Potential of Medicinal Plants to Ameliorate Retinopathy Events in Diabetes: A Systematic Review

Diabetes, or Type 2 Diabetes Mellitus (T2DM), is currently considered an epidemic that is expected to affect up to 4 million people globally by 2025.¹ Diabetic Retinopathy (DR) is one of the most common diabetic complications, alongside nephropathy and neurodegeneration and it raises significant clinical concern as it can eventually lead to visual loss without proper management. DR is a clinical manifestation of diabetic microvascular complications, involving alterations to retinal cells that result in the formation of microvascular lesions, inter-retinal edema, exudation, haemorrhage and even new growth of intraocular blood vessels.²

An advanced stage of DR is characterized by retinal neovascularization and vitreous haemorrhage.³ If untreated, it may progress to tractional retinal detachment, where the retina separates from the underlying retinal pigment epithelium due to scar tissue contraction from damaged retinal blood vessels. The most common conventional treatment for DR is laser photocoagulation, which stabilizes visual acuity but can cause retinal damage and scarring.^2,4 Other treatments include intravitreal pharmacological agents and vitreous surgery. The former, such as anti-VEGF agents, are preferred as they can be applied to both mild and severe stages of DR. Surgical intervention would be the last resort for DR patients if they fail to respond to both laser treatment and pharmacotherapy (Mansour et al., 2020). Although the use of pharmacological agents has become the mainstay therapy for DR, there are studies that demonstrate the insignificant response and improvement in DR patients yielded from anti-VEGE treatment.^4,5 Drugs like thiazolidinediones (an insulin sensitizer) have even been withdrawn or restricted for use due to their severe side effects.⁶ The combination treatment comprising pharmacotherapy and laser therapy is also suggested but there is still lack of clinical evidence to prove its efficacy while concurrently not increasing burden of patients.⁷ All these certainly prompt the need to discover new alternative or complementary therapeutic intervention to ameliorate DR in diabetic patients.

When there is no single strategy established for highly effective Diabetic Retinopathy (DR) management owing to high cost and adverse effects of pharmacotherapy and laser therapy on retinal tissue, researchers have resorted to seek medicinal plants for their potent therapeutic and preventive functions against DR to delay as much of its progression.^8–10 This is because natural products are inexpensive and have minimal toxicity that have been widely used in different parts of the world to treat various diseases since centuries ago. Many have shifted their focus from conventional drug discovery to identification of natural products to ameliorate the diabetes-induced pathological changes in retina.^11,12 There are also different plant-derived bioactive compounds identified to demonstrate potentials in treating or preventing DR that are desired to reduce the incidence of this complication in diabetic patients.^11–15

There are many primary research studies investigating the therapeutic effects of medicinal plants or natural products, of in vivo and/ or in vitro study design on diabetes and its complications in human, animals and/ or cell lines. However, there is very limited centralized discussion to integrate and elucidate the types and functional status of medicinal plants or natural products in amelioration of Diabetic Retinopathy (DR) in different study models together with their underlying therapeutic mechanisms.

Therefore, this systematic review aimed to summarize the phytochemical properties of medicinal plants and to collect evidence on the potential therapeutic effects of medicinal plants or natural products on DR in preclinical studies on animals as well as clinical trials. In other words, this systematic review aims to establish a constructive database on diverse therapeutic properties of various natural products and medicinal plants in amelioration of DR in different experimental models based on various outcome measures, as a direction for future research.

This systematic review was carried out and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.¹⁶ The PRISMA checklist was utilized to streamline the reporting process and improve the quality of the protocol. The study explicitly identifies itself as a systematic review in the title. An abstract is included, summarizing the primary objectives, methods and results of the review. The review’s objective is clearly and succinctly stated. Inclusion and exclusion criteria, as well as methods for grouping studies in the synthesis, are specified. Documentation includes all consulted databases, registers, websites, organizations and reference lists. Comprehensive search strategies, including any filters and limits, are presented for all sources. Methods for determining study inclusion, including the number of reviewers and records screened, are explained. All sought outcomes are listed and defined. The process for assessing risk of bias in included studies, including tools used, number of reviewers and independence, is detailed.

