Introduction

Almost 6% of the global population is affected by diabetes mellitus. This is a multifaceted and chronic metabolic disease that is also becoming prevalent in low to middle-income countries (Forid et al. 2021; Deore et al. 2022). It is reported that 422 million of the global population are diabetic and 1.5 million patients died due to diabetes (Nazir et al. 2018). However, according to the International Diabetes Federation (IDF) in 2021, 537 million people diagnosed with diabetics and projected to reach 643 and 784 million by 2030 and 2045, respectively. The IDF also reported that diabeties was responsible for 6.7 million deaths in 2021 (International Diabetes Federation 2021).

One of the main ways that diabetes causes damage to tissues is through the reactive oxygen species (ROS) generated by hyperglycemia (Dewanjee et al. 2009). Moreover, chronic hyperglycemia can lead to damage to the kidneys, eyes, arteries and nerves due to the glycation of proteins (Foretz et al. 2019). To defend and repair against such damage, researchers have been investigating many natural products that contain antioxidants, which are substances that can scavenge ROS and reactive nitrogen species (RNS), thus reducing the risk of diabetes complications and enhancing the immune defense (Deore et al. 2011).

At present, several drugs are available in market, such as hormone replacement (insulin) and glucose-lowering agents such as thiazolidinediones, biguanides, sulfonylureas and alpha-glucosidase inhibitors, but most of them have side effects like vomiting, anorexia, skin rashes, heartburn and gastrointestinal discomforts (Mawa et al. 2019; Forid et al. 2021). Thus, many researchers are investigating the use of herbal remedies in treating a wide varieties of diseases, including diabetes, that have less or no side effects and toxicity while still being efficient and cost-effective.

Actinodaphne angustifolia (A. angustifolia) Nees is an Asian plant (Family- Lauraceae) (Uddin et al. 2020). The Actinodaphne is an important genus both pharmaceutically and medicinally. The trees are usually 3 to 25 m in height. The leaves are clustered or nearly verticillate, rarely alternate or opposite, unlobed, pinninerved and rarely triplinerved. They have small, greenish star-shaped flowers.  It is widely distributed in the forests of Bangladesh, India, Malaysia and Indonesia (Purkayastha 1985). Traditionally, a decoction of A. angustifolia leaf is used in kidney trouble due to stone (Lokendrajit et al. 2011). The leaf is also used as a folk medicine to treat diabetes and urinary disorders in the Khagrachari district of Chittagong (Uddin et al. 2020). It is very little available information on the pharmacological activities which only include antidiarrheal, antioxidant and thrombolytic activities (Uddin et al. 2020). Several phytochemical studies of the leaves showed the presence of hydrocarbons, friedelin, vitexin, quercetin-3-O-rhamnoside and β-sitosterol (Rastogi & Mehrotra 1992; Asolkar et al. 1992). Despite of the traditional antidiabetic application of A. aungustifolia leaf, there has been no study conducted to verify its folklore use. Therefore, the current study was carried out to evaluate the antidiabetic activity of A. angustifolia in rat model.

Materials and methods

Chemicals and Reagents 

All chemicals and reagents were analytical grade except stated otherwise. Ethanol and alloxan were from Sigma-Aldrich Chemicals, CA, USA. The standard drug glibenclamide was purchased from Chadwell Heath Essex, England.

Plant Collection and Identification 

The fresh leaves of A. angustifolia were obtained from the Chattogram district in March 2018. An expert taxonomist from the Bangladesh National Herbarium Dhaka, Bangladesh was consulted to identify the plant. For future reference, a voucher specimen (accession No. 46052) was submitted to the Bangladesh National Herbarium, Mirpur and Dhaka.

Preparation of Crude Extract 

Distilled water was used to wash the fresh A. angustifolia leaves. Then, shade-drying was used to dry the leaves for 10 days at room temperature (25 + 1°C) to avoid damaging by heat-sensitive phytochemicals. After drying, a mechanical grinder (Miyako, Model No: DL-718 Jiaxing China) was used to grind the leaves into 500 g of powder which was then stored in an airtight container. A simple maceration technique was used for extraction in 2.5 L of ethanol (96%) at room temperature (25 + 1°C) using 400 g of the powder. The extraction was continued for 7 days with occasional stirring, filtered and concentrated under reduced pressure in a rotary evaporator (Barloworld, Berkshire, UK) at 50°C. The resultant concentrated extract was then air-dried to completely remove any remaining solvents and stored at 4°C for test.

