that LB berries have total dietary fiber (TDF) with a concentration of 3.63 g/100 g fresh weight (FW) [27]. One study further confirmed that LB contains soluble (SDF) and insoluble (IDF) dietary fiber at 16 g/100 g fresh weight [52]. Another study demonstrated that 100 g of dried LB fruit (both organic and conventional) contains 9.88% and 11.27% TDF, respectively. In these dried samples, IDFs, such as cellulose, hemicellulose, and lignin, were predominant in both LB samples, comprising approximately four times more than SDF. Additionally, it was found that 100 g of dried LB fruit supplies 28% of the recommended dietary fiber intake for women and 42% for men [53].

Adults should consume 25 g of dietary fiber per day. About 14% of adults required daily fiber intake can be obtained from 30 g of dried fruits. Dried LB can have the label “high fiber content”, per European law (Regulation CE 1924/2006), as it has a minimum of 6 g of fiber per 100 g [54].

Various extraction techniques yield different LB dietary fiber contents, with the amount varying depending on pre- or post-harvesting procedures, environmental factors, and processing techniques. For instance, the TDF content obtained using an enzymatic TDF assay kit is higher in red and yellow LB berries than in black ones: red (3.63 ± 0.25), yellow (3.34 ± 0.17), and black (2.76 ± 0.21) g/100 g FW. IDFs are also higher in red and yellow LB berries than in black ones: red (2.73 ± 0.16), yellow (2.68 ± 0.10), and black (2.17 ± 0.15) g/100 g FW [27].

The LBDF content determined using the Association of Official Agricultural Chemists extraction method revealed significant amounts of dietary fiber, including both IDF (8.8%) and SDF (2.6%) types, in dried LB fruits. A ratio of approximately 3:1 was observed between soluble and insoluble fiber [54]. Hot-acid extraction boosts GalA levels in LB berries (46.9%) and raspberries (65.2%), signifying the presence of homogalacturonans. High-pressure processing also enhances rhamnogalacturonan areas. Together, these methods (hot-acid extraction and high-pressure processing) effectively increase the fiber content in LB [55].

LBDFs, whose content varies with extraction methods, modulate digestion by delaying gastric emptying and influencing insulin response through reduced carbohydrate absorption and enhanced short-chain fatty acid synthesis by gut bacteria. The dietary fiber content of LB, including cellulose, hemicellulose, pectin, and resistant starches, is beneficial for individuals with diabetes, as it regulates blood sugar, supports gut health, and manages cholesterol levels.

3.5. LB fatty acids

Different researchers have investigated the fatty acid profile of LB, reporting varying values due to differences in extraction methods and geographical factors. However, linoleic acid, oleic acid, and palmitic acid consistently emerge as significant components. For instance, Pires et al. [29] identified the fatty acid content of LB fruit using the Soxhlet extraction method as 4.1 g/100 g of dry weight, with linoleic acid (53.4%), oleic acid (16.5%), and palmitic acid (12.77%) being predominant.

Skenderidis et al. [56] documented concentrations of 37.89%–43.96%, 16.71%–20.07%, and 15.08%–21.79% for linoleic acid, oleic acid, and palmitic acid, respectively, using fatty acid methyl ester (FAME) synthesis. Similarly, Ilić et al. [27] reported that linoleic acid (52.1%), oleic acid (23.6%), and palmitic acid (17.6%) are the most abundant fatty acids, constituting 95% of the total LB fatty acids, as determined by gas chromatography.

Moreover, the FAME method revealed that palmitic acid (8.23%), oleic acid (21.69%), and linoleic acid (60.77%) are significant components in LB fruit oil [30]. Comparable Soxhlet extraction findings were reported by Chi et al. [57] for Lycium berries collected from the Qinghai-Tibet Plateau, showing linoleic acid (54.11%) and oleic acid (20.70%) as primary fatty acids.

Various LBFA extraction methods may impact digestion differently due to lipid composition and bioavailability variations, influencing absorption rates and enzyme activity. LBFAs can affect diabetes management through effects on insulin sensitivity, inflammation, and lipid metabolism [56].

3.6. LB vitamins and minerals

While the explicit composition of vitamins in LB remains somewhat unclear, most reports have consistently determined ascorbic acid (AA; vitamin C) as the most prevalent vitamin in LB berries [14]. According to previous studies, LB also contains twice as much retinol (vitamin A) as an individual needs daily. Other studies further revealed that LB may contain thiamine (vitamin B1) and riboflavin (vitamin B2), but it is unknown how many of these vitamins are present [58].

According to one study, LB contains a significant amount of tocopherol (vitamin E), nearly 0.33 mg/100 g [29]. Regarding the mineral composition, previous studies found that potassium, calcium, and sodium are the main minerals found in LB. However, there is still debate about these findings.

Various extraction methods can influence the concentration of vitamins and minerals in LB. Inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis revealed that new big LB berries exhibited the highest vitamin C concentration at 5.8 mg/100 g FW, with levels ranging from 4.3 to 5.8 mg/100 g FW [59]. Other studies found LB berries contain approximately 48.9 mg/100 g FW of vitamin C using atomic absorption spectrometry and solid-phase extraction [28,60].

The mineral contents of LB fruit were analyzed using inductively coupled plasma atomic emission spectrometry. The potassium (K), magnesium (Mg), iron (Fe), phosphorus (P), calcium (Ca), and sodium (Na) contents of LB fruit were determined to be 13447.35 mg/kg, 806.88 mg/kg, 45.77 mg/kg, 1103.30 mg/kg, 1003.40 mg/kg, and 28.27 mg/kg, respectively [30]. Ilić et al. [27] quantified potassium (445.12 mg/100 g DW), phosphorus (231.52 mg/100 g DW), sodium (74.57 mg/100 g DW), and calcium (29.02 mg/100 g DW) through ICP-OES. Llorent-Martínez et al. [61] reported more significant amounts of potassium (1460 mg/100 g), sodium (550 mg/100 g), and calcium (50 mg/100 g) by plasma-mass spectrometry after microwave digestion.

As extraction methods can significantly influence the yield of vitamins and minerals in LB, variations in nutrient content may, in turn, affect digestion and impact insulin response. These LB nutrients are essential for overall health and can affect managing conditions such as diabetes [30].

4. Mechanisms of LB in the treatment of diabetes and its related complications

LB demonstrates multifaceted mechanisms in treating diabetes and its related complications. Through its bioactive components, LB modulates various pathways involved in glucose metabolism, insulin sensitivity, and inflammation, thereby improving glycemic control and reducing insulin resistance. Moreover, LB's antioxidant properties help mitigate oxidative stress, a key contributor to diabetic complications, such as nephropathy, neuropathy, and retinopathy. Additionally, the immunomodulatory effects of LB may attenuate autoimmune-mediated damage to pancreatic β-cells, while its influence on gut microbiota composition holds promise in improving metabolic health. Table 2 (Antioxidant, immune activity, gut microbiota, and anti-diabetic) summarizes important experimental and clinical studies on diabetes [[8], [12], [15], [56], [62], [63], [64], [65], [66], [67], [68], [69], [70], [71], [72], [73], [74], [75], [76], [77], [78], [79], [80], [81], [82], [83], [84], [85], [86], [87], [88], [89], [90], [91], [92], [93], [94], [95], [96], [97], [98], [99], [100], [101], [102], [103], [104], [105]].

Table 2.

Summary of important experimental and clinical studies on diabetes: Indirect (antioxidants/anti-inflammatory, immune activity, gut microbiome) and direct impact.