A systematic search was conducted using four database search engines, namely PubMed, Science Direct, Scopus and Google Scholar. Manual search was performed using references of included articles to obtain additional studies and information related to the search outcomes.

The literature search was conducted through catalogued descriptors in MeSH (Medical Subject Headings) in English. The literature searches for each step starting from identification of studies, screening, eligibility and inclusion of studies was presented in a flowchart as shown in Figure 1, along with the inclusion and exclusion criteria used for study selection.

The full-text article for each study was independently screened, studied and analysed to determine the eligibility of studies to be selected.

Studies conducted over the past ten years (January 2011 to June 2020) were considered. We included studies that utilized rats as study subjects and conducted at least three of the following assays: retinal vascular permeability, Western blot analysis for VEGF, Real-Time Polymerase Chain Reaction (RT-PCR) for VEGF and body weight measurement. We excluded duplicate studies, case reports, reviews, editorials, surveys and those not involving medicinal plants from this systematic review. Additionally, phytochemical studies, in vitro studies, human studies, studies unrelated to Diabetic Retinopathy (DR) and studies not employing common assays for investigating DR amelioration were also excluded.

Each of the searches was independently conducted by three reviewers. Disagreements were resolved by a designated coordinator. Following removal of duplicates, the titles and abstracts were independently analysed by the three reviewers to exclude studies not meeting the inclusion criteria. Data from the selected studies were extracted and tabulated using a standardized format. Any discrepancies or disagreements were adjudicated by a fourth reviewer to determine adherence to the inclusion and exclusion criteria. The study subjects used, special treatment given, plant extraction methods and methods used for each assay with the corresponding findings were collected from the 10 selected studies.

Figure 1:
Flowchart of systematic literature search (VEGF*: vascular endothelial growth factor).

Animal intervention studies differ from Randomized Controlled Trials (RCTs) in several aspects. Therefore, the Systematic Review Center for Laboratory Animal Experimentation (SYRCLE)’s Risk of Bias (RoB) tool, was utilized for assessing the risk of bias in the selected studies. This approach ensured consistency and minimized discrepancies in evaluating the methodological aspects of all 10 chosen studies.^16,17 The quality of the article was assessed based on the following criteria: (1) Was the allocation sequence adequately generated and applied? (2) Were the groups similar at baseline or were they adjusted for confounders in the analysis? (3) Were the animals randomly housed during the experiment? (4) Was incomplete outcome data adequately addressed? (5) Are reports of the study free of selective outcome reporting? Each question was independently evaluated by three reviewers and categorized as ‘high,’ ‘low,’ or ‘unclear risk of bias.’ Discrepancies among the reviewers were identified and resolved through consensus.

All the relevant information included for all 10 selected studies were summarized accordingly in Supplementary Table 1.

Among the 7 studies evaluating the rat retinal vascular permeability by Evans Blue (EB) dye injection, all showed amelioration in EB leakage in retinas of treated diabetic rats, with different levels of significance at p<0.05, p<0.01 or p<0.001 in treatment groups as compared to diabetic control model and normal rats. Four out of the 5 studies comprising positive control group (treated with calcium dobesilate, CD or fenofibric acid, FA) revealed greater amelioration of EB leakage in retinas of diabetic rats by plant treatment in comparison to synthetic drug treatment.^18–21

