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Introduction

Honey and stingless bees (Apidae Meliponini), called ‘lukut’ in Philippines, and ‘damar’ in India (1), generate propolis, a naturally occurring resinous substance, by gathering exudates and materials from plant parts, including flower buds, tree bark and leaf buds, and combining them with beeswax and enzymes (2-4). The active constituents of propolis are determined by the local flora (5). Bees use propolis to build and maintain their hive to seal holes and fissures and smooth the internal walls because of its waxy structure and mechanical characteristics. Propolis has been an essential part of apitherapy. More recently, it has also been used as a food additive or supplement in alternative and traditional medicine (6). Natural products produced by stingless bees, such as propolis, honey, beehives and pollen exert pharmacological effects and used as traditional medicine by many Asian cultures (4). Propolis exerts antibacterial, antioxidant, antiseptic, anti-inflammatory, antifungal, hepatoprotective, and immunomodulatory effects (7). The present systematic review aimed to analyse the possible usefulness of propolis as a preventive and therapeutic method based on in vitro and in vivo research.

Materials and methods

Google Scholar (8), Science Direct (7), and Web of Science (WoS) (6), were searched for studies assessing and reporting the biological activities of Malaysian propolis. The search strategy was ‘propolis’ AND ‘Malaysian’ for Science Direct and WoS. For Google Scholar, the ‘Advance Search’ option was used to discover papers that contained the words ‘propolis, Malaysian,’ ‘Malaysian,’ or ‘Malaysia,’ but not ‘systematic review, meta-analysis, or review.’ Studies on the biological activities of Malaysian propolis conducted in vitro and in vivo were included. PRISMA 2020 flow diagram for new systematic reviews which included searchers of databases and registers was used (7). Only full-text studies published in English between January 2012 and June, 2023 were included. Review papers and research that included meta-analyses were not included in. The present review did not include any studies on non-Malaysian propolis. Two authors completed the data extraction, which included the main author, year, bee species, location, propolis preparation, study type and biological activities.

Results and Discussion

Database search

WOS is considered the global leading platform for scientific citation search and analytical information (9). To ascertain whether Google Scholar can be utilized as a reliable source of scientific information and data for scientific evaluation, a previous study (10), reviewed 91 comparative articles from 2005 to 2016 that compared Google Scholar with various databases, particularly WOS, and revealed that Google Scholar is a powerful database of scholarly literature, having broadened its scope over the years. PubMed is the most commonly searched database for systematic reviews (11). However, Pubmed resulted in a very small number of articles (n=19) in the present preliminary search and most of the articles were identical to with the results of Google Scholar, ScienceDirect and Web of Science. Similar to PubMed Single Citation Matcher, the ScienceDirect advanced search function allows user to search by author, title, volume, issue, and page (12). Therefore, the present review utilized three databases for literature search, namely Google Scholar, ScienceDirect and Web of Science.

In total, 780 records were identified from the first primary database search, of which 777 remained after duplicates were eliminated (Fig. 1). After the 777 titles were filtered, 746 were removed (irrelevant article title); 31 selected for further inspection and the full texts were then obtained. Then, 30 entries were chosen from a full-text review to be included in the review, and one was omitted (non-English language article). Of these, 23 studies were conducted in vitro and seven were conducted in vivo or on animals (Table I).

Figure 1 Flowchart of identification and selection of study articles.

Table I In vitro and in vivo biological activity of Malaysian propolis.

Table I

In vitro and in vivo biological activity of Malaysian propolis.