Antioxidant activity	Refs.	Immune activity	Refs.	Gut microbiome	Refs.	Anti-diabetic activity	Refs.
Prevented and treated reactive oxygen species (ROS)-induced diseases	[8]	Stimulated lymphocyte proliferation	[62]	Stimulated the growth and proliferation of probiotic strains	[56]	Restored normal glycemia and insulinemia	[12]
Provided defense by delaying, inhibiting, or preventing the free radicals from damaging proteins, DNA, and lipids	[63]	Enhanced RAW264.7 murine macrophage activity, suggesting potential for diabetes therapy	[64]	Modified gut microbiota and intestinal barrier function	[65]	Reduced insulin resistance enhancing glucose consumption	[15]
Reduced ischemia-reperfusion injury	[66]	Enhanced macrophage viability and polarization	[67]	Modified gut microbiota and SCFAs corrected diabetic abnormalities in rats	[68]	Promoted functional maturity of pancreatic β-cells	[69]
Reduced inflammatory reactions providing hepatoprotective properties	[70]	Stimulated TNF-α and IL-1β in peritoneal macrophages of wild-type (C3H/HeN) mice, but not TLR4-deficient mice	[71]	Decreased potentially harmful bacteria (e.g., Allobaculum and Tannerella spp.) and increased SCFA-producing bacteria	[72]	Showed an improvement in insulin resistance through the translocation of GLUT4	[73]
Inhibited the expression of inflammatory genes	[74]	Enhanced immune response in vitro (RAW264.7) by increasing TNF-α production and upregulating pro-inflammatory cytokines	[75]	Promoted probiotics proliferation	[76]	Improved glycaemic management and hypolipidemia	[77]
Improved radical scavenging activity	[78]	Exhibited an anti-proliferative effect against cancer cells	[79]	Demonstrated a beneficial impact on the growth of L. acidophilus and B. longum strains	[80]	Inhibited glucagon secretion, reducing glucose uptake and postprandial blood glucose levels	[81]
Enhanced antioxidant enzymes	[82]	Up-regulated p53 and p21, down-regulated CDK2, CDK1, cyclin A, and cyclin B; potentially impacts diabetes	[83]	Lowered intestinal permeability and modulated the gut microbiome	[84]	Improved glucose tolerance and insulin resistance during pregnancy	[85]
Provided neuroprotection and neuroplasticity	[86]	Showed immunomodulatory effects on cancer cells	[87]	Improved cognitive performance, immunity, and gastrointestinal health	[88]	Lowered serum glucose and enhanced the insulinogenic index	[89]
Enhanced mitochondrial biogenesis in the retina	[90]	Lowered blood glucose, TC, TG levels; increased HDL-c; potential immunomodulation and diabetes management	[91]			Possessed protective properties in patients with diabetic nephropathy	[92]
Reduced ER stress, alleviating the consequences of hyperglycaemia-induced oxidative stress	[93]	Decreased ALT and AST; possesses immunomodulatory properties	[94]			Regulated liver insulin resistance, glucose metabolism disorders	[95]
Improved vascular health and decreased oxidative stress	[96]	Improved vaccination response and immunological protection without overburdening the immune system	[97]			Improved in renal function and inflammation in diabetic rabbit	[98]
Reduced lipid peroxidation in subjects at high risk of oxidative stress-related disorders	[99]	Improved immunological responses and overall well-being in 60 healthy older adults	[100]			Possessed the capacity to lower FBG, HbA1C, and enhance the morphology of pancreatic islets	[101]
Increased plasma zeaxanthin and antioxidant levels protecting against hyperpigmentation	[102]
Improved antioxidant efficacy	[103]
Prevented atherosclerosis by improving lipid profiles, oxidative stress, and blood pressure	[104]
Increased fasting plasma zeaxanthin levels	[105]

ER: endoplasmic reticulum; TNF-α: tumor necrosis factor-α; IL-1β: interleukin-1β; TLR4: toll-like receptor 4; CDK: cyclin-dependent kinase; TC: total cholesterol; TG: triglyceride; HDL-c: high-density lipoprotein cholesterol; ALT: alanine aminotransferase; AST: aspartate aminotransferase; SCFAs: short-chain fatty acids; GLUT4: glucose transporter 4; FBG: fasting blood glucose.

4.1. LB inhibits the activity of α-amylase and α-glucosidase

Postprandial hyperglycemia is mainly caused by the enzymes α-amylase and α-glucosidase, which break down carbohydrates. α-amylase breaks down polysaccharides into disaccharides, while α-glucosidase converts disaccharides to monosaccharides, leading to postprandial hyperglycemia. The inhibitors of these enzymes help to manage hyperglycemia by pausing carbohydrate breakdown and lowering postprandial plasma glucose levels [106].

In an in vitro study, LB berry exhibited high α-glucosidase and α-amylase inhibitory activities, with half-maximal inhibitory concentration (IC₅₀) values of 9.9 and 33.4 mg/mL, respectively [107]. Furthermore, lipopolysaccharides (LPS) of LB berries were noted for their inhibitory effects on α-amylase and lipase activities [108].

In a study on LB-enriched bakery products, LB fresh flesh puree exhibited the highest activity against diabetes-linked enzymes, with an IC₅₀ of 48.02 μg/mL, followed by processed flesh puree extract with an IC₅₀ of 86.46 μg/mL. Enriched ciabatta bread showed lipase inhibitory activity, with IC₅₀ values of 6.88 and 6.52 μg/mL, varying in strength depending on the concentration of LB puree [109]. In another study, LB leaf extracts (LB leaf extract (black-fruited variant) (Lbb), LB leaf extract (early-harvest) (Lbe), and LB leaf extract (normal) (Lbn)) from three different Chinese regions inhibited α-glucosidase more effectively than α-amylase. Lbb exhibited the most inhibitory capacity on both enzymes. The inhibitory potential for α-glucosidase and α-amylase was 5.38 and 0.26 mmol ACAE/g, respectively, but wild-grown plant extracts were less effective on α-glucosidase (2.25 mmoL ACAE/g). Lbb and Lbe contain significant phenolic compounds, which bind to the active protein pocket of α-glucosidase [110].

In a study exploring α-glucosidase inhibitory activity in eleven monomeric compounds, rutin (85%), chlorogenic acid (55%), vanillic acid (80%), and ferulic acid (88%) demonstrated the highest inhibition rates. These results indicate the potential of rutin, a major LBF, among others, as a significant anti-diabetic component [111]. LBAs inhibit α-glucosidase activity in Saccharomyces cerevisiae and Caco-2 cells, with IC₅₀ values similar to acarbose (1.32–1.57 μg/mL). LBAs may inhibit the hydrolysis of (1 → 4)-linked short-chain reducing sugars, lowering blood glucose levels via binding to α-glucosidase in the small intestine. This inhibitory effect of LBFAs on α-glucosidase is increased by the GI digesting, presumably due to enhanced hydroxyl group binding in the anthocyanin structure [112].

In the in vitro hypoglycemic tests, the inhibitory capability of LB carotenoids was observed to be 5.7%–15.3% for α-glucosidase enzymes and 9.6%–82.6% for α-amylase [7]. Some in vitro studies using spectrophotometric methods revealed that carotenoids such as lutein, a major carotenoid in LB, may inhibit α-amylase and α-glucosidase activities [113,114]. SDF in LB forms viscous gels in the digestive tract, slowing carbohydrate digestion and absorption, with both SDF and IDF showing dose-dependent decreases in starch hydrolysis velocity. Fermentation increases SDF content, enhancing α-amylase inhibition [54,115]. While the direct impact of LBFAs on enzyme activity is unclear, oleic and linoleic acids have been found to inhibit α-glucosidase activity effectively [116].

Vitamins and minerals in LB, such as vitamin C and zinc, affect glucose metabolism and insulin sensitivity; vitamin C exhibits dose-dependent inhibition of α-amylase, and zinc inhibits the structure and dynamic characteristics of α-amylase [117].

LB can reduce diabetes in vivo by blocking α-amylase and α-glucosidase enzymes responsible for the digestion of glucose. LB inhibits these enzymes, slowing the breakdown of carbs into glucose, resulting in lower glucose absorption and blood sugar levels. This approach may help treat diabetes by regulating postprandial blood glucose levels.

4.2. LB modulates pancreatic α- and β-cells

4.2.1. Regulation of resting membrane potential of α- and β-cells

Pancreatic α- and β-cells use negative feedback to regulate blood sugar and avoid diabetes. The resting potential of cells is determined by hyperpolarization, which inhibits activity, and depolarization, which excites cells. Elevated blood sugar levels after meals promote insulin secretion in β-cells through Akt. Maintaining mature β-cells is crucial for glucose homeostasis. Immature cells secrete excessive insulin, whereas mature cells release insulin correctly [1,106].

An in vivo study on mice demonstrated that treating LBPs (LBP-38, LBP-50, or LBP-60 for 48 h) did not promote β-cells to release more insulin at high glucose conditions (16.7 mM). However, LBPs promoted β-cells to release less insulin at low glucose levels (3.3 mM). Therefore, it can be concluded that LBP promotes the functional maturity of pancreatic β-cells [69]. LB contains phytochemicals, such as quercetin, resveratrol, rutin, and epigallocatechin, which have been demonstrated to regulate ion channel function in different types of cells [118]. In addition, in vivo studies on rats and mice have shown that extracts from LB berries can improve the body's response to insulin and increase glucose uptake in peripheral organs. This could impact membrane potential changes in α- and β-cells [15,73].

