This research was carried out after being approved by the research ethics committee of King Khalid University under the license number (ECM#2024-2004). The issued ethical license is entitled as Effectiveness of Extracts of sugar ants and Apis mellifera jemenitica in the treatment of diabetes mellitus. The Oryctolagus cuniculus rabbits were used as animal models for the treatment of diabetes mellitus. However, the Oryctolagus cuniculus rabbits are not mentioned in title of the ethical license, but it was stated in the proposal sent to the ethical committee. Regarding the informed consent, we are the owners of the honeybees and the rabbits. The honeybees were obtained from our apiaries and the rabbits were bought from the local market private veterinary clinics.

This study followed a pilot true experimental research design. It included a control group (four rabbits) and two experimental groups (two rabbits each). Initially, all four rabbits served as the control group before being divided into two experimental groups of two rabbits each. The four rabbits were from the species of Oryctolagus cuniculus rabbits as stated by the owner and they were confirmed using the Google Lens at: https://lens.google/#shopping.

The study sample consisted of adult workers and drones of A. m. jemenitica with the age of, approximately, 30 days. The workers and drones were collected from a single hive at morning in the summer season and when the bees were actively foraging. To collect bee samples from a hive, specific steps were followed: 1) Smoke was utilized to calm the bees and reduce aggression; 2) The hive was opened gently, ensuring that the queen was absent; 3) A frame was removed from the hive and examined to confirm the absence of the queen; 4) The frame was placed next to a sample collection box; and 5) The bees were gently shaken into the box and quickly closed to prevent them from flying away. The gentle shaking aimed to minimize the level of monoamine neurotransmitters (dopamine, octopamine, and serotonin) in the hemolymph to prepare the honeybees for euthanasia. Euthanasia of the studied honeybees was achieved by transferring them into liquid nitrogen, ensuring rapid death due to the extremely low temperature, similar to using dry ice for killing honeybees ( Mutinelli, 2021).

The deceased honeybees were divided into two parts (10 g each) and soaked in 50 mL of water or ethanol. The soaked honeybee samples were then incubated for 48 hours at 35°C while shaking to facilitate effective extraction and they were immediately analyzed after the incubation period ( Mutinelli, 2021).

The four Oryctolagus cuniculus rabbits were housed in pairs (two rabbits per cage) for a one-week acclimatization period. Following adaptation, all rabbits received an oral glucose (MERCK; G8270-1KG) load to establish baseline glucose tolerance test (GTT) curves, which served as control data. After a one-week washout period, the rabbits were divided into two experimental groups (n=2 per group) to assess the effects of aqueous and ethanolic honeybee extracts on GTT profiles.

2.4.1 Inclusion and exclusion criteria

Only physically healthy, 3 month age and male Oryctolagus cuniculus rabbits were included in this study.

2.4.2 Blinding

Asseri RM and Mohammed MEA were responsible for looking after the rabits and blood sampling. All the rabbits were put in the same conditions in one animal house at room temperature.

Blood samples were collected from the central ear artery after applying a local anesthetic and disinfecting the area with 70% ethanol. A few drops of blood were placed directly onto a Contour TS glucometer (Bayer HealthCare, Germany) and the glucose concentration (mg/100 mL) was recorded.

Four key steps were implemented to conduct the Glucose Tolerance Test (GTT): 1) Overnight Fasting: Rabbits were subjected to fasting overnight to establish a baseline; 2) Fasting Blood Sample: A fasting blood glucose sample was collected following the fasting period; 3) Glucose Administration: Each rabbit was given a glucose load of 1 g/kg orally alone or with 1 g/kg of each honeybee extract; and 4) Post-Load Sampling: After the glucose administration, blood samples were taken every 30 minutes for a duration of three hours to monitor glucose levels ( Eyth et al., 2024; Moro and Magnan, 2025).

The IR scanning was conducted using the Agilent Cary 630 FTIR Spectrometer (Agilent, USA), accompanied by the Agilent MicroLab software suite. A single drop of each extract was placed on the sampler, and the scanning was performed across a spectral range of 7000 to 350 cm ⁻¹. The resulting spectra highlighted specific peak values, which were subsequently recorded for analysis according the procedure of the manufacturer ( Agilent Technologies, Inc, 2022).