First author/s, year	Bee species	Location	Propolis extract	Study type	Biological activity	(Refs.)
Chew et al, 2014	N/A	N/A	2.50% ethanol	In vitro	Increases stem cell proliferation	(32)
Jacob et al, 2015	Trigona spp	Gurun, Kedah	80.00% ethanol	In vitro	Enhance the Wound healing activity against normal human fibroblast cell line CRL-7522	(33)
Ibrahim et al, 2015	Heterotrigona itama; Geniotrigona thoracica	Besut, Terengganu	100.00% methanol	In vitro	Possess antibacterial activity	(34)
Akhir et al, 2017	Heterotrigona itama	Parit Botak, Johor	100.00% hexane and 70.00% ethanol	In vitro	Heterotrigona itama propolis from southern Malaysia have antimicrobial and antioxidant activity	(35)
Yusoff et al, 2016	Trigona spp	N/A	100.00% water	In vitro	Weaker antifungal activity	(29)
Rosli et al, 2016	Trigona apicalis	N/A	100.00% Ethanol	In vitro	Possess high antioxidant activity with higher phenolic and flavonoids contents	(18)
Usman et al, 2016	N/A	Kota Bharu, Kelantan	100.00% Water and ethanol	In vitro	Exhibited higher antioxidant activity	(20)
Ahmed et al, 2017	Tetratrigona spp	Kuband Kerian, Kelantan	70.00% ethanol	In vivo	Direct cytotoxicradical- scavenging activity, which provides cardioprotective action against ISO-induced oxidative stress.	(15)
Azemin et al, 2017	Heterotrigona itama	Terengganu	100.00% Ethanol	In vitro	Unprocessed propolis has considerably higher antioxidant activity, regardless of extraction method	(36)
Ong et al, 2017	N/A	Pahang	100.00% Ethanol and ethyl acetate	In vitro	Chitosan-propolis nano formulation might be considered a possible anti-biofilm agent in fighting infections	(37)
Salim et al, 2018	Geniotrigona horacica	Kuala Kangsar, Perak	80% ethanol	In vitro	Active components in propolis contribute to its antioxidant properties	(16)
Lim et al, 2022	Heterotrigona itama	Besut, Dungun, Terengganu; Tanah Merah, Gua Musang, Kelantan	95% ethanol	In vitro	Propolis and metformin combination in reducing histological features of diabetic cardiomyopathy	(38)
Nna et al, 2018	Heterotrigona itama	Kelantan	70% ethanol	In vivo	Reduces hepatic lesion and has a synergistic protective effect	(39)
Usman et al, 2018	Heterotrigona itama	Kelantan	70% ethanol	In vivo	Improvement in preg- nancy outcomes and placental oxidative stress	(31)
Asem et al, 2020	N/A	Kuala Kangsar, Perak	80% ethanol	In vitro	Demonstrated positive antioxidant activity	(40)
Annisava et al, 2019	Heterotrigona itama	Kelantan; Terengganu	100.00% Ethanol	In vitro	Contains the highest total phenolic, flavonoid content and antioxidant activity.	(41)
Nafi et al, 2019	Heterotrigona itama, Geniotrigona thoracica, Lepidotrigona terminate; Tretrigona apicalis.	N/A	95% ethanol	In vitro	Heterotrigona itama possessed the highest antioxidant activity compared to other species	(42)
Badiazaman et al, 2019	Geniotrigona thoracica	Besut; Dungun, Terengganu; Tanah Merah, Kelantan; Gua Musang, Kelantan	100.00% Methanol	In vitro	Possessed the highest total flavonoid content and antioxidant activity	(13)
Yusop et al, 2019	Trigona itama	Beladin, Sarawak	100.00% Hexane, ethyl acetate and methanol	In vitro	Antioxidant and anti- bacterial effects against both gram-positive and gram-negative bacteria	(30)
Nna et al, 2019	Heterotrigona itama	Kelantan	70% ethanol	In vivo	Reduced testicular oxidative stress, inflam- mation, and apoptosis in diabetic rats	(43)
Mohamed et al, 2020	Tetrigona apicalis	Tanjung Malim, Perak	80% ethanol	In vitro	Demonstrated antioxi- dant activity and suppressed the prolifera- tion of MCF7 cells	(28)
Mohd Suib et al, 2021	Geniotrigona thoracica	Kuala Kangsar, Perak	80% ethanol	In vitro	Inhibition of the forma- tion of THP-1 derived macrophage foam cells	(26)
Zainal et al, 2022	Tetrigona apicalis	Kuantan, Pahang	100.00% Water and 70 and 80% ethanol	In vitro	Exhibited strong anti- oxidant activity	(44)
Syed Salleh et al, 2021	Tetrigona apicalis, Tetrigona binghami; Homotrigona fimbriata	Selangor	100.00% Water	In vitro	Demonstrated greater antioxidant potential, with higher phenolic and flavonoid levels	(19)
Maroof et al, 2023	Geniotrigona thoracica	Negeri Sembilan	100.00% Ethanol	In vitro	Increased antioxidant activity and antibacterial efficacy against gram- positive bacteria	(22)