LB includes 18 different amino acids, including an abundance of taurine. According to research, berry taurine can enhance the function of insulin and alter the insulin receptor, thereby directly affecting these channels. An in vitro study performed on retinal epithelial cells revealed that LB-derived taurine boosts peroxisome proliferator-activated receptor gamma (PPAR-γ) activity and elevates cyclic adenosine monophosphate levels, improving the prognosis of diabetic nephropathy by reversing epithelial barrier impairments. The methanol extract of LB (0.1, 0.5, and 1 mg/mL) demonstrated a concentration-dependent increase of PPAR-γ luciferase activity in human embryonic kidney (HEK293) cells (2.87-, 3.37-, and 4.33-fold, respectively), which was strongly inhibited by the PPAR-γ antagonist GW9226. LB taurine replicated these effects, with methanol extract having the greatest taurine level (10.7%± 1.1%) [119,120].

Although further research is needed to understand the precise in vivo processes by which LB modulates the resting membrane potential of α- and β-cells, we can speculate on several probable pathways based on the current understanding. LB bioactive compounds have been shown to possess antioxidant and anti-inflammatory properties. These bioactive compounds can affect cellular signaling pathways responsible for regulating ion channels and maintaining membrane potential. Understanding how LB affects the resting membrane potential of α- and β-cells holds promise for understanding its therapeutic role in controlling diabetes.

4.2.2. Stimulation of insulin secretion from β-cells

Stimulation of β-cells to secrete insulin is essential for controlling blood sugar levels. In vivo, mechanisms for controlling diabetes via stimulating insulin secretion from β-cells involve intricate regulation by glucose sensing, incretin hormones, and neural inputs. The disruption of this balance in T2DM leads to inadequate insulin secretion and hyperglycemia. Different functional foods, including LB, have the potential to influence insulin synthesis from β-pancreatic cells and start the insulin signaling process, which is necessary to maintain glucose homeostasis, both physiologically and pathologically [106].

Rat insulinoma cells treated with LBPs improved damaged pancreatic cells, increased survival rates, and stimulated insulin secretion. An in vitro study indicated that LBP-s-1 (500 mg/kg/day) stimulated neogenesis and enhanced insulin secretion in the rat insulinoma-M5F (RIN-m5f) pancreatic β-cell line. This effect reduced high blood glucose levels and alleviated insulin resistance (P < 0.05). The mechanism through which LBP-s-1 may promote glucose utilization and alleviate insulin resistance is associated with the enhancement of HK (2.81-fold) and PK (2.83-fold) activity in hepatocytes compared to the control group [15]. In vitro cell experiments, LBP3 supplementation in the IR cell model, with LBP3b solution concentrations ranging from 20 to 1.25 g/mL, increased glucose consumption and facilitated translocation across the human epithelial colorectal adenocarcinoma cell line (Caco-2) intestinal cells. This resulted in a hypoglycemic effect, as evidenced by the significant inhibition of glucose absorption at a concentration of 20 mmol/L (P < 0.05). The decrease in blood glucose levels suggests enhanced insulin sensitivity and secretion, indicating the potential of LBP to positively influence glucose metabolism and insulin function [33].

In an in vitro cellular experiment assessing the insulin secretory response in pancreatic β-cells, it was found that anthocyanins, particularly delphinidin-3-arabinoside from fermented berry beverages, potentially upregulate the incretin hormone glucagon-like peptide-1 (GLP-1) (2–3 fold change), increase insulin secretion (100–233 μIU insulin/mL), and enhance the expression of mRNA and proteins associated with insulin receptors in pancreatic β-cells (INS-1E) [121].

LB is a considerable source of potassium; its daily intake may help prevent hypokalemia by enhancing potassium-dependent insulin release or preventing aberrant losses. Nevertheless, at typical dietary levels, LB does not provide enough potassium to influence insulin secretion or maintain electrolyte balance. However, when used as a medicinal supplement, LB may assist in diabetes management [61].

Evidence indicates that LB stimulates insulin secretion from β-cells through intricate in vivo mechanisms. Bioactive compounds found in LB, such as polysaccharides, have been shown to interact with receptors on β-cell surfaces, initiating signaling cascades that culminate in enhanced insulin synthesis and secretion. Moreover, supplementation with LB has been associated with improved β-cell function, as evidenced by heightened insulin response to glucose challenges.

4.2.3. Inhibition of glucagon secretion from α-cells

Gastric inhibitory polypeptide, glucagon, and GLP-1 are among glucagon-related peptides secreted by pancreatic α-cells. The regulation of blood glucose and hormones depends on the interactions between α- and β-cells. The primary mediator of glucose-inhibited glucagon secretion is insulin. In vivo, glucose usually cannot inhibit glucagon release without insulin. Furthermore, glucose may cause α-cells to secrete more glucagon when β-cells are absent [106].

Al-Fartosy [77] studied the acute (24 h) and chronic (21 days) effects of oral galactomannan supplementation from LB against alloxan-induced diabetes in mice. LBPs (500 mg/kg) promoted glucose utilization in peripheral tissues, activated glycogen synthase and insulin-like growth factor (IGF) release in the liver, and suppressed glucagon release in pancreatic α-cells. After 21 days, there was a reduction in blood glucose levels from 311 mg/dL on day 0 to 126 mg/dL on day 21. LBPs also improve glycemic management and hypolipidemia by inhibiting the cholesterol production pathway and activating low-density lipoprotein (LDL) receptors in hepatocytes.

Research findings suggest that LB components suppress glucagon secretion by altering glucose uptake and intestinal absorption. An in vitro cell experiment assessing the hypoglycemic effect of LBP3b found that a water-soluble glycoconjugate LBP3 reduces glucose uptake in intestinal cells, potentially lowering the stimulus for glucagon release (P < 0.05). This inhibition appears to be a competitive interaction with glucose during absorption, as evidenced by dose-dependent effects [33]. An in vitro study investigated the impact of LBP on glucose absorption in Caco2 monolayer cells and discovered that an LB extract (0–400 μg/mL) could inhibit glucagon secretion from α-cells by modulating the expression of sodium-glucose cotransporter-1 in intestinal cells, thereby reducing glucose uptake and postprandial blood glucose levels. After 120 min, glucose absorption levels were 0.800, 0.850, 0.950, and 0.900 mmol/L for doses of LBP at 0, 100, 200, and 400 μg/mL, respectively [81].

In vivo, LB can decrease glucagon secretion from α-cells by multiple routes, as evidenced by the abovementioned studies. LB berries include bioactive substances such as polysaccharides, flavonoids, and carotenoids, which have been found to regulate glucose metabolism and hormone production. These substances may interact with pancreatic α-cells, resulting in reduced glucagon secretion. LB antioxidant and anti-inflammatory qualities can reduce oxidative stress and inflammation in pancreatic α-cells, preventing excessive glucagon release [63]. Furthermore, LB may improve insulin sensitivity and glucose uptake by peripheral tissues, indirectly decreasing glucagon secretion while enhancing glucose homeostasis.

4.3. LB induces β-cell proliferation

In T1DM, the autoimmune loss of β-cells highlights the importance of identifying immune-mediated mechanisms that regulate β-cell proliferation. Alternatively, in T2DM, understanding metabolic variables and communication pathways that control β-cell proliferation is critical for therapeutic approaches [1]. Targeting growth factor signaling, such as IGF-1 and GLP-1, can potentially increase β-cell proliferation and restore normoglycemia. Weight loss therapies may improve β-cell function and insulin sensitivity, potentially improving T2DM management by reducing chronic inflammation, decreasing circulating free fatty acids, and encouraging β-cell regeneration and proliferation. These findings suggest that restoring β-cell mass could help reduce diabetes complications [106].

Zhu et al. [15] conducted in vitro and in vivo experiments to study the impact of LBP-s-1, a new acidic LBP (MW = 1920 kDa, 1.85% protein content), on rat insulinoma cell (RIN-m5F cells) at doses ranging from 16 to 250 nmol/L for 4 h. The researchers compared the results of cell proliferation and glucose-induced insulin secretion experiments to those of exendin-4 (200 nmol/L), a GLP-1R agonist employed as a positive control. Both experiments showed a dosage-dependent response to LBP-s-1 treatment, with peak β-cell proliferation (135%) occurring at the highest dose tested, similar to the impact of exendin-4. Pure LBPs in pancreatic cells improved glucose uptake, metabolism, insulin secretion, and β-cell proliferation. The enhanced glucose metabolism was linked to increased hepatic HK and PK expression/activity [15,77].