Spectral matching was performed using the spectral database for organic compounds ( https://sdbs.db.aist.go.jp/SearchInformation.aspx) to identify the probable chemical constituents. Two parameters are set by the database developers to control the quality of the obtained search results: 1) the absorption wavelength matching range (Allowance), expressed as ± number (±1, ±2, etc.); and 2) the transmission percentage. Precise search results are obtained by minimizing both the Allowance and the transmission percentage. The set of IR spectra search criteria depends on the nature of the sample, whether it is crude or pure and the processing method. Low values for the Allowance range and transmission percentage are generally set for pure compounds ( SDBS, 2025; Coates, 2006). The SDBS search criteria are presented in a supplementary file (Supplement 1).

The PubChem database ( https://pubchem.ncbi.nlm.nih.gov/) was employed to identify the basic chemical information and functions of the identified compounds. Additionally, some of the biological activities of the suggested compounds were gathered by searching the published literature.

The results of the blood glucose concentration were analyzed using the Statistical Package for Social Sciences (SPSS) version 20. The mean values of the blood glucose concentration of the different study groups were compared using the Analysis of Variance (ANOVA) test, with significant variation determined at a level of p-value ≤ 0.05.

The analysis of the infrared (IR) spectra provided insight into the chemical composition of the extracts.

In the case of the water extract, two primary IR peaks were identified at 1633.36 cm ⁻¹ and 3262.34 cm ⁻¹ [ Figure 1]. The peak at 1633.36 cm ⁻¹ is correlated with C=C stretching or bending vibrations, indicating the presence of carbonyl compounds or conjugated systems, whereas the peak at 3262.34 cm ⁻¹ indicates hydroxyl groups (-OH), proposing a rich content of phenolic compounds and other alcohols ( Coates, 2006).

For the ethanol extracts, a broader range of IR peaks was observed, specifically at 644.32 cm ⁻¹, 879.62 cm ⁻¹, 1045.25 cm ⁻¹, 1087.16 cm ⁻¹, 1324.28 cm ⁻¹, 1377.27 cm ⁻¹, 1459.69 cm ⁻¹, 2881.44 cm ⁻¹, 2972.69 cm ⁻¹, and 3325.92 cm ⁻¹ [ Figure 2]. These peaks provide details about various functional groups present in the ethanol extracts. Notably, the peaks around 1045.25 cm ⁻¹ and 1087.16 cm ⁻¹ are characteristic of C–O stretching vibrations, often associated with sugar derivatives and alcohols. Additionally, the peaks at 2881.44 cm ⁻¹ and 2972.69 cm ⁻¹ relate to C–H stretching, suggesting the presence of aliphatic hydrocarbons, while the peak at 3325.92 cm ⁻¹ indicates the presence of hydroxyl groups ( Coates, 2006).

This comparative analysis highlights the differences in the chemical profiles of the water and ethanol extracts, which may contribute to their varying biological activities as observed in the glucose tolerance test.

As previously mentioned, the identification of the corresponding compounds for the IR peaks was accomplished by searching the spectral database of organic compounds (SDBS). The chemical structures and their functions were further elucidated using the PubChem database and relevant literature.

In the case of the water extract, the two IR peaks identified were found to correspond to a total of 39 compounds within the SDBS. A comprehensive list of these compounds is provided in ( Table 1- Table 7) (Supplement 2).

For the ethanol extract, the spectral analysis revealed that its IR peaks corresponded to 12 compounds, as detailed in ( Table 1- Table 7) (Supplement 3).

The compounds identified in both extracts were categorized into various groups based on their chemical nature and biological potential. The classifications include: sugars and sugar derivatives ( Table 1), phenolic compounds ( Table 2), alkaloids ( Table 3), quinones ( Table 4), dipeptides ( Table 5), amino acid derivatives ( Table 6) and organometallic compounds and pesticides ( Table 7).

This classification not only aids in understanding the chemical complexity of the extracts but also provides insight into their potential biological activities, which may be relevant for their applications in medicinal and therapeutic contexts.

3.3.1 Patterns of GTT curves

The glucose tolerance test (GTT) curve for glucose alone exhibited two distinct phases: an initial increase followed by a decrease. In contrast, the GTT curve for glucose mixed with the water extract demonstrated a decrease initially followed by an increase. The GTT curve involving glucose mixed with the ethanol extract was more complex, displaying three phases: a slight increase, a slight decrease, and then a subsequent increase [ Figure 3]. These variations in the GTT curves can likely be attributed to the different constituents present in the water and ethanol extracts of the A. m. jemenitica drones and workers.