Experimental Animals and Their Husbandary

A total of 24 male and female Long-Evans rats (5-6 weeks old; average body weight 92-9 g) were purchased from the International Centre for Diarrheal Disease Research, Bangladesh (ICDDR, B), located in Mohakhali, Dhaka, Bangladesh. A typical rat pellet diet obtained from the ICDDR,B was given along with access to fresh drinking water during the experiment. The rats were kept in plastic cages with bedding made of soft wood shavings in an adequately ventilated chamber at a constant temperature of 25°C, a relative humidity of 55-65% and a 12 hours cycle of day and night. Animals were handled in accordance with ethical guideline of Southeast University animal ethics committee (Approval. No.: SEU/Pharm/CECR/102/2019).

Acute Toxicity of A. angustifolia 

A modified protocol has been adopted based on OECD guideline to evaluate the preliminary toxicity profile of the A. angustifolia  (OECD 2000). Long-Evans rats were distributed into two groups (two rats in each group) and fasted overnight (water only). Group-I was given a single oral dose of 500 mg/kg body weight of A. angustifolia extract, and Group-II received a single oral dose of A. angustifolia (1000 mg/kg body weight). For a total of 10 days, each animal was checked following oral administration of A. angustifolia, at the first 30 minutes and periodically throughout the first 24 hours, with extra care given during the first 4 hours and every day after that. The rats were closely monitored and individual records of each rat were kept for obvious behavioral, neurological, and autonomic alterations, and each animal’s unique records were kept. Additional records on tremor, convulsion, salivation, diarrhea, lethargy, sleep, coma, and death were also noted if any. 

Diabetes Induction and Design of the Experiment

For the diabetes induction, 16 Long-Evans rats of both sexes with the average body weights of 92 + 9 g were used. Alloxan monohydrate (150 mg/kg) was administered intraperitoneally (IP) once to overnight-fasted rats. Rats with consistent fasting serum glucose levels of >7.5 mmol/L for 3 days after alloxan injection were considered as diabetic as measured by a portable glucometer (Accu-Chek, Japan). After 72 hours of alloxan injection, A. angustifolia treatment was begun. Feeding cannulas were used to deliver the plant extract, standard drug glibenclamide and saline. Fasting blood glucose level was estimated on the 3rd, 5th and 7th day of the study. The following groups were divided for the animals: (i) Normal control (I): Only saline water was given; (ii)Diabetic control (II): Diabetic rats with no treatment (alloxan; 150 mg/kg; IP); (iii) Positive control (III): Diabetic rats treated with standard drug (alloxan;150 mg/kg; IP) + glibenclamide (5 mg/kg; PO); (iv) Treatment group (IV): Alloxan induced (150 mg/kg; IP) + A. angustifolia (250 mg/kg; PO); and (v) Treatment group (V): Alloxan induced (150 mg/kg; IP) + A. angustifolia (500 mg/kg; PO).

Biochemical Analysis

After receiving treatment for 7 days, the animals’ fasting serum glucose levels were assessed. Diethyl ether was used to anesthetise the rats before they were decapitated. In accordance with the heart puncture procedure of Kärber et al. (1931), blood was drawn into a dry test tube by cardiac puncture with the help of a disposable syringe and then centrifuged at 112 g for 15 minutes. The plasma was kept at 4°C until further biochemical evaluations. Wet reagent diagnostic kits together with a biochemical analyser (BAS 100TS, Spectronics Corporation, LA, U.S.A) were used in accordance with the manufacturer’s instructions to measure the total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) (Hodge et al. 2020; Parthasarathy et al. 2009; Mueller et al. 1977).

Histopathological Studies 

Histopathological studies were carried out following the method described by Forid et al. 2021. The comparative effect of the leaf extract of A. angustifolia and standard glibenclamide on the pancreatic tissue architecture was observed. The pancreas of treated rats was collected from each group when sacrificed and cleaned with saline water and preserved in formalin solution (10%). After washing, the pancreas was washed and dehydrated with alcohol followed by cleaning with xylene and paraffin blocks. The blocks were subsequently sliced into serial sections (4-5 µm thickness) using a semi-automated microtome (Biobase BK-MT390S, Jinan, Shandong, China). After that, the sections were hydrated with lowering concentrations of alcohol and deparaffinised with xylene. The slides were then rinsed with water after being dyed with hematoxylene for 10 minutes. These were inspected, stained with eosin once again, washed with xylene, dehydrated with increasing alcohol concentrations and then mounted. An Optica DP20 system was used to view the histopathological pictures, pancreatic tissue shape and cellular state.