Zhang et al. [62] postulated that LBP-a4 (10.2 KDa), a particular portion of LBPs, stimulates lymphocyte proliferation by secreting pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). The treatment with LBP-a4 (400 mg/L for four days) boosted cells in the G0/G1 phase from 49.21% to 69.65%, while cells in the synthesis (S) and gap 2/mitosis (G2/M) phases fell from 40.53% to 10.26%–24.79% and 5.56%, respectively. They further suggested that a spherical molecular shape enhances LBP apoptosis induction, while the flocculent shape lacks this function.

An in vivo study found that diabetic rats treated with LBP-4a (10 mg/kg/day for four weeks) experienced lower blood leptin levels (from 3.05 to 2.07 ng/dL), higher melatonin levels (from 4.34 to 4.90 ng/dL), decreased body weight (from 294 g to 258 g), and reduced epididymal fat (from 1.41 g to 1.07 g). Furthermore, a morphological in vitro study of pancreatic tissue indicated that LBP extract has an ability to minimize histopathological damage caused by diabetes. LBP-4a may restore normal glycemia and insulinemia while preserving β-cell mass, function, and proliferation by normalizing melatonin MT2 receptor mRNA levels in epididymal adipose tissue [12,73]. A study on berry beverages found that phenolic compounds, particularly anthocyanins, primarily delphinidin-3-arabinoside, extracted from these berries were utilized in fermented beverage formulations, showing no increase in deleterious adipokines such as TNF-α and dipeptidyl peptidase-IV. These findings, verified by Western blot analysis, underline the importance of direct effects on protein expression (P < 0.05) within pancreatic β-cell proliferation [121].

LB promotes β-cell proliferation in many ways. It contains bioactive substances, such as polysaccharides and anthocyanins, which interact with cellular receptors and signaling pathways within pancreatic islets. These interactions initiate intracellular cascades that activate transcription factors and increase gene expression in cell cycle progression and proliferation. LB contents may influence the release of growth factors and cytokines from surrounding cells or tissues, promoting β-cell expansion. Similarly, LB antioxidant qualities may protect β-cells from oxidative stress, promoting survival and proliferation.

4.4. LB improves glucose tolerance and insulin resistance

Glucose tolerance shows the body's ability to manage blood sugar and is frequently assessed through glucose tolerance tests. Insulin resistance disturbs blood sugar management, causing cells to respond inadequately to insulin, resulting in hyperinsulinemia. Over time, this can strain pancreatic β-cells, lowering insulin production and leading to T2DM. Furthermore, decreased glucose tolerance and insulin resistance promote inflammation and oxidative stress, exacerbating metabolic dysfunction and diabetic consequences [106].

The glucose tolerance test (LB 1 g/kg body weight (BW)) revealed higher glycemic and insulinemic indices in rats on a high-carbohydrate diet (HCD) compared to those on a conventional diet, indicating lower glucose tolerance in the HCD group. In vivo LB supplementation (250 mg/kg BW for 60 days) improved insulin sensitivity, reducing HCD-induced central/visceral obesity and increasing hepatic lipid content. Total lipids decreased from 7 to 6.2 g/100 g, triglycerides decreased from 1.9 to 1.4 g/100 g, and cholesterol decreased from 0.25 to 0.23 g/100 g [122]. In diabetic rats treated with LBPs (100, 250, and 500 mg/kg), glucose levels dropped dose-dependently from 16 mmol/L in the rat model to 13, 9, and 8 mmol/L, respectively, without inducing cytotoxicity [123].

LBPs improved insulin resistance and reduced inflammation, as evidenced by lower serum levels of insulin, IL-6, and TNF-α in diabetic rats treated with 30 and 60 mg/kg LBPs, respectively, for 12 weeks compared to untreated diabetic rats used as controls [124].

LBP-1 (100–500 μg/mL) protects RIN-m5F cells from alloxan-induced damage, improving cell survival and pancreatic function. Additionally, LBP-1 (1–100 μg/mL) reduces insulin resistance in hepatocellular carcinoma G2 (HepG2) cells, enhancing glucose consumption, as evidenced by in vitro and in vivo hypoglycemic experiments. Exposure to varying concentrations of LBPs-1 resulted in a significant increase in glucose consumption in HepG2 and 3T3-L1 cells, peaking at a concentration of 62.5 nmol/L [15]. In an in vivo study involving 75 female rabbits, supplementation with 1% or 3% LB berries two months before artificial insemination resulted in improved glucose tolerance and insulin resistance during pregnancy compared to the control group, which received only a standard diet (30.0% wheat bran, 42.0% dehydrated alfalfa meal, and 9.5% barley) without LB supplementation. This improvement was evidenced by reduced peak glucose levels post-bolus (from 25 to 18 mmol/L) despite no change in offspring insulin sensitivity [85].

LB possesses both SDFs and IDFs, and it was shown that IDFs are linked to better insulin resistance, as seen by stable blood sugar and insulin levels. Its influence on protein absorption may slow the activation of the mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase 1 (mTOR/S6K1) pathway, which is linked to insulin resistance. Due to its thickness, SDF forms gel-like structures in the stomach, decreasing nutrient and carbohydrate absorption and lowering blood sugar levels after meals. Furthermore, LB soluble fiber can bind to cholesterol and bile acids, reducing inflammation and insulin resistance and lowering blood pressure [54,125].

The high abundance of bioactive substances in LB, including polysaccharides, carotenoids, and antioxidants, improves insulin sensitivity and glucose absorption. LB also contains dietary fibers, which delay food absorption and stabilize blood sugar levels. Fatty acids, such as linoleic acid and oleic acid, increase insulin sensitivity while inhibiting glucose absorption. Additionally, LB has anti-inflammatory characteristics that can improve insulin sensitivity and blood sugar management.

4.5. LB inhibits the production of inflammatory cytokines

The inhibition of inflammatory cytokine production is directly related to diabetes, as chronic inflammation plays a vital role in the origin and progression of the disease. In vivo investigations have shown that high levels of inflammatory cytokines, including IL-6 and TNF-α, contribute to insulin resistance and pancreatic β-cell malfunction. This pro-inflammatory condition compromises insulin signaling and accelerates the death of insulin-producing β-cells in the pancreas, increasing hyperglycemia and resulting in diabetes. Therefore, therapies aiming at decreasing the generation of inflammatory cytokines show promise for reducing insulin resistance, preserving β-cell function, and eventually improving glycemic control in individuals with diabetes [126].

LB, renowned for its potent anti-inflammatory properties, has attracted considerable interest for its potential medicinal advantages. Yang et al. [66] demonstrated the efficacy of LBPs (50 mg/kg for two days) in reducing ischemia-reperfusion injury by inhibiting oxidative stress and inflammation in rats. LBPs inhibited polymorphonuclear neutrophil accumulation (activity decreased from 16 U/g to 9 U/g) and intercellular adhesion molecule 1 (ICAM-1) expression (relative densitometric protein expression decreased from 3.5 to 2.0), thereby influencing TNF-α levels and nuclear factor-kappa B (NF-κB) activation. In vivo studies on rats demonstrated that the LB extract (2 mL/day/animal for 60 days) protects against LPS-induced inflammation. Specifically, groups treated with LB extract showed significantly reduced C-reactive protein levels (from 2.5 mg/L to 1.3 mg/L) compared to the control group, which received only a palatable diet (composed of 25% standard diet, 34% condensed milk, and 23% corn starch) [127].

Xiao et al. [70] emphasized the hepatoprotective properties of LBPs (1 mg/kg or 10 mg/kg), reducing oxidative stress and inflammatory reactions induced by carbon tetrachloride (50 μL/kg) hepatotoxicity. Following CCl4 treatment, CYP2E1 protein levels increased from 40% to 55% of the control, nitrotyrosine formation increased from 90 to 100, malondialdehyde (MDA) levels decreased from 270 to 220, and catalase (CAT) mRNA expression decreased from 110 to 80 after LBP administration. Meanwhile, untreated mice showed extensive necrosis.

Additionally, Nardi et al. [25] investigated the anti-inflammatory properties of LB berries in mice in vitro and in vivo. The berries were administered orally at doses of 50 and 200 mg/kg twice daily for 10 days. The study yielded promising results in an acute inflammation model. Similarly, an in vivo LBP treatment (100, 250, and 500 mg/kg for four weeks) in diabetic rat models revealed renoprotective benefits, lowering albuminuria and inflammatory markers, including IL-2, IL-6, TNF-α, interferon-α (IFN-α), monocyte chemoattractant protein (MCP-1), and ICAM-1, while downregulating nuclear factor-kappa B expression. LBPs at 500 mg/kg caused a significant reduction in the serum levels of IL-2 (28.4%), IL-6 (35.9%), TNF-α (38.8%), IFN-α (34.8%), MCP-1 (36.1% reduction), and ICAM-1 (40.9% reduction) [123].