Phytochemicals properties of propolis

Generally, propolis is rich in various bioactive compounds, such as fatty, aliphatic and aromatic acids, flavonoids, terpenoids, sugars, alcohol and esters. The chemical composition of propolis is affected by its geographical location as well its botanical origin as the resin from different plant species may contain various compounds (13). In Malaysia, 78 stingless bee species have been discovered, including Geniotrigona thoracica, Heterotrigona itama, Tetrigona apicalis and Tetragonilla atripes (14). H. itama is the most common stingless bee species in Malaysia (15). H. itama favours Averrhoa carambola and Antigonon leptopus because both flowers produce notable amounts of nectar, and their morphology is compatible with H. itama tongue morphology (16). Hydrocarbons and oxygenated sesquiterpenes derivatives, such as β-caryophyllene, copaene, cyclohexane, 1H-cycloprop[e]azulen-7-ol and β-caryophyllene oxide, are detected in T. apicalis propolis from Malaysia by gas chromatography-mass spectrometry (GC-MS) analysis. T. apicalis propolis also contains triterpenoids, such as α-amyrin and β-amyrin (17). The GC-MS chromatographic analysis of G. thoracica propolis from Malaysia demonstrates presence of phenol, benzoic acid, trimethylsilyl ester, hydroginkgol, resorcinol, Δ-cadinene, nootkatone, β-amyrenol, friedelany-al, cycloeucalenol and myristic, palmitic linoleic and octadecanoic acid (18). A recent study by Syed Salleh et al (19) revealed the prevalence of some classes of compounds in T. apicalis, T. binghami and Homotrigona fimbriata propolis from Malaysia. The identified components included phenolic (Gallic acid, Vanillin, p-coumaric acid, Quercetin, Pinocembrin Artepillin C methyl ester, Naringenin, Catechin, Epicatechin), terpenoids (mangiferolic acid, Cycloartenol, Ambonic acid, Mangiferonic acid, Ambolic acid, amyrin), Prenylated benzophenones (7-epi-nemorosone, Xanthochymol, Guttiferone C, Gambogenone and Aristophenone A), carboxylic acids, sugar alcohols, hydrocarbon, aldehydes and amino acids when analysed using GC-MS. Therefore, Malaysian propolis is considered as terpenoid-type propolis. The chemical structures of bioactive compounds in propolis are shown in Fig. 2.

Figure 2 Representative chemical compounds founds in Malaysian propolis.