3.3.2 Comparison of the blood glucose results

The glucose concentration in the blood samples was measured from the rabbits at two distinct time points: at zero time (fasting) and after breaking the fast at intervals of 90, 120, 150, and 180 minutes. Blood samples were collected from three different groups involved in the glucose tolerance test (GTT): a control group that received glucose alone, a group that received glucose combined with honeybee water extract, and another group that received glucose combined with honeybee ethanol extract. This setup aimed to assess the effects of the honeybee extracts on glucose metabolism compared to the control group. The results of the blood glucose concentration in the three groups and in the different time intervals are presented in Table 8.

4.1.1 Sugars and their derivatives

The water extract contained one sugar, L-fucose, whereas the ethanol extract contained two sugars, L-fucose and D-fucose, along with two sugar derivatives. ((S)-1,2-O-(2,2,2-trichloroethylidene)-alpha-D-glucofuranose and methyl 2,3,4-tri-O-acetyl-1-deoxy-1-(propoxy (thiocarbonyl))amino-beta-D-glucopyranuronate).

4.1.1.1 L-fucose (6-deoxy-beta-L-galactopyranose)

Fucose was identified in both the water and ethanol extracts, and it stands out as the only deoxy monosaccharide present in mammals in the L-form, while most other monosaccharides exist in their D-form. Known as 6-deoxy-beta-L-galactose, fucose typically adopts a pyranose configuration and exhibits a crystalline white appearance, with a molecular formula of C ₆H ₁₂O ₅ ( Table 1 and Table 2). This compound is commonly found at the terminal positions of oligosaccharides, polysaccharides, and glycolipids. L-fucose is abundant in various organisms, such as brown algae, marine microalgae (which include both green and red algae, as well as diatoms), bacteria, and fungi ( Wang et al., 2024).

In terms of functionality, L-fucose plays significant roles in both medicine and industry. It has been recognized for its antitumor and anti-aging properties, and it can also alleviate intestinal pain. Industrially, L-fucose serves as an emulsifier in the food industry and is incorporated into various skin care products ( Adhikari et al., 2022; Garber et al., 2021; Al-Baarri et al., 2018; Fiume et al., 2019).

4.1.1.2 D-fucose (6- deoxy-D-galactose)

D-fucose is a stereoisomer of L-fucose. Unlike L-fucose, which is more prevalent in human fluids, D-fucose has been reported to exhibit anti-acrosin activity. Acrosin is a sperm protease that plays a crucial role in the fertilization of ova ( National Center for Biotechnology Information, 2024a; Klemm et al., 1991). Notably, D-fucose was identified in the ethanol extract but was not detected in the water extract, as shown in Table 2.

4.1.1.3 (S)-1,2-O-(2,2,2-trichloroethylidene)-alpha-D-glucofuranose (beta-Chloralose)

This derived monosaccharide is detected in the ethanol extract. It was historically used as a sedative drug, but due to its side effects and limited effectiveness, it has been largely replaced by safer and more effective sedatives. Nevertheless, it still finds application as a general anesthetic for animals. In a medical context, it is utilized to relieve psychological excitement by inducing drowsiness and promoting sleep (see Table 2 for further details) ( National Center for Biotechnology Information, 2024b).

4.1.1.4 Methyl 2,3,4-tri-O-acetyl-1-deoxy-1-(propoxy (thiocarbonyl))amino-beta-D-glucopyranuronate

As a glucuronic acid derivative, Methyl 2,3,4-tri-O-acetyl-1-deoxy-1-(propoxy (thiocarbonyl))amino-beta-D-glucopyranuronate may enhance bone matrix deposition and decrease bone resorption. This compound could achieve these effects through the activation of osteoblastic cell differentiation while simultaneously inhibiting osteoclastic cell differentiation. This dual action may contribute to improved bone health and density ( Nagaoka et al., 2012). The Methyl 2,3,4-tri-O-acetyl-1-deoxy-1-(propoxy (thiocarbonyl))amino-beta-D-glucopyranuronate was not detected in the water extract ( Table 2).

4.1.2 Phenolic compounds

4.1.2.1 2,4,6-trimethylpyridinium p-toleunesulfonate

The 2,4,6-trimethylpyridinium p-toluenesulfonate was detected in the water extract. It is a white to pale yellow or orange solid with the molecular formula C ₁₅H ₁₉NO ₃S. This compound is commonly used in chemical synthesis, serving as a significant reagent in various organic reactions ( Chemical Book. 2023). Its presence in the extracts underlines the varied chemical constituents that could affect the GTT curve.