Based on the study outcome by Liu et al. (2016), zerumbone (isolated from Zingiber zerumbet Rhizomes, ZZR) of higher dose (40 mg/kg/day) was considered to exhibit high potential in amelioration of blood retinal barrier disruption by demonstrating 58.1% (p<0.01) of reduction in EB leakage in retinas of STZ (streptozotocin)-diabetic rats.²⁰ As reported by Hong et al. (2016) and Tzeng et al. (2015), EEZZR (Ethanol Extract of Zingiber zerumbet Rhizomes) or ZZRext (Zingiber zerumbet rhizome ethanol extracts) also demonstrated high potential in amelioration of retinal vascular leakage induced by diabetes.^19–21 RRP (aqueous extract of Radix Astragali, Radix angelica sinensis and Panax notoginseng), as reported by Gao et al. (2013), also exhibited almost similar effect of about 53.2% reduction in EB leakage. The same effect was also found to be very significant in the study of Zhang et al. (2020) that TLFA-H (total lignans from Fructus arctii at high dose of 1.38 g/kg/day) treatment reduced the 3-fold increase in EB leakage of 18-19 ng/mg (p<0.001) seen in the diabetic control rats by approximately 52.6-55.6% to 8-9 ng/mg (p<0.001) in treatment group. The remaining two plant treatments-LT (Lycopus lucidus turcz) extract and PSEE (Plantaginis semen ethanol extract) in the studies by Liu et al. (2019) and Tzeng et al. (2016) demonstrated moderate and low potential, respectively but with dose-dependent manner in amelioration of EB leakage in diabetic rats.^22,23

Vascular Endothelial Growth Factor (VEGF) is known to be an important angiogenic factor associated with Diabetic Retinopathy (DR) pathogenesis and thus, can be used to study the underlying mechanism behind vascular leakage into retinas. Among the 6 studies involving Western blot for analysis of VEGF protein expression, all demonstrated significant reduction in VEGF protein level in diabetic rats’ retinas following treatment as compared to diabetic model. Three of the studies by Gao et al. (2013), Yang et al. (2020) and Yao et al. (2018) validated the high potential of the studied medicinal plants in ameliorating DR by means of reducing the tendency of VEGF in mediating increased retinal vascular permeability.^18,24,25

The medicinal plants used included RRP (aqueous extract of Radix Astragali, Radix angelica sinensis and Panax notoginseng) treatment for 12 weeks, Essential Oil extract from Fructus Alpiniae zerumbet (EOFAZ) treatment for 8 weeks and Lycium barbarum Polysaccharides (LBP) treatment for 20 weeks respectively on diabetic rats and cell samples. RRP particularly restored VEGF relative density in diabetic rats’ retinas from 2.0-2.5 units (p<0.05) in diabetic control to 1.0-1.5 units (p<0.05), compared to ~1 unit in normal rats. LBP, particularly of 400 mg/ kg, downregulated VEGF protein level in diabetic rats’ retinas. EOFAZ and Apocynin (16 mg/kg/d) were also found to give an obvious downregulation of VEGF protein level in diabetic rats (p<0.05 versus diabetic model).

The medicinal plant with moderate potential in alleviation of retinal VEGF protein expression at a dose-dependent manner was PSEE (Plantaginis semen Ethanol Extract). Another plant treatment possessing potential lower than other plant treatments in downregulating VEGF protein expression was 300 mg/kg EEZZR (Ethanol Extract of Zingiber zerumbet Rhizomes).

To further confirm Vascular Endothelial Growth Factor (VEGF) expression at molecular level, Real Time-Polymerase Chain Reaction (RT-PCR) is useful for analysis of its mRNA level which firstly involves reverse transcription of retinal total RNA into cDNA followed by the amplification process. All 10 selected studies performed this assay for quantification of VEGF mRNA level. Different medicinal plants demonstrated low, moderate to high potential in amelioration of VEGF mRNA expression depending on the level of reduction of VEGF mRNA level in diabetic treatment groups. Three of the studies using 3-level dosages of plant treatments yielded a dose-dependent effect in VEGF mRNA reduction.^22,23,26 Tzeng et al. (2015) established the amelioration effect of the plant treatment like that of the positive control group using Calcium Dobesilate (CD) treatment,²¹ while Liu et al. (2016) demonstrated a plant treatment with greater amelioration effect in VEGF expression as compared to that of Fenofibric Acid (FA) treatment.²⁰

According to Tzeng, et al. (2016), PSEE gave a moderate effect in amelioration of VEGF gene expression. EEZZR,²³ as examined by Hong et al. (2016), demonstrated 30.8% (p<0.05) of reduction in VEGF mRNA level in diabetic rat retinas as compared to that in diabetic model,¹⁶ which was considered to exhibit lower potential in improving retinal vascular leakage when compared to zerumbone and PSEE. The remaining five studies demonstrated rather low potential in alleviation of increased VEGF expression induced by diabetes.^{18,20,21,24,26}

