Glucose Tolerance Test (GTT) Curves of Water and Ethanol Extracts of Whole Body Apis mellifera jemenitica

2.1 Ethical clearance and informed consent

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.

2.2 Research design and the study groups

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.

2.3 Preparation of the Honeybee samples and the extraction in water and ethanol

The study sample consisted of adult workers and drones of Apis mellifera jemenitica. 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 water and ethanol. The soaked honeybee samples were then incubated for 48 hours at 35°C while shaking to facilitate effective extraction (Mutinelli, 2021).

2.4 The experimental Oryctolagus cuniculus rabbits and the blood sampling

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.

2.5 Blood sampling and measurement of blood glucose

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.

2.6 GTT procedure

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; 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 & Magnan, 2025).

2.7 IR spectrometry

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.

2.8 Searching the spectral database for organic compounds (SDBS)

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.

2.9 Statistical analysis

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.

3.1 IR Spectral analysis

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).

Figure 1. Shows the IR spectrum of the water extract, highlighting two distinct peaks.

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).

Figure 2. Displays the IR spectrum of the ethanol extract emphasizing 10 noticeable peaks.

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.

3.2 Compound identification

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).

Table 1. The sugars and their derivatives in the water and ethanol extract of the workers and drones of Apis mellifera jemenitica.

	Class	Compound and formula	Chemical structure	Biological activity	Extract	Pubchem ID
1	Sugars and their derivatives	6-deoxy-beta-L-galactopyranose (L-Fucose) C6H12O5		Antitumor Anti-aging Relief of intestinal disease Component in skin care products Emulsifier in food industry (Wang et al., 2024; Adhikari et al., 2022; Garber et al., 2021; Al-Baarri et al., 2018; 21. Fiume et al., 2019).	Water and ethanol	Pubchem: 444863 SDBS:32641
		6- deoxy-D- galactose (D- Fucose) C6H12O5		Anti- human acrosin which causes male infertility (National Center for Biotechnology Information, 2024a; Klemm et al., 1991).	Ethanol	Pubchem: 444200 SDBS:2561
		(S)-1,2-O-(2,2,2-trichloroethylidene)-alpha-D-glucofuranose C8H11Cl3O6		Sedative Anesthetic in animal experiments Induction of drowsiness and sleep (National Center for Biotechnology Information, 2024b).	Ethanol	Pubchem: 5284343 SDBS:15171
		methyl 2,3,4-tri-O-acetyl-1-deoxy-1-(propoxy (thiocarbonyl))amino-beta-D-glucopyranuronate C17H25NO10S		May increase bone matrix deposition (Nagaoka et al., 2012).	Ethanol	Pubchem: 273075659 SDBS:32735

Table 2. The phenolic compounds in the water and ethanol extract of the workers and drones of Apis mellifera jemenitica.