4.1.2.2 2,4-dihydroxybenzoic acid

2,4-Dihydroxybenzoic acid was identified in the water extract of Apis mellifera jemenitica. It has the molecular formula C ₇H ₆O ₄, appears as a solid, and is characterized by a white color. This compound is commonly found in various plants ( Table 2) and is utilized in the food industry as a flavoring agent. In the plant kingdom, 2,4-dihydroxybenzoic acid is known to play a crucial role in enhancing disease resistance ( National Center for Biotechnology Information, 2024c; Lu et al., 2024). Additionally, it possesses notable antioxidant and antimicrobial properties ( Kalinowska et al., 2021).

4.1.2.3 2,3-naphthalenediol

2,3-naphthalenediol is a white powder with the molecular formula C ₁₀H ₈O ₂. It is recognized as a human metabolite and is utilized as a hair dyeing material ( Table 2) ( National Center for Biotechnology Information, 2024d). Naphthalenediols exhibit a range of biological activities, including antioxidant, antilplatelet aggregation, anti-inflammatory, antimicrobial, and anti-protozoal effects ( Ibrahim and Mohamed, 2016).

4.1.2.4 6-methyl-2-benzothiazolamine

6-methyl-2-benzothiazolamine was identified in the water extract. It is solid in nature with the molecular formula of C ₈H ₈N ₂S ( Table.2) ( National Center for Biotechnology Information, 2024e). Benzothiazole derivatives are proven to exhibit various biological activities, including anticancer, anti-tuberculosis, antimicrobial, anti-inflammatory, anti-convulsant, and anti-diabetic properties ( Ali and Siddiqui, 2013; Dhadda et al., 2021).

4.1.2.5 3-amino-4-hydroxybenzenesulfonic acid

The compound mentioned is a brown solid with the molecular formula C ₆H ₇NO ₄S, identified as 3-amino-4-hydroxybenzenesulfonic acid ( Table.2) ( National Center for Biotechnology Information, 2024f). This compound exhibits inhibitory effects on a variety of enzymes, including Coenzyme A dehydrogenase, Aldehyde dehydrogenase, and Apurinic/apyrimidinic endonuclease. Additionally, it has been reported to have anticancer properties, particularly in the context of leukemia. The compound also acts as an antagonist for certain receptors, specifically retinoid-related orphan receptor gamma, while functioning as an agonist for some signaling pathways, notably the peroxisome proliferator-activated receptor delta signaling pathway ( National Center for Biotechnology Information, 2024f).

4.1.2.6 5-(p-aminophenyl)-2-thiazolamine

5-(p-aminophenyl)-2-thiazolamine is a notable water extract with the molecular formula C ₉H ₉N ₃S. This compound features an aminophenol moiety and is incorporated into nano-pigments used for printing applications ( Table 2) ( National Center for Biotechnology Information, 2024g). In addition to its use in printing, aminophenol derivatives are recognized for their antimicrobial and anti-diabetic activities. Specifically, compounds that possess thiazolamine and aminophenol structures have demonstrated antimicrobial properties, with sulfathiazol being a prominent example ( Rafique et al., 2022; Ali and Sayed, 2021).

4.1.2.7 3’-hydroxy-2’-acetonaphthone

3’-Hydroxy-2’-acetonaphthone has the molecular formula C ₁₂H ₁₀O ₂ and has been identified in the water extract of Apis mellifera jemenitica. As a naphthene derivative, acetonaphthone is noted for its potential antimicrobial and anti-convulsant properties, as indicated by Karakurt et al. (2010) and Center for Advancing Translational Sciences (2020).

4.1.2.8 4-nitro-2-(trifluoromethyl)aniline

The compound 4-nitro-2-(trifluoromethyl) aniline, with the molecular formula C ₇H ₅F ₃N ₂O ₂, is primarily utilized in the synthesis of mono-azo dyes ( Dickey et al., 1953). Additionally, trifluoromethyl compounds have shown potential in the treatment of various diseases, serving as effective agents in anti-tuberculosis, antiviral, anticancer, and antidepressant applications ( Nair et al., 2022).