When the development of diabetes is characterized by decrease in body weight, an increase in body weight following treatment of medicinal plants indicates the amelioration of diabetic condition. Of the 10 selected studies within years 2011-2020, 60% (n=6) of them demonstrated positive result in alleviation of body weight loss induced by diabetes. Three of the studies by Hong et al. (2016), Tzeng et al. (2015) and Tzeng et al. (2016) demonstrated insignificant body weight reduction in diabetic rats while the other three studies by Liu et al. (2016), Liu et al. (2019) and Wang et al. (2019) demonstrated obvious increment in body weight following treatment. The remaining 40% (n=4) of the selected studies by Gao et al. (2013), Yang et al. (2020), Yao et al. (2018) and Zhang et al. (2020) did not demonstrate significant difference in body weights between control and treatment groups. This indicated that treatments involved in these studies had no effect on alleviation of body weight reduction caused by hyperglycaemia.

Most studies investigating the treatment for Diabetic Retinopathy (DR),^27–32 as well as the 10 selected animal studies involved the assays to assess retinal vascular permeability together with protein and gene expressions of Vascular Endothelial Growth Factor (VEGF) in the diabetic study subjects besides monitoring other parameters such as body weight.

Inflammatory response because of oxidative stress and protein glycation implicated by hyperglycaemia can be generated through signaling of various pro-inflammatory cytokines and lead to leukostasis. These events can include several biochemical pathways.^1,33,34 Occlusion of the retinal capillaries followed by retinal ischaemia might then occur which stimulate the release of several growth factors and cytokines. This is where VEGF, one of the important angiogenic factors, plays a key role in mediating the increased retinal vascular permeability and angiogenesis.^35–37 The resulting disruption of Blood-Retinal Barrier (BRB) which later increases the permeability of serum constituents into the neural tissues signifies the hallmark of DR.³⁸

In the selected studies, the breakdown of BRB or retinal vascular permeability was quantified as the concentration of Evans Blue (EB) dye in rat dry retinas. The potential of plant treatments used in the studies was based on the high, moderate and low extent of EB dye reduction in treated diabetic rats as compared to normal rats and diabetic controls. According to all 7 studies performing this assay on the Streptozotocin (STZ)-induced diabetic rats, all used medicinal plants demonstrated potential in attenuation of BRB disruption. Of the five studies compared against synthetic drug, four of them demonstrated high potential of medicinal plants in attenuation of DR by causing greater amelioration of retinal vascular permeability than that of synthetic drugs (calcium dobesilate, CD and fenofibric acid, FA). These plants included: RRP (aqueous extract of Radix Astragali, Radix Angelica sinensis and Panax notoginseng),¹⁸ EEZZR (ethanolic extract of Zingiber zerumbet rhizomes) or ZZRext (Zingiber zerumbet Rhizome ethanol extracts)^19,21 and zerumbone studied.²⁰ Besides that, as reported by Zhang et al. (2020), TLFA (total lignans of Fructus arctii) also demonstrated highly significant reduction of vascular permeability at its highest dose of 1.38 g/kg/day.²⁶

The two selected studies by Hong et al. (2016) and Tzeng et al. (2015) examining the same type of plant-ethanol extracts of Zingiber zerumbet rhizomes, together with the study by Liu et al. (2016) on zerumbone (an important phytochemical of Zingiber zerumbet rhizomes, shared the similar outcome that they achieved greater attenuation of DR than drugs (CD and FA). In fact, Zingiber zerumbet rhizomes rich in medicinal properties have long been extensively studied for its anti-cancer, anti-microbial, anti-inflammatory and antioxidant properties but not for attenuation of diabetic complications (Vasant, et al., 2017). Many studies with positive outcomes as reviewed by Chen et al. (2013) and Haque et al. (2019) have postulated its protection against diabetic microvascular complications.^39,40