Pharmacological activity and mechanism of action of propolis. Cytotoxic activity

Cytotoxicity refers to the capacity of a molecule or compound to result in cellular damage, which may involve damage to specific cell structures or the essential functions that keep cells alive, such as cell division, survival and normal physiology (20). Extracts of H. itama propolis from different locations possess low to moderate cytotoxic effects against HeLa cells with half-maximal inhibitory concentration (IC50) value ranging from 14 to 60 µg/µl (21). Propolis extract induces apoptosis in HeLa cells in a dose-dependent manner. Cytotoxic activities of propolis extract are also species-dependent. Propolis produced by H. itama, G. thoracica, L. terminate and T. apicalis have been extracted and evaluated for their cytotoxicity against three cancer cell lines (22). H. itama propolis extract demonstrates the highest cytotoxic activity against MDA-MB-231, SK-UT-1 and HeLa cells, with IC50 values of 5, 4 and 8 µg/ml, respectively (22). It is proposed that the capacity of terpenoid compounds to impede proliferation, induce apoptosis and inhibit metastasis makes them useful against tumours and inflammation (22). Another study demonstrated that propolis extract from T. apicalis exerts cytotoxic activity against breast cancer cell lines in a dose- and time-dependent manner (17). IC50 values of T. apicalis propolis extract were reduced with longer incubation time in MCF7 cells. However, in MCF 10A cells, longer incubation time increased the IC50 values of the propolis extract (17). MCF7 is an Estrogen Receptor, Progesterone Receptor (PR)-positive, and Human Epidermal Growth Factor Receptor 2 (HER2)-negative breast cancer cell line while MCF 10A is originally non-tumorigenic cells. The effects of propolis on cell proliferation of MCF7 and MCF10A were cell type-dependant, thus the activity is reduced in MCF7 cells and increased in MCF 10A cells. At incubation up to 72 h, T. apicalis propolis extract demonstrated selectivity, with a high selectivity index (SI) of 2.20(17). SI is an important indicator to evaluate the toxicity of a compound or extract against normal cells, and to predict their therapeutic potential on cancer cells (23,24). FITC Annexin V with flow cytometry is one of the most powerful tools for quantitative determination of the percentage of cells that are actively undergoing apoptosis within a population (25). A previous study utilized this method to evaluate apoptosis induction by T. apicalis propolis extract in MCF7 cells (26). At IC50 of 32.70 µg/ml and 72 h incubation with MCF7 cells, the percentage of apoptosis induction by propolis extract in viable, early and late apoptotic and necrotic or dead cells corresponded to 48.39±2.06, 14.02±0.98, 35.25±1.16 and 2.34±0.14%, respectively (26). It was suggested that the antioxidant properties of propolis extract are partly responsible for apoptosis induction in cancer cells (26). Apart from cytotoxicity study in mammalian cells, there was also a study that tested H. itama propolis extracts using the Brine shrimp lethality test; extracts showed a low level of toxicity (15).

Antimicrobial activity. An antimicrobial substance generally eliminates or prevents the growth of bacteria. Antimicrobial substances can be microbiostatic, which prevents microbial development, antibacterial, which fights bacteria, or antifungal, which fights fungi (27). Propolis extract from Trigona spp. exerts antifungal activities against oral Candida albicans, C. tropicalis and C. glabrata with minimal inhibitory concentration (MIC) of 500 mg/ml (28). Limited studies have reported the antifungal activities of propolis extracts (28,29). However, there are numerous of studies that evaluated their antibacterial effects (30,31).

A study utilized ethanol and water as solvents to extract T. thoracica propolis, then evaluated the antimicrobial activity of the extracts against Staphylococcus aureus using broth microdilution method (31). Ethanolic extract of the propolis exhibited the strongest antimicrobial effect due to its higher content of phenolic compounds, such as quercetin (31). At 1 mg/ml, H. itama propolis methanolic extract demonstrated the highest antimicrobial activities against S. aureus and E. coli, both with an inhibition zone of 10 mm, in comparison with hexane extract and ethyl acetate extract (<10 mm) (15).

Antibacterial activity of H. itama propolis and G. thoracica propolis extracts has been evaluated against Gram-positive bacteria, such as S. aureus, Bacillus subtilis, Enterococcus faecalis and Listeria monocytogen, as well as Gram-negative bacteria, such as Acinetobacter baumannii, Salmonella typhi and E. coli (30). It was found that the extract from H. itama propolis demonstrates better inhibition against the strains (S. aureus, B. subtilis, E. faecalis and L. monocytogen, A. baumannii, S. thyphi and E. coli) with an inhibition zone of 6-14 mm compared with G. thoracica propolis extract (6-7 mm) (29). Both propolis extracts exhibited greater inhibitory effect against S. aureus (Gram-positive) than E. coli and S. thyphi (Gram-negative) (30). The aforementioned study suggested that the antibacterial activities of propolis were species-dependent and affected by the polar phenolic compounds of the extracts (30). These results were supported by another study that reported better antibacterial activity shown by propolis extract against Gram-positive bacteria (B. cereus and S. aureus) in comparison with Gram-negative bacteria (E. coli and Salmonella) (45). Extracts of Acacia mangium and Garcinia mangostana propolis demonstrated promising antibacterial effects against S. aureus with an inhibition zone of 20.00±0.1 and 24.00±0.52 mm, respectively, compared with erythromycin (24.80±0.72 mm) (35). However, there are no antibacterial activities shown on Gram-negative bacteria (E. coli and P. aeruginosa) by both propolis extracts (35). It is suggested that the lower susceptibility of Gram-negative bacteria may be due to lipopolysaccharides of the outer membrane that hinder the penetration of propolis antibacterial components into bacterial cells (35).