	Class	Compound	Chemical structure	Biological activity	Extract	ID
2	Phenolic compounds	2,4,6-trimethylpyridinium p-toleunesulfonate C15H19NO3S		It is used for chemical synthesis (Chemical book, 2023).	Water	Pubchem: NA SDBS:18269
		2,4- dihydroxybenzoic acid C7H6O4		Plays a role in plant immunity Antioxidant and antimicrobial (Lu et al., 2024; Kalinowska et al., 2021).	Water	Pubchem: 1491 SDBS:3084
		2,3-naphthalenediol C10H8O2		Antioxidant, antiplatelet aggregation, anti-inflammatory, antimicrobial and anti-protozoa (Ibrahim and Mohamed, 2016).	Water	Pubchem: 7091 SDBS:1626
		6-methyl-2-benzothiazolamine C8H8N2S		Anti-tubercular, antimicrobial, anti-inflammatory, anti-convulsion, anti-diabetic and anticancer (Ali and Siddiqui, 2013; Dhadda et al., 2021).	Water	Pubchem: 17335 SDBS: 3579
		3-amino-4-hydroxybenzenesulfonic acid C6H7NO4S		Anti- acute myeloid leukemia inhibitor of some enzymes antagonist and agonist of some pathways (National Center for Biotechnology Information, 2024f).	Water	Pubchem: 7385 SDBS: 10121
		5-(p-aminophenyl)-2-thiazolamine C9H9N3S		Collectively, the amino phenol and the thiazoleamine have antimicrobial and anti-diabetic activities (Rafique et al., 2022; Ali and Sayed, 2021).	Water	Pubchem: 605642 SDBS:32115
		3'-hydroxy-2'-acetonaphthone C12H10O2		It may act as antimicrobial and anti-convulsion (Karakurt et al., 2010; National Center for Advancing Translational Sciences, 2020).	Water	Pubchem: NA SDBS: 28941
		4-nitro-2-(trifluoromethyl)aniline C7H5F3N2O2		It is used for the synthesis of monoazo dyes (Dickey et al., 1953). Anti-tuberculosis, antiviral, anticancer and antidepressant (Nair et al., 2022).	Water	Pubchem: 67128 SDBS:3261
		5,6-dihydro-4H-benzo(6,7)cyclohepta(1,2-d)thiazol-2-amine hydrobromide C12H12N2S HBr	NA	Anti-angiogenesis (Bhat et al., 2013).	Water	Pubchem: NA SDBS:26013
		O-(N-(dimethylcarbamoylmethyl)acetamido)-N-N-dimethylbenzamide C15H21N3O3		Anticancer, Anti-epilepsy, antiviral, anti-Alzheimer and urease inhibitor (Ghosh and Brindisi, 2019; Matošević and Bosak, 2020; Ahmad et al., 2023a).	Water	Pubchem: 600891 SDBS:32336
		8-cyano-3,3-diphenyl-3,3a-dihydrocyclohepta(b)furan-2-one C22H15NO2		Anti-tumor, antimicrobial, antioxidant and anti-inflammatory (Miao et al., 2019).	Water	Pubchem: 275779763 SDBS:30670
		2-ethylthio (thiocarbonyl)amino-phenylpropionic acid cyclohexylamine salt C12H15NO2S2 C6H13N		Phenylprpoanoid derivatives Antimicrobial, antioxidant, anti-inflammatory, antidiabetic and anticancer (Neelam et al., 2020). Cyclohexylamine derivatives Analgesic activity and decrease the the motor activity (Glushkov et al., 2006).	Water	Pubchem: NA SDBS:29929
		Tiropramide hydrochloride C28H41N3O3 HCl		Antispasmodic for hepatobiliary and urinary tract diseases (National Center for Biotechnology Information, 2024h, Lee et al., 2013; Center for Advancing Translational Sciences, 2024).	Water	Pubchem: 134448 SDBS: 53486
		2-methyl-1,2,3,4-tetrahydro-2-naphthol C11H14O		Antioxidant and inhibitor of acetylcholinesterase (Erdoğan et al., 2021).	Ethanol	Pubchem: NA SDBS: 31692
		(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 C15H26O2	NA	As a benzocycloheptane derivative it can act as antihistamine and anti-hepatoma (Abounassif et al., 2005; Liang et al., 2020).	Ethanol	Pubchem: NA SDBS: 32776
		ethyl p-((2-chloroethoxy) carbonylamino) benzoate C12H14CINO4		benzoate derivatives have antioxidant, anticancer, antimicrobial, anti-Alzheimer and they are used as pesticides Zou et al., 2019; Haroon et al., 2023; Lee et al., 2023; Elabasy et al., 2019).	Ethanol	Pubchem: 273078333 SDBS: 33657
		Lasalocid Sodium Salt C34H53NaO8		Veterinary antimicrobial and ionophore (National Center for Biotechnology Information, 2024i).	Ethanol	Pubchem: 6426773 SDBS: 21356
		methyl 4-(3,5-dichloro-4-methoxyphenyl)-3-ethyl-1-pyrazoline-3-carboxylate C14H16Cl2N2O3		Methoxyphenyl dervitives act as anti food spoilage bacteria and antioxidant (Orlo et al., 2021; Ahmad et al., 2023b). Pyrazole derivatives exhibit a wide range of pharmacological activities including anticancer, antimicrobial, antioxidant, anti-obesity and antihypertension (Karrouchi et al., 2018; Hassan, 2013).	Ethanol	Pubchem: 274969331 SDBS: 39016
		2',7'-dihydroxyspiro (isobenzofuran-1(3H),9'-(9H)xanthen)-3-one C20H12O5		Antitumor, antibacterial, antiviral and antioxidant (Miao et al., 2019). Anticancer, anti-proliferative (Zukić et al., 2020).	Ethanol	Pubchem: 625532 SDBS: 35197

Table 3. The alkaloids in the water and ethanol extract of the drones and workers of Apis mellifera jemenitica.