Phytochemical Profiling of Bioactive Extract

UPLC-MS was used to determine the phytoconstituents of the A. angustifolia extract. Waters ACQUITY UPLC IClass/Xevo and a Waters Xevo G2 Q-TOF mass spectrometer were coupled and integrated with positive electrospray ionisation mode for the UPLCMS analysis (Milford, MA, USA). About 100 mg of the extract was dissolved in 1 mL of ethanol to create extract samples. A Zorbax Eclipse plus Acquity UPLC BEH C18 column (1.7 m particle size) 2.1 mm x 50 mm in diameter was used for separation. From m/z 50 to 1000, a complete scan mode was run while maintaining a 120°C source temperature. A gradient elution system was created using solvents A and B, solvent A was water with 0.1% formic acid and solvent B was acetonitrile with 0.1% formic acid. For the first 15 minutes, the elution conditions were 99% solvent A and 1% solvent B, then 65% solvent A and 35% solvent B for 1 minute, then a progressive increase in solvent A to 100% over 2 minutes, and lastly a gradual increase in solvent B to 99% and solvent A 1% over 2 minutes. For nebulising and collision gases, ultra-high-pure nitrogen (N2) and helium (He), respectively, were employed. A 2.0 kV capillary voltage in positive electrospray mode was used. Other inferred instrument settings were a 100 V source offset, 550°C desolvation temperature, 50 L/h cone gas flow with 120°C temperature desolvation gas flow and 800 L/h.

Statistical Analysis 

All experimental data obtained were expressed as mean ± standard error of mean (SEM) where n=4. Data were normalised by One way-ANOVA statistical tool was used and Tukey’s post hoc test was done to analyse the difference, using GraphPad Prism ,version 6 (GraphPad Software, USA). The p-value less than≤0.05 was considered as significant value.

Results and Discussions

Acute Oral Toxicity and Dose Selection

Animals are used in acute oral toxicity studies to determine the safety of products based on plants and other formulations for humans. The oral treatment of A. angustifolia to rats did not affect any changes in their behavioral characteristics in the acute toxicity investigation. Even at the greater dose of 1000 mg/kg body weight of A. angustifolia ethanol leaves extract, no detrimental and abnormal effects of the experimental animals were seen. As very little pharmacological studies had been conducted on this plant species and its potential acute toxicity at higher doses was needed to be carried out along with sub-acute and chronic toxicity profile. 

Effect of A. angustifolia on Blood Glucose Levels

Figure 1 illustrated how the A. angustifolia leaf extract affected the blood sugar level. The administration of alloxan raised blood glucose levels significantly which remained stable over the course of the study in comparison to the normal control group (4.45 + 0.03 mmol/L). When comparing with the diabetic group, the A. angustifolia extract at 250 and 500 mg/kg body weight doses were able to substantially lower the increased blood glucose level (11.8 +±0.5 mmol/L, p<0.001 and (11.92 +±0.62 mmol/L, p<0.001 respectively) over the duration of the treatment after each. Throughout the course of the 7-days intervention, a progressive reduction in blood glucose was seen (Figure 1). In this study, only fasting glucose level was measured. Therefore, acute oral glucose tolerance test and insulin level measurement needed to be done to determine how quickly glucose is cleared from the blood and to diagnose insulin resistance.

Effect of A. angustifolia extract on Lipid Profiles 

Serum lipid profiles were measured as depicted in Figure 2. In comparison to the diabetic control group, both dosages of A. angustifolia extract (250 and 500 mg/kg body weight) reduced LDL levels (5.69 + 0.14 mmol/L, p<0.001) while continued to enhance the HDL levels (9.41 +±0.53 and 7.76 + 0.66 mmol/L, respectively, p<0.001). Reduced levels of total blood cholesterol of 8.58 + 1.07 mmol/L (p<0.001) by 250 mg/kg and 7.76 + 0.66 mmol/L (p<0.001) by 500 mg/kg were also seen. In comparison to the diabetic control group (4.78 + 0.05 mmol/L), the triglyceride level was considerably reduced (p<0.01 only at the dosage of 500 mg/kg body, or 2.9 + 0.32 mmol/L).