Antioxidant activity. Oxidative stress is an imbalance between the production and accumulation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), and the capacity to neutralize and eliminate them. Moderate concentrations of ROS and RNS are key for many physiological processes within the human body. Key endogenous antioxidant enzymes are superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px). SOD converts superoxide anion to H2O2, a substrate for CAT and GSH-Px (46). When reacting with GSH, CAT metabolizes H2O2 in water and oxygen while GSH-Px lowers H2O2 and organic hydroperoxide levels (46). Several studies have investigated the antioxidant activity of propolis in in vitro and animal models (32,47,48). Propolis from UniSZA Apiary, Besut (BST-1) has the highest total phenolic and flavonoid content (TPC and TFC, respectively) and antioxidant activity (35). BST-1 extract with the highest phenolic content had higher antioxidant activity than the other localities (35). Another study compared ethanolic extract of propolis samples was produced from three different stingless bee species, T. apicalis, H. itama and G. thoracica, collected from bee farms in Perak, Malaysia (33). Among the species tested, G. thoracica had the highest antioxidant activity, with IC50 values of 206.27 for 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 64.98 mg/ml for 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay, respectively. This was consistent with another study on A. mangium and G. mangostana-derived propolis (35). The is no significant difference in the ABTS+ scavenging effect (A. mangium, 0.05±0.00; G. mangostana, 0.05±0.00 mg/ml) and metal chelating activity (A. mangium, 51.44±4.99; G. mangostana, 52.12±1.61 mg/ml) between the two propolis samples. However, SOD enzyme-like activity is considerably higher in G. mangostana propolis (0.17±0.01 mg/ml) compared with A. mangium (0.26±0.00 mg/ml) (35). The ethanolic extracts from propolis produced by H. itama in Terengganu, Malaysia, have the strongest antioxidant activity with an IC50 of 30 µg/ml and the highest percentage of inhibition with 85.69%, followed by G. thoracica with an IC50 of 40 µg/ml and 82.22% inhibition at 150 µg/ml concentrations. The H. itama scavenging activity is comparable to quercetin and trolox, with IC50 values of 10 and 9 µg/ml, respectively. On the other hand, Lepidotrigona terminate has poor antioxidant activity with an IC50 of 128 µg/ml and an inhibition percentage of 80.47% at a dosage of 500 µg/ml. T. apicalis demonstrates antioxidant activity with IC50 values >500 µg/ml. The differences in antioxidant activity may be attributed to the phenolic, flavonoid or other components of propolis extracts, which have been associated with antioxidant capabilities (22). This finding was consistent with that of H. itama propolis from Besut, which had the best antioxidant activity with the lowest IC50 values (10.000±2.623), followed by Dungun (84.000±2.623) and Gua Musang (151.000±2.623 µg/ml) (21). The aforementioned experiment demonstrated that propolis from Besut had the highest antioxidant activity while propolis from Tanah Merah had the lowest antioxidant activity of DPPH scavenging radicals.

Soft propolis of H. itama (found inside the beehive) contains more phytochemicals, specifically flavonoids, phenols and terpenoids, than hard propolis (which forms part of the wall of the hive) (47). In the DPPH assay, the propolis samples demonstrate a dose-dependent increase in radical scavenging activity, with soft propolis H. itama exhibiting a significant effect (IC50, 79.90±11.75) compared with hard propolis H. itama (180.00±16.67 µg/ml) (47). In addition, at a higher concentration of 1,000 g/ml, soft propolis H. itama demonstrated stronger H2O2 scavenging activity (53.94±1.88) than hard propolis H. itama (43.34±0.51%) (47).