	Class	Compound	Chemical structure	Biological activity	Extract	Pubchem ID
3	Alkaloids	2-(5-methyl-3-pyrroly) piperidine hydrochloride C10H16N2 HCl		Anticancer, antimicrobial, anti-Alzheimer, antioxidant, anti-neuropathic pain, anti-hypertension, anti-asthma, anti-inflammation (Frolov and Vereshchagin, 2023; Abdelshaheed et al., 2021).	Water	Pubchem: NA SDBS: 37497
		3-methyl-4-oxo-3,4-dihydro-1-phthalazinecarbohydrazide C10H10N4O2		Phthalazine derivatives Anticancer, anti-diabetes, anti-hypertension, anti-microbes, anti-depression and they have analgesic activity (Sangshetti et al., 2019). Carbohydrazide derivatives Antibacterial, antifungal, anti-inflammatory and anti-tuberculosis (Onyeyilim et al., 2022).	Water	Pubchem: 604579 SDBS: 26308
		N-(5-chloro-6-oxo-1-phenyl-1,6-dihydro-4-pyridazinyl)acetamide (Alkaloid and phenolic) C12H10ClN3O2		Pyridazine derivatives Antitumor, antibacterial and antifungal (He et al., 2021; Butnariu and Mangalagiu, 2009). Acetamide derivatives Antioxidant and anti-inflammatory (Autore et al., 2010).	Water	Pubchem: 614997 SDBS: 34810
		2-hydrazino-3,5,6,7-tetrahydrocyclopentapyrimidin-4-one C7H10N4O		Hydrazine derivatives Carbonic anhydrase inhibitors Goff and Ouazzani, 2014; Shirinzadeh and Dilek, 2020). Cyclopentapyrimidin-4-one derivatives Phosphodiesterase 10A inhibitors (Al-Nema et al., 2022; Bhawale et al., 2023).	Water	Pubchem: 273075486 SDBS:32423
		3'-(9-methyl-9H-pyridazino(3,4-b)indol-3-yl)acetanilide C19H16N4O		Pyridazine derivatives Antitumor, antibacterial and antifungal (He et al., 2021; Butnariu and Mangalagiu, 2009). Indole derivatives Antimicrobial, anti-malarial, anti-diabetes, anti-inflammatory and anti-tuberculosis (Kumar and Ritika, 2020). Acetanilide derivatives Analgesic, anti-inflammatory, antipyretic, antioxidant, anticonvulsant, antimicrobial, an-ti-cancer, anti-hyperglycaemia and antimalarial (Singh et al., 2019).	Water	Pubchem: 273078537 SDBS:34070
		Cytochalasin E C28H33NO7		Inhibitor of angiogenesis and increases the sensitivity of lung cancer to chemotherapy (National Center for Biotechnology Information, 2024j; Takanezawa et al., 2018).	Water	Pubchem: 5458385 SDBS:13826
		1,4'-bipiperidine C10H20N2		Anticancer, antimicrobial, anti-Alzheimer, antioxidant, anti-neuropathic pain, anti-hypertension, anti-asthma, anti-inflammation (Frolov and Vereshchagin, 2023; Abdelshaheed et al., 2021).	Ethanol	Pubchem: 78607 SDBS:22415

Table 4. The quinones in the water extract of the drones and workers of Apis mellifera jemenitica.

	Class	Compound	Chemical structure	Biological activity	Extract	ID
4	Quinones	4,5-dianilino-O-benzoquinone C18H14N2O2		Antimalarial, anti-Alzheimer, antiviral, antifungal, antibacterial and antitumor (Huang et al., 1993; Zhang et al., 2021).	Water	Pubchem: 274966116 SDBS:34956
		1-amino-2-bromo-4-hydroxyanthraquinone C14H8BrNO3		Antioxidant, anticancer, anti-inflammation and anti-aging, hepato-protective and neuro-protective (Zhao and Zheng, 2023).	Water	Pubchem: 8320 SDBS:18790
		2,5-bis(2-hydroxypropylamino)-p-benzoquinone C12H18N2O4		Antimalarial, anti-Alzheimer, antiviral, antifungal, antibacterial and antitumor (Zhao and Zheng, 2023).	Water	Pubchem: 620240 SDBS:25209
		2,5,6-trihydroxy-1,4-naphthoquinone C10H6O5		Anticancer, antibacterial, cytotixic, anti-infammatory and antioxidant (Nematollahi et al., 2012; Li et al., 2018).	Water	Pubchem: 273072755 SDBS:31333

Table 5. The dipeptides in the water extract of the drones and workers of Apis mellifera jemenitica.