Dyslipidemia is the major consequence of diabetes mellitus which is characterised by abnormal HDL, LDL, TG or TC blood level in patients and increases the risk of cardiovascular disease (CVD) and mortality. Current therapies for this condition include the use of statins. However, statins possess adverse effects, including paralysis, cataracts, memory loss, myositis, myalgias, hepatotoxicity, polyneuropathy, myopathy and tendinopathy (Muhammad et al. 2019; Abbaszadeh et al. 2018). These problems could be minimised by using alternative remedies such as herbal medicines to treat dyslipidemia without the potential negative effects of pharmaceutical medicines (Amani et al. 2018). Herbs are rich in many bioactive components such fibers, antioxidants, flavonoids, glycosides, plant sterols, saponins, catechins, stanols and triterpenoids, and can be used as functional foods (Amani et al. 2018; Enkhmaa et al. 2015; Alipour et al. 2018). In hyperlipidemia, there is less glucose transport into the cells. Therefore, lipids are more available in the form of LDL fat and deposited to the arterial wall as fatty plaques. These fatty plaques are transported to the liver by HDL for elimination (Virmani et al. 2006). Therefore, the level of HDL and LDL in blood in an inversely proportional manner which is expected to be the therapeutic application of A. angustifolia extract. 

Effect of A. angustifolia Extract on Pancreatic Tissue Architecture

The hematoxylin and eosin staining method was used to examine the morphology of the pancreatic tissues of the different experimental groups. The histopathology of the pancreatic islets were shown in Figure 3. Figure 3A showed a normal tissue morphology of the pancreas and islet cells in normal rats (group I). However, the size and number of the β islet cells were found to be reduced in the diabetic-induced (150 mg/kg) group II rats, that resulted in shrinkage. On the other hand, the morphology of pancreatic islets was restored and improved in the animals with diabetes in groups IV and V that were treated with A. angustifolia extract (250 mg/kg and 500 mg/kg body weight, respectively). This outcome was also similar to those given glibenclamide (5 mg/kg). The pancreas helps to control metabolism. Changes in the production, sensitivity, and control of insulin during metabolic activities are reflected in the structural changes in the pancreas. The characteristics of pancreatic destruction include islet atrophy, a decline in number of β-cells, and cellular damage (Jaiswal et al. 2017). Alloxan-injected rats might have recovered from the effects of the drug mostly due to the regeneration of β islet cells after they were destroyed by the alloxan. The increased number of β-cells in the pancreas in Figure 3 D, E might be an indication of the recovery effect of A. angustifolia leaf extracts. This possible mechanism could be the survival of a greater number of β-cells upon A. angustifolia treatment to release more insulin.

Identification of Flavonoids and Triterpenoids from A. angustifolia Leaf Extract by UPLC-QTOF/ESI-MS

Due to the potential antidiabetic activity, the bioactive extract was then further subjected to phytochemical profiling by using UPLC-QTOF/ESI-MS that can separate and analyse the compounds. This technique can provide the inclusive mass of different ions and accurate chemical formulae (Yousefi et al. 2013). In our study, the positive mode ((+) ESI-MS) and negative mode ((-) ESI-MS) managed to identify the presence of flavonoids based on the pattern of mass fragments, and ion response that were automatically elucidated and verified. The identified compounds were classified either as good match with an error of + 5 mDa or as poor match with an error of + 10 mDa by the UNIFY software.

A total of 45 flavonoids and 109 triterpenoid compounds were identified from the ethanolic extract of A. angustifolia as presented in Table 1. The biological activity of plant extracts is due to the presence of different chemical constituents in it. The numerous bioactive compounds could be presented as a mixture which plays a synergistic role for the activity. Some of the identified flavonoids and terpenoids in A. angusifolia extract have been reported to show various biological activities and might be responsible for its antidiabetic activity. Flavonoids have a wide range of beneficial effects on metabolic illnesses including diabetes (Middleton et al. 2000). Flavonoids’ can regulate insulin signaling and production, carbohydrate digestion glucose absorption and adipose deposition (Vinayagam & Xu 2015) by targeting pathways, related to cell proliferation, stimulating insulin secretion, decreasing apoptosis, and alleviating hyperglycemia through regulating glucose metabolism in the liver (Graf et al. 2005).

Among the identified flavonoids in this study, 3,4-dihydroxyphenothyl-3-O-β-D-glucopyranoside has been reported to exhibit antioxidant activity (Ukaegbu et al. 2018). Another compound, episappanol, has an anti-inflammatory effect (Mueller et al. 2016). Meanwhile, cyclomorusin is an extended flavonoid, and it is a moderate inhibitor of acetylcholinesterase and a strong inhibitor of platelet-activating factor induced platelet aggregation and has a potential role as an anti- hypoglycemic agent (Abd-EL-Mawla et al. 2011; Fernández-Rojas et al. 2022). Jaceosidin has a role as an anti-inflammatory agent (Kim et al. 2008), an apoptosis inducer (Lv et al. 2008), an anti-allergic, antineoplastic (Nam et al. 2013) and antidiabetic agent (Kang et al. 2007). Apocynin B has been reported for its hepato and neuro protective activities (Petrônio et al. 2013; Simonyi et al. 2012) and neuroprotective activity. Irisflorentin can suppress allergic inflammation (Li & Meng et al. 2019). Bowdichione is a hydroxyisoflavone and it exhibits antineoplastic and anti-inflammatory activities (Umehara et al. 2009). 