4.1.2.9 5,6-dihydro-4H-benzo(6,7)cyclohepta(1,2-d)thiazol-2-amine hydrobromide

5,6-Dihydro-4H-benzo(6,7)cyclohepta(1,2-d)thiazol-2-amine hydrobromide is classified as a tricyclic thiazole compound. This compound features derivatives of benzene, cycloheptane, and thiazole, making it a unique structure. It has been noted for its biological activity, particularly as an anti-angiogenesis agent, which suggests it may play a role in inhibiting the growth of new blood vessels that can contribute to tumor development and other pathological conditions. Studies exploring its potential therapeutic applications could be of great interest in drug development and cancer treatment ( Bhat et al., 2013).

4.1.2.10 O-(N-(dimethylcarbamoylmethyl)acetamido)-N-N-dimethylbenzamide

O-(N-(dimethylcarbamoylmethyl)acetamido)-N,N-dimethylbenzamide is classified as a urea derivative. Urea derivatives, which include compounds like acetamide and benzamide, are commonly utilized in drug design. Notable examples include glibenclamide and cariprazine, which are used for treating various conditions. These derivatives have applications in the treatment of certain cancers, epilepsy, viral infections such as hepatitis C and HIV, as well as Alzheimer’s disease ( Ghosh and Brindisi, 2019; Matošević and Bosak, 2020). Additionally, compounds containing acetamide and benzamide are known to function as urease inhibitors, highlighting their significance in pharmacological research and potential therapeutic uses ( Ahmad et al., 2023a).

4.1.2.11 8-cyano-3,3-diphenyl-3,3a-dihydrocyclohepta(b)furan-2-one

The eleventh phenolic compound identified in the water extract has the molecular formula C ₂₂H ₁₅NO ₂. Recognized as a benzofuran, the compound 8-cyano-3,3-diphenyl-3,3a-dihydrocyclohepta(b)furan-2-one exhibits potential biological activities such as antimicrobial, anti-tumor, anti-inflammatory, and antioxidant properties. This highlights its significance in terms of therapeutic applications and its potential benefits in health and medicine ( Miao et al., 2019).

4.1.2.12 2-ethylthio (thiocarbonyl)amino-phenylpropionic acid cyclohexylamine salt

The twelfth compound of the water extract is composed of phenylpropanoid derivative and cyclohexylamine. Phenylpropanoids are known for their various biological activities, including antimicrobial, antioxidant, anti-inflammatory, anti-diabetic, and anticancer properties. Additionally, they play a protective role for the kidneys, neurons, heart, and liver ( Neelam et al., 2020). Conversely, cyclohexylamine derivatives demonstrate analgesic effects and can result in decreased motor activity ( Glushkov et al., 2006).

4.1.2.13 Tiropramide hydrochloride

Tiropramide hydrochloride is a phenolic compound with the molecular formula C ₂₈H ₄₂ClN ₃O ₃. It is well-known for its anti-spasmodic properties and is commonly used in the treatment of various conditions related to the hepatobiliary and urinary tracts, including Irritable Bowel Syndrome (IBS) ( National Center for Biotechnology Information, 2024h, Lee et al., 2013; Center for Advancing Translational Sciences, 2024).

4.1.2.14 2- methyl-1,2,3,4-tetrahydro-2-naphthol

2-Methyl-1,2,3,4-tetrahydro-2-naphthol has the molecular formula C ₁₁H ₁₄O. Naphthol derivatives have been shown to act as antioxidants and acetylcholinesterase inhibitors, which are important since acetylcholinesterase is a marker for degenerative neurological diseases. The antioxidant properties of these compounds may provide protective effects against oxidative stress, while the inhibition of acetylcholinesterase suggests potential therapeutic applications in managing neurological disorders ( Erdoğan et al., 2021).

4.1.2.15 (4aalpha, 7alpha, 9alpha, 9aalpha)-9-9a-epoxy-1,1,41,7-tetramethyl-2,3,4,4a,5,6,7,8,9,9a-decahydro-1H-benzocyclohepten-7-ol

As a benzocycloheptane derivative, (4aα, 7aα, 9aα, 9aα)-9-9a-epoxy-1,1,4,7-tetramethyl-2,3,4,4a,5,6,7,8,9,9a-decahydro-1H-benzocyclohepten-7-ol has the potential to exhibit antihistamine activity, in addition to demonstrating effectiveness in killing hepatoma cells ( Abounassif et al., 2005; Liang et al., 2020).