Furthermore, a comparative investigation found that LB berry extracts (LrND, LrD, LbND, and LbD 10 mg/mL) exhibited higher anti-inflammatory efficacy in cyclooxygenase-2 (COX-2) in vitro peroxidase endpoint assay. The most significant level of COX-2 inhibition was achieved with LrND, resulting in approximately 80% inhibition, followed by LrD, LbND, and LbD [128]. LBAs administered at doses of 50, 100, and 200 mg/kg for eight weeks in obese rats were shown to reduce body weight gain and inhibit the expression of inflammatory genes, such as IL-6, TNF-α, IFN-γ, NF-κB, and inducible nitric oxide synthase (iNOS). The administration of LBAs at doses of 50, 100, and 200 mg/kg reduced body weight gain by 17.4%, 18.7%, and 38.3%, respectively. In contrast, the non-supplemented control group exhibited higher food utilization and increased expression of inflammatory genes [74].

Pro-inflammatory cytokines, such as IL-1β, IFN-γ, and TNF-α, cause apoptosis in pancreatic β-cells, resulting in decreased insulin production and glucose metabolism. LB extracts, specifically LBPs, can block cytokine production, preventing β-cell death and maintaining insulin sensitivity. LBPs can inhibit IFN-γ-induced apoptosis by influencing the P53 signaling pathway. Additionally, LBPs significantly suppress apoptosis caused by IL-1β, TNF-α, and IFN-γ [129]. Furthermore, the anti-inflammatory actions of LB are systemic, lowering pro-inflammatory cytokine levels in the bloodstream. This reduction in systemic inflammation reduces insulin resistance, improves glucose tolerance, and can help prevent diabetic complications, such as CVD and nephropathy.

4.6. LB reduces oxidative stress

Oxidative stress occurs when an imbalance between free radicals and antioxidants in the body causes cellular damage. In diabetes, oxidative damage exacerbates insulin resistance and decreases pancreatic β-cell activity, which is critical for blood sugar control. Reducing oxidative stress has enormous potential in controlling diabetes, a disease characterized by high blood sugar levels [106].

LB is high in antioxidants, notably polyphenols such as caffeic acid, which are essential in neutralizing free radicals and activating antioxidant mechanisms. According to studies, these antioxidants, including superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GPx), CAT, and erythroid-derived 2-like 2 (Nrf2) expression, contribute significantly to the overall antioxidant activity of LB berries. For example, the presence of antioxidants increases the expression of SOD, CAT, and GPx enzymes [78].

LB berries have shown that they can protect hepatic cells from oxidative stress-induced damage. One study demonstrated that LB berry extracts (500 μL/mg) effectively prevent lipid oxidation, DNA damage, and protein carbonylation by reducing intracellular ROS levels and enhancing the activity of antioxidant enzymes such as SOD, CAT, and GSH in liver cells. An in vitro study revealed that Lycium extract increased SOD activity from 37 to 40 U/mg protein, which subsequently decreased to 33 U/mg protein after H₂O₂ treatment. Furthermore, CAT activity increased from 41 U/mg (in the control group) to 47 U/mg protein [130].

LBPs (100, 200, and 300 mg/kg/day by gavage for 30 days) were investigated for their antioxidant properties. They have been shown to decrease oxidative stress following exercise in mice by enhancing antioxidant enzymes such as SOD, CAT, and GPx. SOD increased from 33 to 39 U/mg, CAT from 2.1 to 3 U/mg, and GPx from 2.0 to 2.8 U/mg. Meanwhile, the untreated control group exhibited reduced levels of these biomarkers compared to the treated group [82]. LBP (300 μg/mL for 24 h) reduces ultraviolet B (UVB)-induced oxidative stress in the human keratinocyte cell line (HaCaT), maintaining cell viability, decreasing ROS generation (from 380 to 140 intracellular ROS% of the control), and minimizing DNA damage compared to untreated controls. This effect involves the activation of the Nrf2/antioxidant response element (ARE) pathway, suggesting the potential of LBP as a skincare agent against environmental oxidative damage [131].

One in vitro study highlighted the antimutagenic characteristics of LB berry aqueous extracts (25 and 100 μg/mL), demonstrating their potential to protect against DNA damage (IC₅₀ ranging from 0.69 to 6.90 mg/mL) caused by peroxyl and hydroxyl radicals. Additionally, these extracts exhibited significant antioxidant actions in mouse muscle cells (C2C12), with higher GSH levels (189.5%) and lower lipid peroxidation (21.8%) [78]. A comparative in vitro analysis of two distinct types of Lycium berries revealed noteworthy antioxidant properties. LB showed effectiveness with IC₅₀ values of 830 ± 5.4 μg/mL for 1,1-diphenyl-2-picrylhydrazyl (DPPH·) and 195 ± 3.5 μg/mL for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS·⁺). In contrast, Lycium chinese had values of 950 ± 4.7 μg/mL and 220 ± 6.1 μg/mL, respectively [56].

The antioxidant properties of LB berries showed promise for diabetes control by protecting pancreatic cells from oxidative stress and improving microcirculation to enable glucose and insulin delivery. Furthermore, the presence of antioxidant and anti-inflammatory components in LB helps to combat oxidative stress and chronic inflammation linked with diabetes, which improves immunological function and insulin sensitivity.

4.7. LB modulates the function of cell mitochondria

Mitochondria, or cellular powerhouses, play an essential role in diabetes by controlling energy production, ROS formation, and insulin release. Malfunction in these organelles leads to insulin resistance, β-cell malfunction, inflammation, and cell death, which worsens hyperglycemia. In addition, mitochondrial abnormalities play a critical role in developing both T1DM and T2DM, whether through reduced adenosine triphosphate generation, increased ROS, or inflammatory responses [106].

The study administered LB (30 mL/kg) to 18-month-old rats for 60 days and found that it restored age-related alterations in biomarkers associated with mitochondrial function and cellular health. In vitro experiments demonstrated that LB lowered glial fibrillary acidic protein, caspase-3 (Casp-3), 3-nitrotyrosine (3-NT), Nrf2, and ROS levels while enhancing synaptophysin staining and mitochondrial activity in the prefrontal cortex (PFC) and hippocampus regions (cornu ammonis 1 (CA1) and dentate gyrus (DG)) compared to the control group (vehicle water 1 mL/kg for 60 days). The ROS analyses revealed reductions in PFC, CA1, and DG regions by 34.7%, 49.5%, and 66.8%, respectively. Mitochondrial retention significantly increased by 260% in the PFC and 473% in the CA1 region in LB supplemented groups [86]. Yu et al. [90] found through in vitro experiments that 1% (kcal) LB supplementation increased carotenoid metabolic genes for zeaxanthin and luteolin while also improving mitochondrial biogenesis in the retina of db/db diabetic mice.

Taurine, abundant in LB extract and essential for retinal health, has been shown to help prevent diabetic retinopathy. Studies have shown that it can reduce retinal cell apoptosis by inhibiting mitochondrial malfunction and apoptotic pathways, potentially providing therapeutic benefits for diabetes-related retinal diseases [132,133]. In another in vitro trial, rats in the HCD group, aged 90 days, were given either a vehicle (0.1 mL/100 g BW) or LB (250 mg/kg BW) extract for 60 days. Liver examination of HCD rats revealed increased mitochondrial ROS (from 35 to 60 pmol 2',7'-dichlorofluorescin (DCF)/min × mg protein) and lipid peroxidation, as well as lower mitochondrial (from 1.25 to 1.0 mg GSH/mg protein) and cytosolic GSH levels (from 5.5 to 4.5 μg GSH/mg protein) [122].

A recent study on mice discovered three LB berry cyclic peptides (GCPs). The administration of GCP-1 (1 mg/kg every four days) significantly elevated ROS levels (increased by 37.4% and 254%), suggesting its involvement in apoptosis induction. Furthermore, GCP-1 decreased mitochondrial membrane potential (Δψm), increased cytosolic cytochrome c (Cyt c), reduced mitochondrial Cyt c, and altered the Bax/Bcl-2 ratio in HeLa cells (80.2 and 401 Μm for 48 h), indicating the activation of the mitochondrial apoptotic pathway [134]. In an in vitro study, LB juice (LBJ) at 1 or 10 μg/mL concentrations inhibited mitochondrial dysfunction in human salivary gland cells. LBJ preserved mitochondrial membrane potential (2.47 ± 0.48) while suppressing apoptotic pathways, suggesting antiapoptotic properties relevant to diabetes. In contrast, the Coptidis Rhizoma sample solution (used as a negative control) did not show protective effects against mitochondrial potential decline [135].