The impact of processing and extraction methods on chemical profiles and antioxidant activity of propolis has been studied. In comparison with processed propolis (maceration, sonication, maceration-sonication), unprocessed propolis has more potent antioxidant activity with the lowest IC50 value. For the processed sample, raw propolis was heated at 1 h at 37˚C (42). Meanwhile, the unprocessed sample was retrieved fresh from the hives (21). Another study reported the antioxidant activity of G. thoracica propolis from different locations in Terengganu, Malaysia. It was demonstrated that propolis from Besut has the lowest IC50 value of 53 µg/ml, whereas propolis from Dungun had the highest IC50 value of 190 µg/ml and propolis from Lundang was inactive. The stronger the radical scavenging activity, the lower the IC50 value (22). Thus, propolis from Besut exerts the strongest antioxidant and radical scavenging properties. These differences could be attributed to changes in the chemical composition of the propolis extracts (49). In addition, the hexane extract of H. itama propolis from Johor, Malaysia, exhibits the highest Ferric Reducing Antioxidant Power value of 6.64 mM Ferrous Equivalent/g). These findings demonstrate that propolis is a potent natural antioxidative agent (45). In another study, ethanolic extract of T. apicalis inhibited ABTS+ radical with an IC50 of 1.68 mg/ml while the positive control (Trolox) used as a standard reference compound had a lower IC50 of 0.31 mg/ml (17). Another study demonstrated that the ethanolic extract of G. thoracica propolis in Perak, Malaysia has an IC50 value of 48.3±0.2 µg/ml using DPPH assay (50).

Furthermore, antioxidant properties of T. apicalis propolis extract are dose-dependent, with IC50 value for DPPH test of 4.27 mg/ml (51). The antioxidant properties of propolis extract are regulated primarily by its phenolic and flavonoid content. A similar pattern has been identified in which TPC and TFC concentrations were related to the antioxidant activity of T. apicalis propolis extract (14). For all extraction solvents, a significantly high correlation between antioxidant activity and TPC and TFC has been detected using maceration and ultrasound-assisted extraction. Furthermore, propolis extracted with 70% ethanol gave the highest extraction yield and had significantly higher radical scavenging activity, TPC and TFC than water extract of propolis (52). In addition, methanol (IC50, 17.18 µg/ml) extract has the highest percentage of antioxidants compared with hexane (32.10), ethyl acetate (21.05) and ascorbic acid (30.63) (52).

In vivo, pre-treatment with propolis significantly improved SOD, GRx, GPx, and GST enzyme levels in rats (47). The effects of propolis supplementation on antioxidant levels and its mode of action in the aorta of diabetic rats have been examined; the propolis-treated group showed lower SOD/(CAT + GPx-1) ratios than the control group, indicating that the propolis has an antioxidative capability in avoiding hydrogen peroxide accumulation (53). In another animal study, propolis treatment in diabetic rats led to a significant decrease in the antioxidant status of pancreatic tissue. Specifically, the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione S-transferase (GST), glutathione reductase (GR), and catalase (CAT) were notably reduced compared to the control group (47).

Anti-inflammatory activity. Inflammation contributes significantly to the development of cardiovascular illness and other comorbidities, such as hypertension, hypercholesterolemia, type 2 diabetes, chronic renal disease and obesity (34). Another study aimed to investigate the anti-inflammatory effect and possible mechanisms of propolis in Sprague Dawley rats models. In vivo, proliferating cell nuclear antigen and IL-10 increased while malondialdehyde, NF-κB, TNF-α, IL-1 and cleaved caspase-3 decreased significantly in the propolis-treated diabetic groups compared with the diabetic control group (48). It has been reported that the elevated IL-17 levels are associated with better outcomes in patients with myocardial infarction (MI) caused by atherosclerosis (37). In vitro study also confirmed anti-inflammatory effects; cytokine secretion of TNF-α and IL-1b in supernatant of treated THP-1-derived macrophages was measured using ELISA. TNF-α and IL-1b secretion levels were significantly reduced in THP-1-derived macrophages treated with both oxidized Low-Density Lipoprotein (oxLDL) and ethanolic extract of propolis compared with THP-1-derived macrophages treated with only oxLDL at 6, 24, and 48 h. Ethanol extract of ≤200 ug/ml was used to avoid toxic effect on THP-1 derived macrophages cells. This finding indicates that ethanol extract inhibited the release of both of these pro-inflammatory cytokines (41).