	Class	Compound	Chemical structure	Biological activity	Extract	ID
5	Dipeptides	DL-alanyl-L-phenylalanine C12H16N2O3		ACE and Renin inhibitor Anti-hypertension DPP IV inhibitor Anti-hyperglycemia (anti-diabetes) (Gallego et al., 2019; Messerli et al., 2018; Deacon, 2020; Fisher and Meagher, 2011).	Water	Pubchem: 2080 SDBS:4845
		n-isovaleryl-L-alanine anilide C14H20N2O2		Anilide derivatives with glycine Anti-convulsions of Epilepsy (Soyer et al., 2013). Anti-hypertension, anti-diabetes and antioxidant (Gallego et al., 2019).	Water	Pubchem: 564405 SDBS:32067

Table 6. The amino acids derivatives in the water extract of the drones and workers of Apis mellifera jemenitica.

	Class	Compound	Chemical structure	Biological activity	Extract	ID
6	Amino acid derivatives	L-glutamic acid 5-hydrazide C5H11N3O3 H2O		It has strong mutagenic activity on E coli (Maeda et al., 2021). Ii is involved in the anabolism of fosfazinomycin and kanamycin (Wang et al., 2018).	Water	Pubchem: 92165 SDBS:29556
		(R)-noradrenaline C8H11NO3		It is a neurotransmitter and vasoconstrictor used for the treatment of hypotension (Smith and Maani, 2024).	Water	Pubchem: 439260 SDBS:3536
		N (alpha)-benzoyl-DL-arginine-p-nitroanilide hydrochloride C19H22N6O4 HCl		Enhance proteolytic activity of plasmin and trypsin (Christensen and, Müllertz, 1974; Dulay et al., 2005).	Water	Pubchem: NA SDBS:12532
		1-nitroguanidine CH4N4O2		Component of insecticides and explosives (National Center for Biotechnology Information, 2024k).	Water	Pubchem: 86287517 SDBS:3695

Table 7. The organometallic compounds and pesticides in the water extract of the drones and workers of Apis mellifera jemenitica.

	Class	Compound	Chemical structure	Biological activity	Extract	ID
7	Organometallic compounds	Pentaammine (chloroacetato) cobalt (III)diperchlorate C2H17Cl3CoN5O10	NA	Used in synthesis of explosive material (Ilyushin et al., 2010).	Water	Pubchem: NA SDBS:35464
		Potassium diaquabis (malonato) manganite (III) C6H8KMnO10 2H2O	NA	synthetic Inorganic compound with magnetic activity (Delgado et al., 2006).	Water	Pubchem: NA SDBS:26382
		alpha-chloralose C8H11Cl3O6		Pesticide, anesthetic, hypnotics and sedatives (National Center for Biotechnology Information, 2024l).	Ethanol	Pubchem: 7057995 SDBS:3495

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 GTT results

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 Apis mellifera jemenitica drones and workers.

Figure 3. The GTT Curves of glucose alone (a), glucose with water extract (b) and glucose with ethanol extract (c).

The water extract altered the GTT curve from a convex shape to a concave shape, indicating a significant effect on blood glucose levels. In contrast, the ethanol extract demonstrated a more variable response, showing a slight increase, followed by a slight decrease, and then a subsequent increase in blood glucose levels. This suggests different mechanisms of action or efficacy between the two extracts in influencing glucose tolerance.

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 Chemical composition of the water and ethanol extracts

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 C₆H₁₂O₅ ( 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 C₁₅H₁₉NO₃S. 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 C₇H₆O₄, 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 C₁₀H₈O₂. 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 C₈H₈N₂S (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 C₆H₇NO₄S, 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 C₉H₉N₃S. 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 C₁₂H₁₀O₂ 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 C₇H₅F₃N₂O₂, 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 C₂₂H₁₅NO₂. 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 C₂₈H₄₂ClN₃O₃. 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 C₁₁H₁₄O. 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 C₃₄H₅₃NaO₈. 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).

4.2 Effect the water and ethanol extracts on the GTT curves

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).