Numerous studies have shown that LB has mitochondrial modulation effects on various disorders, including in vivo studies highlighting its potential therapeutic applications beyond diabetes [25,136]. The regulation of mitochondria by LB has tremendous potential to affect diabetes. LB may improve insulin sensitivity, reduce oxidative stress, and increase cellular energy generation by preserving mitochondrial function. Its antiapoptotic characteristics may protect pancreatic β-cell against apoptosis, preserving β-cells bulk and function.

4.8. LB ameliorates diabetes through epigenetic modification

Epigenetic modifications significantly impact diabetes by altering gene expressions related to insulin secretion and sensitivity. Changes in DNA methylation, histone modifications, and non-coding RNA expression disrupt crucial pathways in glucose metabolism and insulin action [106].

Research into the possible advantages of LB for diabetes through epigenetic changes is ongoing. Initial findings indicate that its bioactive components, like polyphenols and flavonoids, may alter DNA methylation and histone modification, potentially improving diabetic outcomes. Experimental investigations show that LBPs (1% (kcal) for eight weeks) increase mitochondrial biogenesis by upregulating particular metabolic genes, potentially protecting against diabetic retinopathy [90]. Furthermore, 1% LBPs can reduceER stress, which is critical for alleviating hyperglycemia-associated oxidative stress affecting protein synthesis and folding inside the ER, resulting in apoptosis. LB significantly mitigated retinal ER stress, demonstrated by lowered expression of ER stress biomarkers: binding immunoglobulin protein (BiP)/b-actin decreased from 0.42 to 0.25), protein kinase RNA-like ER kinase (PERK)/b-actin decreased from 0.41 to 0.25, activating transcription factor-6 (ATF6)/b-actin decreased from 0.7 to 0.15, and active caspase-12 (active caspase-12)/b-actin decreased from 0.39 to 0.18 in db/db mice [93].

In a study on pregnant rabbits, LB supplementation (C, 1% G1, and 3% G3) was evaluated for its influence on maternal insulin sensitivity and potential epigenetic consequences on offspring health. Higher doses increased maternal insulin responsiveness, but no substantial improvements were found in offspring insulin sensitivity. This is evidenced by reduced peak glucose levels post-bolus (from 25 mmol/L to 18 mmol/L) [85].

Numerous studies have shown that LB berries favorably impact epigenetic regulation in various illnesses [137,138]. The data indicates that LB supplementation can change epigenetic pathways in vivo, leading to better outcomes in controlling diabetes. This modulation may include changes in DNA methylation patterns, histone modifications, and non-coding RNA expression, which eventually influence gene expression associated with insulin sensitivity and glucose metabolism. Furthermore, while immunological activities and epigenetic regulation are independent, they can overlap because epigenetic changes can directly affect immune cell function, potentially affecting overall immunity.

4.9. LB ameliorates diabetes through immune modifications

Immune senescence, the term for age-related immune system remodeling, is marked by a reduction in the body's ability to fight infections, a weakened immune response, a higher risk of developing chronic illnesses, a rise in the frequency of autoimmune diseases, and constant low-grade inflammation [1]. An inflammatory response is brought on by the body's reaction to high blood glucose levels and the production of inflammatory mediators by adipocytes and macrophages in adipose tissue. This low-grade, continuous inflammation damages pancreatic β-cells and prevents enough insulin from being synthesized, leading to hyperglycemia. In obese adipose tissue, interactions between pathogenic CD4⁺ and CD8⁺ T cells and CD11c + M1 macrophages exacerbate the inflammatory immune response caused by adipocyte death and macrophage infiltration. This exacerbates inflammation in the adipose tissue and peripheral insulin resistance [126]. To counteract peripheral insulin resistance and hyperinsulinemia, pancreatic cells generate more insulin. Eventually, chronically increased insulin resistance leads to worn-out β-cells and an insulin shortage. Moreover, the accumulation of inflammatory cytokines, amyloids, and free fatty acids causes cell death, which leads to persistent hyperglycemia and T2DM [106].

In recent years, LBPs have attracted a lot of attention from researchers. The properties of LBP as a natural active ingredient include non-toxicity, safety, lack of residue, and drug resistance. It raises high-density lipoprotein (HDL) levels, works as an antioxidant, improves insulin sensitivity, and encourages the pancreas to make more cells that secrete insulin. This increased insulin allows cells to absorb more glucose and thus benefits diabetes treatment. The digestion of carbohydrates also improves, and they get stored in the form of glucose in the cells, thereby maintaining blood sugar levels [5]. LBPs are organic immune modulators that can stimulate immune cells, such as T/B lymphocytes, macrophages, and NK cells, triggering the release of several cytokines and antibody formation. Numerous immunomodulatory activities and immunological adjuvant effects of LBPs are well-established [139,140].

One in vitro study demonstrated that LBP3 (<350 kDa) enhances RAW 264.7 murine macrophage activity, suggesting potential for diabetes therapy. LBP3 (100 μg/mL) improved immunological modulation in these macrophages for 24 or 48 h. Conversely, LPS (100 μg/mL) significantly increased the expression levels of major histocompatibility complex class II (MHC-II) (by 50%) and CD86 molecules (by 75%) on RAW 264.7 macrophages [64]. In another in vitro study, three different cell models, tumor cells, kidney cells, and murine macrophages, demonstrated that LBP fractions (>10 and <10 kDa) enhanced macrophage viability and polarization (274.07% ± 19.83% at 1000 μg/mL). The >10 kDa fraction regulated NO, TNF-α, IL-6, and ROS, leading to dual inflammatory actions. Dye-labeled LBP was internalized through clathrin-mediated endocytosis, revealing its immunomodulatory characteristics [67].

One in vitro study focusing on LBPF4-OL identified its relationship with the toll-like receptor 4-mitogen-activated protein kinase (TLR4-MAPK) pathway. LBPF4-OL (50,100 μg/mL) stimulates TNF-α and IL-1β in peritoneal macrophages of wild-type (C3H/HeN) mice but not in TLR4-deficient mice (C3H/HeJ). LBPF4-OL also increases TLR4/MD2 expression (from 400 to 3000 cpm at 200 μg/mL). It activates p38-MAPK while suppressing c-Jun N-terminal kinase and extracellular signal-regulated kinases (ERK1/2) phosphorylation, indicating that it functions as a TLR4/MD2-MAPK pathway activator, which ultimately plays a vital role in the immunomodulation of diabetes [71].

Certain LBFs, such as rutin and flavonols, can boost immunological responses by increasing radicals, generating antibodies, and acting cytotoxically on cancer cells. These benefits may assist in reducing immunological dysfunction, inflammation, and oxidative stress, all of which contribute to diabetes development and progression. One study demonstrated that rutin presence in a complex great powerful blend (GPB), derived from the significant component LB, contributes to its phytochemical profile. This blend enhances immune response both in vitro (RAW 264.7 cells at concentrations of 50 or 200 mg/mL for 24 h) and in vivo (mice at doses of 300 or 600 mg/kg once per day for 30 days) by increasing TNF-α production (from 1.5 to 4.8 pg/mL at 300 mg/kg), upregulating pro-inflammatory cytokines, and activating the NF-κB and activator protein-1 (AP-1) pathways [75].

Similarly, a phenolic-rich extract from LB fruits exhibited an antiproliferative effect (IC₅₀ < 100 μg/mL) in vitro against head and neck human papillomavirus type 16 (HPV16) squamous cell cancer measured after 24 and 48 h. LB extract at 1.0, 10, and 100 μg/mL inhibited HPV type 16 cell lines. The researchers used chemotherapeutic cisplatin as a reference point (control) to compare its effectiveness against the cell lines. These observed effects were attributed to anticancer and immunomodulatory phenolics, particularly flavonols/flavan-3-ols and tyramine-conjugated hydroxycinnamic acid amides found in the extract [79].

A study explored LBCs and developed a carotenoid nanoemulsion to investigate its inhibitory mechanism on HT-29 colon cancer cells and assess immunomodulatory effects. In vitro experiments demonstrated that three doses of carotenoid nanoemulsion or extracts (2, 4, and 6 μg/mL) effectively suppressed HT-29 cell growth (IC₅₀ = 4.5 and 4.9 μg/mL) and arrested the cell cycle at G2/M. These findings suggest that LB nanoemulsion may influence the immune system, as evidenced by the up-regulation of p53 and p21 expression and the down-regulation of cyclin-dependent kinase (CDK2 and CDK1), cyclin A, and cyclin B expression, potentially impacting diabetes [83].