4.1.2.16 Ethyl p-((2-chloroethoxy) carbonylamino) benzoate

Benzoate derivatives are well-known for their diverse biological activities, including functions as local anesthetics, anticancer agents, anti-Alzheimer compounds, and antimicrobial, antioxidant, and anti-inflammatory agents ( Zou et al., 2019; Haroon et al., 2023). Additionally, these derivatives are utilized in agricultural applications as pesticides ( Lee et al., 2023; Elabasy et al., 2019).

4.1.2.17 Lasalocid Sodium Salt

Lasalocid Sodium Salt is a benzoate derivative that contains lasalocid. Its molecular formula is C ₃₄H ₅₃NaO ₈. In veterinary medicine, it serves dual functions as an antibacterial agent and an ionophore. As an ionophore, it enhances calcium influx in muscle fibers, which can have various therapeutic effects ( National Center for Biotechnology Information, 2024i).

4.1.2.18 Methyl 4-(3,5-dichloro-4-methoxyphenyl)-3-ethyl-1-pyrazoline-3-carboxylate

The above compound contains two bioactive moieties: methoxyphenyl and pyrazoline. Compounds with methoxyphenol exhibit antibacterial activity against food spoilage bacteria and possess antioxidant properties ( Orlo et al., 2021; Ahmad et al., 2023b). Pyrazole derivatives are well known for their range of pharmacological activities, which include anticancer, anti-inflammatory, antiviral, antifungal, antioxidant, anti-obesity, antidepressant, antipsychotic, and analgesic effects ( Karrouchi et al., 2018; Hassan, 2013).

4.1.2.19 2’,7’-dihydroxyspiro (isobenzofuran-1(3H),9’-(9H)xanthen)-3-one

The nineteenth phenolic compounds contain two bioactive moieties: benzofuran and xanthen-3-one. Benzofuran derivatives are well-known for their antitumor, antiviral, antibacterial, and antioxidant properties ( Miao et al., 2019). On the other hand, xanthen-3-one derivatives exhibit notable anticancer and anti-proliferative activities ( Zukić et al., 2020).

4.1.3 Alkaloids

4.1.3.1 2-(5-methyl-3-pyrroly) piperidine hydrochloride

Natural and synthetic piperdines are known for their diverse biological activities, including anticancer, antioxidant, anti-Alzheimer, antimicrobial, and anti-neuropathic pain properties ( Frolov and Vereshchagin, 2023). Additionally, piperidine exhibits effectiveness against hypertension, asthma, and inflammation ( Abdelshaheed et al., 2021).

4.1.3.2 3-methyl-4-oxo-3,4-dihydro-1-phthalazinecarbohydrazide

The compound contains two bioactive moieties: phthalazine and carbohydrazide. Phthalazine derivatives are recognized for their activity against several health issues, including cancer, diabetes, hypertension, microbial infections, and depression, as well as their analgesic properties ( Sangshetti et al., 2019). On the other hand, carbohydrazide derivatives exhibit a range of biological activities, including antibacterial, antifungal, anti-inflammatory, and anti-tuberculosis effects ( Onyeyilim et al., 2022).

4.1.3.3 N-(5-chloro-6-oxo-1-phenyl-1,6-dihydro-4-pyridazinyl)acetamide

The bioactive moieties of this compound are the pyridazine and acetamide. Pyridazine derivatives are reported to possess antitumor ( He et al., 2021), antibacterial, and antifungal properties ( Butnariu and Mangalagiu, 2009). In contrast, acetamide derivatives are known for their antioxidant and anti-inflammatory effects ( Autore et al., 2010).

4.1.3.4 3’-(9-methyl-9H-pyridazino(3,4-b)indol-3-yl)acetanilide

The compound 3’-(9-methyl-9H-pyridazino(3,4-b)indol-3-yl) acetanilide is notable for containing three significant bioactive groups: pyridazine, indole, and acetanilide. Pyridazine derivatives are particularly recognized for their anticancer, antibacterial, and antifungal activities ( He et al., 2021; Butnariu and Mangalagiu, 2009). Indole derivatives, on the other hand, exhibit a wide range of biological activities including antimicrobial, anti-malarial, anti-diabetic, anti-inflammatory, and anti-tuberculosis effects ( Kumar and Ritika, 2020). Furthermore, acetanilide derivatives are known for their diverse therapeutic actions, which encompass analgesic, anti-inflammatory, antipyretic, antioxidant, anticonvulsant, antimicrobial, anticancer, anti-hyperglycemic, and antimalarial activities ( Singh et al., 2019).