Anti-hyperglycemia and MI activity. Type 2 diabetes mellitus (T2DM) is a type of diabetes characterized by high blood glucose levels, insulin resistance and a poorer insulin-stimulated response in the presence of high blood glucose levels compared with other forms of diabetes (39). In a rat study where diabetes was induced using intraperitoneal streptozotocin (60 mg/kg), the effects of propolis extract were investigated. The rats were administered either propolis alone (300 mg/kg/day), metformin alone (standard diabetes medication), or a combination of both (DM + M + P). The study aimed to assess their impact on blood sugar levels. The results showed significant improvements in glycemic control. Specifically, the fasting blood glucose (FBG) levels were reduced to 8.9 (2.7) mM, 11.9 (0.5) mM, and 5.6 (0.8) mM in the propolis-treated, metformin-treated, and combined treatment groups, respectively. By contrast, the FBG value in the diabetic group was substantially higher at 27.0 (5.8) mM (32). Combination treatment of metformin and propolis results in the highest FBG improvement in comparison with metformin or propolis treatment alone. In the aforementioned study, all treatment arms significantly improved acetylcholine-induced relaxation compared with the DM group. Another study examined the effects of single oral dose of metformin, soft propolis H. itama methanol extract (MP) and their combination on the blood glucose levels of fasting rats over 9 h. MP had no discernible impact on blood sugar levels of normal rats compared with the control (47). Furthermore, the aforementioned study also reported that the most potent inhibitor of α-glucosidase is soft H. itama with lower IC50 value (1.23±0.32 mg/ml) than acarbose (1.48±0.13 mg/ml) (positive control). Therefore, one of the mechanisms used by soft H. itama to lower blood sugar levels may involve restricting the digestion of ingested carbohydrates to prevent glucose absorption. A previous study examined the impact of propolis, both individually and in combination with insulin treatment, on the maternal condition, pregnancy outcomes, and placental oxidative stress in streptozotocin-induced diabetic rats (29). The final FBG in the propolis-treated diabetic rat group was comparable to the insulin-treated diabetic rat group, indicating that propolis and insulin are equally effective in producing an antihyperglycemic effect (29). Furthermore, the antihyperglycemic effect was more pronounced in the combined group (propolis + insulin)-treated diabetic rats compared with the propolis- and the insulin-treated diabetic rat group groups, indicating that propolis in combination with insulin produced a more significant antihyperglycemic effect than propolis or insulin monotherapy (29). This suggests propolis may protect against DM-induced poor pregnancy outcomes and placental oxidative stress, with more significant effects when supplemented with insulin.

MI, also known as a heart attack, is caused by disruption in the delivery of blood to heart tissue. Necrosis of the myocardium occurs because of coronary artery blockage. The primary cause of myocardium necrosis after MI is an imbalance between coronary blood supply and myocardial demand (47). Pre-treatment with propolis significantly decreases levels of creatine Kinase-MB, Lactate Dehydrogenase, Aspartate Aminotransferase (AST), and Alanine Aminotransferase (diagnostic markers of MI in rats), Total Cholesterol (TC), Triglyceride and Very Low-Density Lipoprotein Cholesterol while increasing the level of High-Density lipoprotein Cholesterol. Furthermore, compared with control group, rats receiving prior propolis treatment exhibit substantially decreased serum cardiac troponin levels (47). Receptor for advanced glycation end products (RAGE) exists as a full-length receptor that is attached to the cell membrane. The soluble(s)RAGE isoform is produced by either alternate splicing of the pre-mRNA (endogenous secretory RAGE; esRAGE) or shedding of RAGE by sheddase (cleaved RAGE; cRAGE). The cRAGE portion is large while the esRAGE portion is minor. sRAGE (cRAGE and esRAGE) binds and sequesters RAGE ligands or competes with RAGE binding, shielding the cell from the damaging effects of AGE-RAGE signalling. Serum sRAGE levels were observed to be greater or lower in people with T2DM (43). Another study reported that propolis-treated diabetic rats have higher heart/serum esRAGE levels than diabetic control groups, indicating a higher concentration of protective decoy receptor esRAGE in the heart compared with the serum. Because esRAGE binds to excess AGE and eliminates it, the AGE/esRAGE ratio may be used as a biomarker in diabetic cardiomyopathy. The combination of propolis and metformin results in significant cardiac AGE/esRAGE ratio alterations, implying a synergistic impact in preventing diabetic cardiomyopathy. The cardioprotective activity of propolis requires more research into whether propolis directly stimulates esRAGE formation or indirectly increases esRAGE by lowering AGE, as in hyperglycemia improvement (40). Furthermore, combination of propolis and metformin has synergistic cardioprotective activity in the heart, as demonstrated by lower cardiac AGE/esRAGE ratio (40).