L5178Y lymphoma cells and HTB-26 breast cancer cells showed similar immunomodulatory effects when treated with LB zeaxanthin extract and caper lutein extract. Treatment with β-cryptoxanthin, lutein, and lycophyll decreased cell size by less than 5%. Shrinkage was detected in the presence of zeaxanthin [87].

Only a few previous studies have directly demonstrated the link between LB vitamins, minerals, and immunomodulatory effects. In a study, LB extracts lowered blood glucose, TC, and TG levels while increasing HDL cholesterol (HDL-c) in rabbits. Administered doses included water decoction (0.25 g/kg/day), crude LBP (10 mg/kg/day), and purified polysaccharide fractions (LBP-X) (10 mg/kg/day) for 10 days. Mean decreases in blood glucose levels caused by fruit water decoction, crude LBP, and LBP-X were 8.04, 8.47, and 14.13 mmol/L, respectively. Average reductions in TC and TG caused by LB extracts/fractions were 3.39 and 2.77 mmol/L for fruit water decoction, 3.82 and 1.69 mmol/L for crude LBP, and 4.27 and 3.50 mmol/L for LBP-X. In vitro, these extracts also demonstrated antioxidant activity, potentially contributing to immunomodulation and diabetes management, attributed to components such as vitamins C, B1, and B2 [91].

2-O-β-d-Glucopyranosyl-l-ascorbic acid (AA-2βG) from LB exhibits superior antioxidant properties to L-AA (AA), particularly in scavenging radicals and preventing hemolysis. An in vivo study showed that LB AA-2βG, administered at 100, 200, and 300 mg/kg doses for seven days, protects against carbon tetrachloride-induced liver injury in mice. Serum ALT and AST levels notably decreased. Following AA-2βG administration at 300 mg/kg, ALT levels decreased from 131 to 61.9 U/L, and AST levels decreased from 281 to 137 U/L. These findings suggest that AA-2βG possesses immunomodulatory properties and may aid in diabetes management through antioxidant mechanisms [94].

A healthy immune system is essential for managing diabetes because it fights infections and controls inflammation, which may improve insulin sensitivity and glycemic control. The presence of anti-inflammatory and antioxidant compounds in LB aids in the fight against chronic inflammation and oxidative stress, which are frequently connected with diabetes. As a result, immune function is improved by maintaining a balanced response between pro-inflammatory and anti-inflammatory activities. Moreover, consuming LB improves immunological function, particularly phagocytes and lymphocytes, which are essential in fighting diabetes-related infections and inflammation. This effect on immunomodulation promotes a resilient immune system, which is vital in diabetes management since it reduces inflammation and promotes general health. LB can also improve immunomodulation because of its numerous bioactive components and essential minerals (Fig. 4).

Fig. 4.

Potential immunomodulatory and microbial antidiabetic effects of bioactive compounds found in Lycium barbarum (LB).

4.10. LB ameliorates diabetes through gut microbiota modifications

Microorganisms inhabit numerous parts of the human body, most of which live in the GI tract, which benefits metabolism, immunity, and the nervous system. Based on the residential microbial genetic material (microbiome) that goes along with the human genome, humans are now called “superorganisms”. It is well recognized that human gene expression changes in response to environmental adaptation as our microbiota changes and evolves [141]. Additionally, the gut microorganisms support effective energy metabolism, which offers specific advantages during hunger. It has been suggested that the human gut contains up to 1,000 different phyla of bacterial sp. Firmicutes and Bacteroidetes are the two phyla that comprise most of the bacterial sp.; Actinobacteria, Proteobacteria, and Verucomicrobia are also found, though in fewer numbers. Alterations in microbial compositions, which are often influenced by dietary habits, antibiotics, probiotics, and lifestyle, can impact the overall health of the gut ecosystem [142].

The microbiotic community is rebuilt and maintained during early development to adulthood by changes in dietary compositions from a high-fat diet through the consumption of mother's milk in newborns to the introduction of a carbohydrate-rich, solid and complex diet. Additionally, the decreased expression of tight junction proteins, which may facilitate the unchecked transit of antigens, is a result of the increased intestinal permeability. It makes it possible for bacterial LPSs to go from the intestine to the bloodstream and produce metabolic endotoxemia and insulin resistance [141]. Through providing energy and support for microbial development, a diet containing LB could significantly influence the gut microbiota. The microbiota can digest non-digestible polysaccharides and produce various metabolites, such as SCFAs, which support the gut ecology.

Bioactive compounds in LB, such as polysaccharides, flavonoids, and carotenoids, could significantly influence gut microbiota composition. They promote beneficial bacteria and inhibit harmful microbes, supporting a balanced gut environment [56]. LBP has the potential to aid diabetes management through microbiota modulation and cellular mechanisms, impacting pathways linked to insulin sensitivity and β-cell function, as indicated by various studies. Similarly, one study revealed that flavonoids such as quercetin inhibit pathogen growth while boosting beneficial genera, such as Bifidobacterium and Lactobacillus, potentially enhancing gut health by reducing endotoxin production, aiding bile acid conversion, supporting immune balance, and facilitating nutrient absorption [143]. In another study, consuming carotenoids, particularly astaxanthin, relieved inflammation and reduced lipid accumulation by decreasing Bacteroidetes and Proteobacteria abundance while increasing Verrucomicrobiota and Akkermansia sp. population density [142].

LB, rich in dietary fibers, impacts diabetes management by modulating the microbiota through in vivo mechanisms. Gut bacteria ferment fiber into SCFAs, activating G protein-coupled receptors (GPCRs), such as GPR41 and GPR43, which regulate inflammation and metabolic balance. GPR41 activation increases GLP-1 release, enhancing satiety and insulin sensitivity. GLP-1 triggers insulin synthesis by binding to pancreatic β-cell receptors. Exendin-4, a GLP-1 analog, upregulates ephrin receptor exchange protein directly activated by cAMP 1 (Epac1) and Epac2 isoforms, which are crucial for diabetes therapy and glucose homeostasis [144].

LB is a considerable source of vitamin C. Ang et al. [145] found that vitamin C influences the proliferation of helpful bacteria while inhibiting detrimental microbial strains, resulting in a more balanced gut environment. This modulation has been linked to improved gut barrier function, decreased inflammation, and increased insulin sensitivity, all essential variables in diabetes management. Likewise, minerals can modify intestinal microbiota composition, metabolic inflammation, and glucose metabolism control [146]. Although no direct study of LB micronutrients separately assessed their effect on gut microbiota modulation, various studies have suggested that LB micronutrients could contribute to positive changes in gut microbiota [137,147].

In one in vivo study, eight weeks of dietary insulin treatment, either alone or combined with LBP (400 mg/kg), corrected diabetic abnormalities in rats by lowering glucose (P = 0.0397), LPS (P = 0.0127), and inflammation while modifying gut microbiota and SCFAs [68]. LB, rich in polysaccharides and polyphenols, modifies gut microbiota and intestinal barrier function. In male C57BL/6J mice fed diets with 1.5% or 3% LB for 10 weeks, high-throughput sequencing of 16S ribosomal DNA (HiSeq 16S rDNA) analysis revealed the significant impact of LB on the microbial profile, promoting Verrucomicrobia, Bacteroidetes, and SCFA-producing bacteria while suppressing Firmicutes and ammonia concentrations [65].

In C57BL/6J mice, LBP (750 mg/kg for 15 days) shifted epithelial immunity toward anti-inflammatory responses, decreased potentially harmful bacteria (Allobaculum stercoricanis, Parasutterella excrementihominis, and Tannerella spp.), and increased SCFA-producing bacteria (Ruminococcus sp., Lachnoclostridium clostridium xylanolyticum, and Clostridium sulfatireducens). These findings indicate that LB health effects may involve manipulation of the gut microbiota, as evidenced by the upregulation of SCFA-sensing receptors (GPRs 41, 43, and 109a) [72].

Prebiotics are indigestible substances that can modify the gut microbiota by promoting the growth and activity of bacteria. The prebiotics included in LB can potentially enhance gut flora's health, improving glucose metabolism and insulin sensitivity.