RRP is a modified Dang Gui Bu Xue Tang (DBT) comprising Astragali Radix (AR) or “huang-qi” and Angelicae sinensis Radix (ASR) or “dang-gui” combined with bitter ginger-Panax notoginseng. It was newly found by Gao et al. (2013) to significantly reduce retinal vascular leakage by 53.2% through inhibiting the expression of various pro-inflammatory factors and chemokines. Its anti-diabetic effect against the complications has also been studied since a decade ago that it not only exerts anti-inflammatory effect in diabetic atherosclerosis of rat model as reported by Zhang et al. (2006) but also antioxidant property in amelioration of diabetic nephropathy according to Tzeng et al. (2013) and Zhang et al. (2019).^32,41 P. notoginseng with its different ginsenoside (Rg1, Rb1, Rd, Re14) and notoginsenoside (R1) constituents also exhibited anti-inflammatory and antioxidant properties.^15,42 This could most likely explain the reason RRP exhibited high potential in attenuation of retinal vascular leakage, based on the inhibitory effect of each plant included in RRP on the inflammatory processes and oxidative stress induced by diabetes. For TLFA, in the study by Zhang et al. (2020), its highest dose at 1.38g/kg/day reduced the 3-fold increase in EB. The Lycopus lucidus turcz (LT) aqueous extract and Plantaginis semen ethanol extract (PSEE) used in the selected studies were found to exhibit significant but lower potential than other plants aforementioned in amelioration of retinal vascular leakage. LT extract was first studied for DR attenuation by Liu, et al. (2019) and was found to decrease BRB permeability in diabetic rats through amelioration of inflammation and angiogenesis via p38-MAPK/NF-кB signalling pathway. ²² This finding matches with a previous study by Lee et al. (2008) which demonstrated the inhibitory effect of the aqueous extract of LT leaves on vascular inflammatory process induced by high glucose in Human Umbilical Vein Endothelial Cells (HUVEC).⁴³ Its protective effects on rat retinas demonstrated in the selected study²³ can be supported by a recent study conducted by Lei, et al. (2018).⁴⁴

Among the studies, there were six plants being assessed for both VEGF protein and mRNA expressions. All the studied plants demonstrated potential in suppression on the overall expression of VEGF in diabetic rat retinas. In one of the selected studies, Gao et al. (2013) reported that RRP demonstrated significant downregulation of increased VEGF expression levels with its protein levels being restored close to normal in treated diabetic rats.¹⁸ A recent study by Xie et al. (2020) also found that Ginsenoside Re (Re), an active ingredient of P. notoginseng, demonstrated inhibition on VEGF expression responsible for hyperglycaemia-induced retinal angiogenesis via “the Phosphoinositide 3-Kinase (PI3K)/AKT mediated Hypoxia-Inducible Factor-1-alpha (HIF-1α)/VEGF signal pathway” which in turn downregulated oxidative stress precipitating DR.⁴⁵

In the selected study by Yao et al. (2018), Lycium barbarum also reduced the overall increased VEGF expression with its protein expression close to its normal value.²⁵ For this, Wang et al. (2017) have established similar outcome that it was effective in decreasing the oxidative damage of retinal nerve cells in diabetic rats via decreasing VEGF protein and mRNA levels.⁴⁶ A recent study by Wang et al. (2019) even demonstrated the downregulation of VEGF-induced retinal vascular hyperpermeability and angiogenesis by LBP.⁴⁷ As for the Essential Oil from Fructus Alpiniae zerumbet (EOFAZ) in the selected study by Yang et al. (2020), was found to restore VEGF protein expression back to normal level and significant reduction of VEGF gene expression in diabetic treatment group.⁴⁷

According to the selected study by Wang et al. (2019), apocynin (“a natural organic compound isolated from Picrorhiza kurroa”) was found to give an obvious downregulation of both VEGF protein and gene expressions in diabetic rats. The ability of apocynin in preserving BRB and normalizing VEGF expression has already been demonstrated earlier.^48,49

According to Tzeng et al. (2016),²³ PSEE (P. semen ethanol extract) of highest dose (300 mg/kg) demonstrated significant lowering effect on VEGF protein and gene expressions. This was also reported by Lei, et al. (2018).⁴⁴

As for LT (Lycopus lucidus Turcz) extract and TLFA (total lignans of Fructus alpiniae) among the studied plants, Liu, et al. (2019) and Zhang, et al. (2020) also successfully demonstrated the lowering effect of increased VEGF expression induced by diabetes in HRECs (human retinal endothelial cells) and diabetic rat retinas, respectively.^22,26 It was the first time both medicinal plants were evaluated for potential in DR amelioration. Therefore, more scientific studies are needed for elucidation of the mechanism in association with the lowering effect of increased VEGF expression in diabetic condition.