Wound healing activity. Wound healing is a dynamic system involving constant cell-cell and cell-matrix interactions in a succession of overlapping phases, including haemostasis (blood coagulation cascade), inflammatory, proliferative, and remodelling (48). Several mediators and cell types regulate this system, including platelets, inflammatory cells, fibroblasts, keratinocytes, cytokines, growth factors and matrix metalloproteinases (48). Propolis at low concentrations (0.005, 0.125, 0.250 and 0.500 mg/ml) maintains or increases stem cell proliferation considerably. Propolis is bioactive and biocompatible at optimal concentrations and may be used to boost stem cell proliferation in culture media (44). In a proliferation assay using propolis, the average number of cells increased, peaking at 500 µg/ml after 48 h and then falling significantly with 1,000 µg/ml (30). This concentration-dependent pattern is the mechanism by which propolis influences the proliferation of fibroblast cells. In addition, propolis at concentrations of 1, 10 and 250 µg/ml results in significantly faster wound closure than controls, but the other concentrations have no notable effect. Nonetheless, only the 250 µg/ml concentration demonstrated a significantly higher migration rate than the control at 30 h. Therefore, propolis showed a generally positive effect on both assays compared with the control, and it followed a concentration-dependent curve, with 250 µg/ml being the most optimal concentration for cell migration and 500 µg/ml for cell proliferation. Doses >500 µg/ml may have toxic effect on proliferation of fibroblast cells. The proposed modes of action of propolis on pharmacological activities are illustrated in Fig. 3. Moreover, to have a better understanding of safe dosages and beneficial effects, it is crucial that future research assess the toxicity effect of propolis.

Figure 3 Summary of the proposed mechanism of action of Malaysian propolis in pharmacological activities such as antimicrobial, anti-hyperglycemia, wound healing, hepatoprotective, anti-inflammatory, antioxidant, atherosclerosis, myocardial infarction and cytotoxicity. ACh, acetylcholine; iNOS, inducible nitric oxide synthase; SPAGE, spatial gene enhancement; SOD, superoxide dismutase; CAT, catalase; GPX, glutathione peroxidase; PBG, postprandial blood glucose; GSH, glutathione; TAC, total antioxidant capacity; ROS, reactive oxygen species; FBG, fasting blood glucose; TC, total cholesterol; TG, triglyceride; VLDL-C, very low-density lipoprotein; HDL-C, high density lipoprotein; CK-MB, creatine kinase-myocardial band; LDH, lactate dehydrogenase; AST, aspartate aminotransferase; ALT, alanine aminotransferase; CE, carcinoembryonic antigen; GRx, glutaredoxin; GPx, glutathione peroxidase.

In summary, the present review provided insight into the therapeutic potential of Malaysian propolis based on in vitro and in vivo studies. Apart from antioxidant activity, propolis exhibits antimicrobial, proliferative, anti-inflammatory, anti-hyperglycaemia, hepatoprotective, wound healing effects and prevents MI and atherosclerosis. Nonetheless, this review only summarised propolis activities based on the limited number of studies available. In the future, more extensive research and clinical studies, as well as meta-analyses, are required.

Acknowledgements

The authors acknowledge the financial and technical support for this Fundamental Research Grant Scheme (FRGS) project provided by the Ministry of Higher Education of Malaysia (MOHE).

Funding

Funding: The present study was funded by the Fundamental Research Grant Scheme (FRGS) provided by the Ministry of Higher Education of Malaysia (MOHE) (grant no. FRGS/1/2022/STG02/UNISZA/02/1/).

Availability of data and materials

Not applicable.

Authors' contributions

NAJ, NAM and KSM contributed to the conception of the study and critically reviewed the article. NAJ wrote and edited the manuscript and constructed figures. NAJ, NAM and AAMB contributed to the data acquisition and analysis and drafted the manuscript. Data authentication is not applicable. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References