Skenderidis et al. [76] conducted an in vitro study where they cultured strains of Bifidobacterium and Lactobacillus in the presence and absence of several encapsulated LB extracts, demonstrating the prebiotic activity of LB. The extracts enhanced the growth of probiotics, increasing Bifidobacterium animalis subsp. lactis (Bb12), Bifidobacterium longum (Bb46), and Lactobacillus casei colonies by 2.0, 0.26, and 1.34 (log cfu/mL), respectively. A different study used the growth of Lactobacillus acidophilus and Bifidobacterium longum to evaluate the prebiotic action of LBPs in vitro. The two strains were grown appropriately, and the experimental group received varying doses of LBP powder (2.5%, 5%, 10%, and 15% (w/v)), while the control group received glucose. Compared to the control group, these trials demonstrated the beneficial impact of LBPs on the growth of both strains (8.23 and 6.62 (log10 cfu/mL)) [80].

In a study on male mice, administering fermented LB juice (20 mL/kg/day) for 30 days showed superior efficacy against ulcerative colitis compared to unfermented juice. It reduced pro-inflammatory cytokines and total SOD (T-SOD) levels while increasing anti-inflammatory cytokines, myeloperoxidase, and GSH levels in serum and colon. Additionally, LB juice lowered intestinal permeability and modulated the gut microbiome, with Bacteroidetes increasing by 0.47 times and Firmicutes decreasing by 0.34 times compared to the ulcerative colitis group [84].

The bioactive compounds found in LB can potentially promote the growth of a healthy gut microbiota, as illustrated in Fig. 4. Therefore, incorporating LB into the diet, either alone or in combination with probiotics, could benefit diabetic individuals by positively impacting their microbial flora.

5. LB clinical trials: Strengthening the case for antidiabetic potential

In recent years, researchers have focused more on the functional properties of LB. However, the number of clinical trials is still limited. Clinical trials are vital because they ensure the successful designation of any functional food for therapeutic use. Future clinical trials have the potential to strengthen the case for the use of LB as an antidiabetic diet. We will explore noteworthy clinical trials concerning LB, focusing on antioxidants, immunomodulation, gut microbiota, and diabetes. These trials hold promise for potentially impacting diabetes or its related symptoms.

One study found that consuming LB (with lutein 6 mg and zeaxanthin 4 mg) daily at 28 g for 90 days in healthy middle-aged individuals increased macular pigment optical density at 0.25 and 1.75 retinal eccentricities. This improvement suggests a potential preventive effect against age-related macular degeneration and highlights antioxidant properties [148]. Adding 15 g of LB daily to a balanced diet for 16 weeks improved vascular health and decreased oxidative stress. Subjects exhibited significantly increased HDL-c (0.08 ± 0.04 mmol/L), reduced CVD risk (−0.8% ± 0.5%), and lowered vascular age (−1.9 ± 1.0 years) post-intervention [96]. In another study, supplementing 15 g of LB daily to a balanced diet for 16 weeks reduced oxidative stress in middle-aged and older adults. MDA levels remained unchanged (0.29–0.28 μmol/L), while the levels of 8-iso-prostaglandin F2α decreased from 73.1 to 53.2 ng/L. Additionally, plasma zeaxanthin significantly increased by 92.4%. This intervention holds promise for mitigating age-related diseases [99].

Consuming 14 g of LB daily for 45 days has demonstrated its potential as a beneficial supplement for preventing CVD in individuals with metabolic syndrome, along with antioxidant properties. The results indicated an increase in serum antioxidant capacity. They reduced GSH by 173% and 5.1%, respectively. In comparison, lipid peroxidation and waist circumference values decreased by 15% and 6.3 cm, respectively [149]. Daily supplementation with 13.7 g of LB for 90 days increased plasma zeaxanthin and antioxidant levels by 26% and 57%, respectively, protecting against hyperpigmentation. In the control group, which consumed a placebo (skim milk 290 mg/g, maltodextrin 200 mg/g, and sucrose 476 mg/g) instead of LB, no change was observed in plasma zeaxanthin and antioxidant levels [102]. Supplementing 50 healthy adults with LB (120 mL/day) improved antioxidant efficacy. SOD and GSH-Px levels showed an increase of 8.1% and 9.0%, respectively, while MDA levels decreased by 6.0% [103]. LBP treatment at 100 mg/kg BW for three months combined with qigong exercise prevented atherosclerosis by improving lipid profiles, oxidative stress, and blood pressure. In men, HDL-c increased from 1.10 to 1.58 mmol/L, LDL cholesterol (LDL-c) decreased from 2.61 to 2.46 mmol/L, and TC decreased from 4.73 to 4.62 mmol/L. In women, HDL-c increased from 1.19 to 1.53 mmol/L, LDL-c decreased from 2.821 to 2.57 mmol/L, and TC decreased from 4.81 to 4.75 mmol/L [104].

In a placebo-controlled study on healthy subjects, the consumption of 15 g of LB per day, which contains almost 3 mg of zeaxanthin, considerably increased fasting plasma zeaxanthin levels (2.5-fold) over 28 days [105]. In a three-month study, dietary lacto-LB supplementation at 13.7 g per day in 150 elderly people improved vaccination response (27.7% vs. 8.8% in the placebo group) and immunological protection without overburdening the immune system [97]. In a 30-day experiment, a daily intake of 120 mL of standardized LB fruit juice improved immunological responses (lymphocytes from 2.09 to 2.23 × 106/mL, IL-2 from 7.48 to 9.36 pg/mL, and IgG from 14.98 to 16.78 g/L) and overall well-being in 60 healthy old adults without side effects compared to the placebo group [100].

Furthermore, ingesting 120 mL of LB daily for 14 days improved overall well-being, cognitive performance, immunity, and GI health in 70% of the subjects [88]. Finally, 300 mg/day of LBP for three months effectively lowered serum glucose from 1.61% to −7.86% and enhanced the insulinogenic index from −0.98% to 0.04% in T2DM patients. No difference was observed in the serum glucose levels of the control group, which received a placebo (300 mg of microcrystalline cellulose), indicating LBP's promise as a therapeutic adjunct [89].

6. Implications

6.1. Limitations

Exploring LB bioactive compounds encompassing polysaccharides, flavonoids, carotenoids, fibers, fatty acids, vitamins, and minerals indicates substantial promise in treating diabetes and its related symptoms. These compounds have properties that suggest they can reduce inflammation, protect pancreatic β-cells, improve insulin sensitivity, and manage blood sugar levels. Furthermore, their impact on immunomodulation and microbial ecology opens a new path for diabetic treatment. However, it is critical to recognize the limits of the present body of research regarding the effects of LB on diabetes. While available evidence suggests that LB has therapeutic potential, much of it comes from preclinical investigations, namely in vitro and animal experiments. While these studies help to reveal potential mechanisms and consequences, they mostly use cell cultures and animal models. Due to the scarcity of existing studies, more in vivo and in vitro studies are required. Extensive human clinical investigations are also necessary to close the gap between preclinical evidence and practical use. Thoroughly and carefully planned trials are essential to determining LB safety, effectiveness, and suitability for treating diabetes in human patients. These studies should focus on determining the mechanisms of action of LB's bioactive components and their impact on insulin sensitivity, inflammatory indicators, glycemic management, and other vital parameters in diabetics.

6.2. Conclusion

In conclusion, while evidence suggests that LB's bioactive compounds can potentially reduce diabetes and its accompanying symptoms, a comprehensive understanding requires future research, most notably human clinical trials. Incorporating LB into a nutritionally balanced diet can be a beneficial adjunctive treatment for diabetes control. Nevertheless, a comprehensive evaluation of its precise impacts, mechanisms of action, and practical implementation necessitates extensive preclinical and, notably, clinical investigations. By addressing these areas of limited understanding, LB can become essential in comprehensive diabetes management, offering inclusive benefits, and regulating glucose levels.

6.3. Future perspectives

Future research should extensively investigate the prolonged consequences, desirable amounts, and possible interaction of LB with current diabetic drugs, in addition to the contiguous therapeutic aspects. An extensive investigation of the concerted benefits of LB in a comprehensive diabetes management approach encompassing dietary and lifestyle modifications could yield valuable insights into its contribution to enhancing overall health outcomes in individuals with diabetes.

CRediT authorship contribution statement

Zeshan Ali: Writing – review & editing, Writing – original draft, Validation, Supervision, Data curation, Conceptualization. Aqsa Ayub: Writing – original draft, Data curation, Formal analysis. Ya wen Lin: Writing – review & editing, Validation. Sonam Anis: Writing – original draft, Formal analysis, Data curation. Ishrat Khan: Writing – original draft, Validation. Shoaib Younas: Writing – review & editing, Writing – original draft. Rana Adnan Tahir: Writing – review & editing, Validation. Shulin Wang: Supervision, Writing – review & editing. Jianrong Li: Supervision, Funding acquisition, Writing – review & editing.

Declaration of competing interest

The authors declare that there are no conflicts of interest.