4.1.3.5 2-hydrazino-3,5,6,7-tetrahydrocyclopentapyrimidin-4-one

2-Hydrazino-3,5,6,7-tetrahydrocyclopentapyrimidin-4-one is classified as both a hydrazine derivative and a cyclopentapyrimidin-4-one derivative. Hydrazine derivatives are known for their potential as carbonic anhydrase inhibitors. These inhibitors play a significant role in treating a variety of conditions, including glaucoma, edema, obesity, osteoporosis, epilepsy, and certain types of cancer ( Goff and Ouazzani, 2014; Shirinzadeh and Dilek, 2020). On the other hand, cyclopentapyrimidin-4-one derivatives are recognized for their ability to inhibit Phosphodiesterase10A, making them potential therapeutic targets for various neurodegenerative disorders ( Al-Nema et al., 2022; Bhawale et al., 2023).

4.1.3.6 Cytochalasin E

It is an alkaloid drug used for the inhibition of angiogenesis and it increases the sensitivity of lung cancer to medication ( National Center for Biotechnology Information, 2024j; Takanezawa et al., 2018).

4.1.3.7 1,4’-bipiperidine

As noted earlier, piperidine derivatives have demonstrated a range of biological activities, including anticancer, antioxidant, anti-Alzheimer, antimicrobial, anti-neuropathic pain, anti-hypertensive, anti-asthmatic, and anti-inflammatory effects ( Frolov and Vereshchagin, 2023; Abdelshaheed et al., 2021).

4.1.4 Quinones

4.1.4.1 4,5-dianilino-O-benzoquinone

4,5-Dianilino-O-benzoquinone is reported to have weak antitumor activity ( Huang et al., 1993). Ortho and para benzoquinones are routinely used for their various therapeutic effects, including antimalarial, anti-Alzheimer, antiviral, antifungal, antibacterial, and antitumor properties ( Zhang et al., 2021).

4.1.4.2 1-amino-2-bromo-4-hydroxyanthraquinone

As an anthraquinone derivative, 1-amino-2-bromo-4-hydroxyanthraquinone may exhibit a range of beneficial pharmacological properties, including antioxidant, anticancer, anti-inflammatory, and anti-aging effects. Additionally, studies suggest that anthraquinone derivatives possess hepatoprotective and neuroprotective activities, contributing to their potential therapeutic applications in various medical fields ( Zhao and Zheng, 2023).

4.1.4.3 2,5-bis(2-hydroxypropylamino)-p-benzoquinone

Benzoquinone derivatives exhibit a range of medicinal properties, making them significant in pharmacology. They have been studied for their antimalarial, anti-Alzheimer, antiviral, antifungal, antibacterial, and antitumor activities ( Zhang et al., 2021).

4.1.4.4 2,5,6-trihydroxy-1,4-naphthoquinone

1,4-naphthoquinones are studied as natural products and have exhibited anticancer and antibacterial activities ( Nematollahi et al., 2012). Additionally, Li et al. (2018) stated that naphthoquinone derivatives possess cytotoxicity, antioxidant, and anti-inflammatory activities.

4.1.5 Dipeptides

4.1.5.1 DL-alanyl-L-phenylalanine

Dipeptides containing alanine from dry cured ham have been reported to have the ability to inhibit angiotensin-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP IV), making them potential candidates for anti-hypertension and anti-hyperglycemic therapeutic agents, respectively ( Gallego et al., 2019; Messerli et al., 2018; Deacon, 2020). Dipeptides that include phenylalanine also exhibit inhibition of ACE and renin, further establishing their suitability as anti-hypertensive compounds ( Gallego et al., 2019; Messerli et al., 2018; Fisher and Meagher, 2011). However, our review of the literature reveals a lack of articles documenting the presence of the Ala-Phe (AF) dipeptide in food, biological fluids, or tissues. Consequently, the functional role of the AF sequence has not been previously addressed.

4.1.5.2 n-isovaleryl-L-alanine anilide

Anilide derivatives with amino acids exhibit potential anti-convulsant properties, as seen with glycine anilide derivatives ( Soyer et al., 2013). Additionally, dipeptides that include valine or alanine have been shown to inhibit enzymes such as ACE, renin, and DPP IV, indicating their possible roles in managing hypertension and diabetes. Furthermore, dipeptides containing valine residues are noted for their antioxidant activity ( Gallego et al., 2019).