The antioxidant flavonoid molecule kaempferol, also known as kaempferol 3 or kaempferide, lowers oxidative stress, possesses antibacterial and geroprotective properties. Rat studies have also shown that kaempferol has a hypoglycemic and hypolipidemic effects (Devi et al. 2015; Imran et al. 2019; Ren et al. 2019; Chandramohan et al. 2014). It can be used to treat a range of acute and chronic inflammatory diseases because of its anti-inflammatory characteristics. Resokaempferol also has anti-inflammatory activity (Yu et al. 2016). Quercetin 3,4’-dimethyl ether is a dimethoxyflavone that is a 3,4’-dimethyl ether derivative of quercetin which is isolated from Combretum quadrangulare and it has roles as an antioxidant and antineoplastic agents (Azeem et al. 2022; Di Petrillo et al. 2022). Meanwhile, quercetin-3-methyl ether shows neuroprotective and antidiabetic activities with promising chemopreventive potential against esophageal carcinogenesis (Dok-Go et al. 2003; Zhao et al. 2018). Another study showed that it can inhibit breast cancer cell growth by inducing G2/M arrest and apoptosis (Li et al. 2013). Quercetin (3,5,7-trihydroxy-2-(3,4-dihydroxyphenyl)-4Hchromen4-one), a common active ingredient in Chinese traditional medicine, is a polyhydroxy flavonoid usually found in  fruits, leaves and flowers of various plants (Boots et al. 2008). It is claimed to have anti-inflammation, anticancer, anti-fibrosis, anti-oxidation effects (Fu et al. 2020; Shi et al. 2008), antidiabetic effects and prevention of neurodegeneration in diabetic retinopathy (Vessal et al. 2003; Ola et al. 2017; Gomes et al. 2015). Due to the biological activities, these flavonoids may prevent cardiovascular and other lifestyle-related diseases (Kobori et al. 2009).

Triterpenes appear to have different pharmacological properties. Previous studies have shown that these substances exhibit  anti-diabetic actions. They have the ability to regulate plasma glucose and insulin levels, inhibit the onset of insulin resistance and block glucose metabolism enzymes. Triterpenes also have potential to be used for the prevention of diabetic complications. Triterpenes have a high antioxidant activity and prevent the production of advanced glycation end products, which have been linked to the pathophysiology of poor wound healing, diabetic neuropathy, embryopathy and nephropathy (Nazaruk & Borzym-Kluczyk 2015).

Loganin is a plant metabolite that has demonstrated pharmacological efficacy as an antioxidant, neuroprotective agent and as treatment for diabetic neuropathy. Loganin also protects against hepatic damage and other diabetes problems induced by oxidative stress and the production of advanced glycation end products (Jiang et al. 2012; Lee et al. 2009; Yamabe et al. 2010). Ginkgolide C, an active constituent of Gingko biloba has been reported to increase both adipose triglyceride and hormone-sensitive lipase enzymes for increased lipolysis with suppression of lipid accumulation in differentiated adipocytes (Liou et al. 2015). Gedunin is a limonoid which is found mostly in the seeds of many Meliaceae genera. Anti-inflammatory, antiallergic, anticancer, antimalarial, insecticidal, antibacterial and neuroprotective properties have been attributed to gedunin. It also has potential for the antidiabetic activity reported by Ponnusamy et al. (2015). Moreover, other species of the Actinidaphne genus have been studied for anti-diabetic activity. Bhaskaran et al. (2019) studied the antidiabetic activity of Actinodaphne hookeri and found a strong hypoglycemic effect with improved lipid profile and restoration of damaged pancreatic β-cells.

Conclusion

Our study suggested that A. angustifolia leaf ethanolic extract may be useful in the management of diabetes and its consequences, as they protected the pancreas by restoring the damaged beta islets and improving the lipid profiles. The presence of a number of bioactive flavonoids and triterpenoids might suggest its potential role for bioactivity. More investigations are required to completely comprehend its mechanism and isolate the bioactive components for further development as an antidiabetic natural product.