Lastly, unexplained weight loss is considered a classic symptom of diabetes with no planned diet or diuretic treatment.^50,51 Based on the results for body weight reported by the authors among the selected studies, zerumbone, LT extract and apocynin were found to give obvious body weight gain in treated diabetic rats. However, when compared to previous existing findings, apocynin did not cause obvious body weight alterations in the animal subjects as observed in other studies.^52,53 Thus, more evidence is needed to support the outcomes of zerumbone, LT extract and apocynin in amelioration of body weight loss induced by diabetes.

As for the two studies on ZZR ethanol extract by Hong et al. (2016) and Tzeng et al. (2015) and one study on P. semen Ethanol Extract (PSEE) by Tzeng et al. (2016), all demonstrated insignificant body weight reduction following treatment in diabetic rats.^19,21,23 This indicates that these plants prevented further body weight loss caused by hyperglycaemia. The findings on ZZR ethanol extract were equivalent to a previous study.⁵⁴ For P. semen, not much literature focuses on the evaluation of its effect in amelioration of body weight.^55,56

As for the remaining 4 plant treatments namely LBP, TLFA, EOFAZ and aqueous extract of RRP, all did not demonstrate significant difference in body weight between diabetic control and treatment group. This finding somehow contradicted with the previous findings regarding each of them. Lycium barbarum L. root bark extract significantly increased body weight of diabetic mice 4 weeks after treatment.⁵⁷ Lycium barbarum Polysaccharide (LBP) of 500 mg/kg was also reported by Du et al. (2016) to specifically cause 40.5% body weight increment in diet-STZ- induced diabetic rats.⁵⁸ But LBP was found to be unsuccessful in increasing the lowered body weight induced by diabetes in the latest study by Lei et al. (2020). For TLFA, previous studies mostly demonstrated its two main anti-diabetic effects: hypoglycaemic activity and inhibition on weight gain.^59,60 This opposed to the negative outcome in body weight amelioration demonstrated in the latest study included in this review.²⁶

To ensure the reliability of the diabetic animal models used for study, the type and duration of treatment for diabetic induction in the selected studies were screened to establish the stability of the diabetic models. All the 10 selected animal studies included male rats of different stocks as study subjects, namely Wistar, Sprague-Dawley (SD) and Goto-Kakizaki rats. Streptozotocin (STZ) was used for diabetic induction in rats in 8 of the selected studies while the remaining two studies by Yang et al. (2020) and Zhang et al. (2020) on EOFAZ and TLFA respectively introduced high-fat-sugar diet (HFSD) with low-dose STZ of 25 mg/kg and 30 mg/kg for diabetic induction.^24,26 The use of HFSD followed by multiple low-dose STZ injections in rats has been found to best mimic the natural development of T2DM in human, with 25 mg/kg to be the optimal dose of STZ as higher dose of 35 mg/kg and above is more likely to resemble T1DM.⁶⁰ Since the selected studies did not use STZ for long-term studies and the remaining two studies adopted better way for diabetic induction, the diabetic models established in all selected studies are considered reliable and thus, the results generated were deemed valid.

In conclusion, all medicinal plants included in the selected studies demonstrated potential in ameliorating diabetic retinopathy by downregulating retinal vascular permeability and reducing the increased VEGF expressions induced by diabetes. Among the studied medicinal plants, zerumbone showed strong potential for its anti-retinopathy effect, significantly improving retinal vascular dysfunction, preventing body weight loss and downregulating increased VEGF expressions induced by diabetes. Further efforts are needed to elucidate the mechanisms behind each therapeutic property of these medicinal plants against DR.