4.1.6 Amino acid derivatives

4.1.6.1 L-glutamic acid 5-hydrazide

L-glutamic acid, 5-hydrazide exhibits significant mutagenic activity on E. coli, which qualifies it to function as an antibacterial agent ( Maeda et al., 2021). It plays a role in the bacterial biosynthesis of important antibiotics such as fosfazinomycin and kanamycin ( Wang et al., 2018). This highlights its potential relevance in both medicinal and microbiological contexts.

4.1.6.2 R-noradrenaline

Noreadrenaline, also known as norepinephrine, is a neurotransmitter and hormone that plays a crucial role in the body’s response to stress and blood pressure regulation. It is synthesized from the amino acids tyrosine and phenylalanine ( Dalangin et al., 2020). Norepinephrine primarily functions as a vasoconstrictor, making it an important agent in the treatment of hypotension ( Smith and Maani, 2024).

4.1.6.3 N (alpha)-benzoyl-DL-arginine-p-nitroanilide hydrochloride

N (alpha)-benzoyl-DL-arginine-p-nitroanilide has the capability of enhancing the proteolytic activity of plasmin and trypsin ( Christensen and, Müllertz, 1974; Dulay et al., 2005). The plasmin is involved in the breakdown of the fibrin fibers in blood clots converting them to soluble products ( Famutimi et al., 2024). This compound may be exploited in the treatment of ischemic vascular diseases. Trypsin is a serine protease which facilitates the digestion of proteins and it is involved in progression of colorectal and ovarian cancers ( National Center for Biotechnology Information, 2024k).

4.1.6.4 1-nitroguanidine

Nitroguanidine is likely synthesized from arginine and plays a significant role in the structure of various insecticides and explosives ( National Center for Biotechnology Information, 2024k; Berlinck and Romminger, 2016). The presence of this compound in the structure of honeybees may be attributed to their exposure to insecticides.

4.1.7 Organometallics and pesticides

4.1.7.1 Pentaammine (chloroacetato) cobalt (III) diperchlorate

Pentaammine (chloroacetato) cobalt (III) diperchlorate is reacted with 4-Amino-1,2,4-triazole to produce (4-amino-1,2,4- triazole-N1(N2) pentaamminocobalt (III) perchlorate; an explosive material with low toxicity ( Ilyushin et al., 2010). The existence of this organometallic compound in the water extract of the honeybees may be attributed to environmental pollution, presence in insecticide composition or similarity in IR spectra.

4.1.7.2 Potassium diaquabis (malonato) manganite (III)

It is as synthetic organometallic compound with magnetic activity ( Delgado et al., 2006). This compound may found in the water extract of honeybees due to external sources.

4.1.7.3 Alpha-chloralose

Alpha-chloralose is used as pesticides and it is classified as ultra-short acting anesthetic that induces loss of consciousness or as hypnotics and sedatives that induces drowsiness or sleep ( National Center for Biotechnology Information, 2024l).

The normal GTT curve exhibits various patterns, including monophasic (characterized by one peak with subsequent increase and decrease), biphasic (featuring two peaks), triphasic (showing three peaks), and multi-phasic responses. This curve serves as an indicator of the physiological, metabolic, or pathological state of the subjects, whether humans or animals ( de Andrade Mesquita et al., 2018; Vejrazkova et al., 2023).

According to de Andrade et al. (2018), a monophasic GTT curve signals pre-diabetes and pre-metabolic syndrome [104].

The water extract’s impact on the GTT curve is likely attributed to its rich content of phenolic acids and alkaloids, as supported by various studies ( Ali and Siddiqui, 2013; Neelam et al., 2020; Sangshetti et al., 2019; Kumar and Ritika, 2020; Singh et al., 2019; Lin et al., 2016; Kumar et al., 2019). Notably, the presence of five specific compounds in the water extract could help elucidate the observed decrease in glucose levels during the second phase of the water extract GTT curve. The five compounds are 6-methyl-2-benzothiazolamine, 5-(p-aminophenyl)-2-thiazolamine, 2-ethylthio (thiocarbonyl)amino-phenylpropionic acid cyclohexylamine salt, 3-methyl-4-oxo-3,4-dihydro-1-phthalazinecarbohydrazide and 3’-(9-methyl-9H-pyridazino(3,4-b)indol-3-yl) acetanilide ( Table.2 and Table.3).