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

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Methods Chemical analysis of the extracts was performed using infrared (IR) spectroscopy alongside the spectral database of organic compounds. Results The water extract revealed a richer diversity of natural products (38 compounds) compared to the ethanol extract (12 compounds). Notably, the water extract comprised various bioactive molecules, including sugars and their derivatives, phenolic compounds, alkaloids, quinones, amino acid derivatives, dipeptides, and organometallic compounds. In contrast, the ethanol extract primarily contained sugar derivatives, phenolic compounds, alkaloids, and pesticides. The water extract decreased the blood glucose level and transformed the GTT curve from a convex to a concave shape. Conclusion The blood glucose-lowering effect of the water extract may be attributed to the anti-diabetic properties of its dipeptides, phenolic compounds and alkaloid contents. 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F1000Research 2025, 14 :872 ( https://doi.org/10.12688/f1000research.166622.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Research Article Glucose Tolerance Test (GTT) Curves of Water and Ethanol Extracts of Whole Body Apis mellifera jemenitica [version 1; peer review: 1 approved with reservations, 1 not approved] Raaih Marwae Ahmad Asseri 1 , Mohamed Adam Ali Ismail 1 , Hamed A Ghramh 2,3 , [...] Wed Mohammed Ali Alarjani 4 , Tarik El-Sayed Ali Ismail 5 , Mogbel A A El-Niweiri 2 , Mohammed Mohammed https://orcid.org/0000-0003-3909-3788 5 Raaih Marwae Ahmad Asseri 1 , Mohamed Adam Ali Ismail 1 , [...] Hamed A Ghramh 2,3 , Wed Mohammed Ali Alarjani 4 , Tarik El-Sayed Ali Ismail 5 , Mogbel A A El-Niweiri 2 , Mohammed Mohammed https://orcid.org/0000-0003-3909-3788 5 PUBLISHED 04 Sep 2025 Author details Author details 1 Department of Chemical Engineering, King Khalid University, Abha, Aseer Province, Saudi Arabia 2 Department of Biology, King Khalid University, Abha, Aseer Province, Saudi Arabia 3 Honeybees and their Products Research Center, King Khalid University, Abha, Aseer Province, Saudi Arabia 4 Department of Chemistry- Preparatory Year Program, Batterjee Medical College, Abha, Aseer Region, Saudi Arabia 5 Department of Chemistry, King Khalid University, Abha, Aseer Province, Saudi Arabia Raaih Marwae Ahmad Asseri Roles: Investigation, Methodology, Writing – Original Draft Preparation Mohamed Adam Ali Ismail Roles: Conceptualization, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing Hamed A Ghramh Roles: Funding Acquisition, Resources, Writing – Original Draft Preparation, Writing – Review & Editing Wed Mohammed Ali Alarjani Roles: Formal Analysis, Methodology, Resources, Writing – Original Draft Preparation, Writing – Review & Editing Tarik El-Sayed Ali Ismail Roles: Formal Analysis, Methodology, Resources, Validation, Writing – Review & Editing Mogbel A A El-Niweiri Roles: Conceptualization, Formal Analysis, Validation, Writing – Original Draft Preparation, Writing – Review & Editing Mohammed Mohammed Roles: Conceptualization, Formal Analysis, Funding Acquisition, Methodology, Project Administration, Resources, Software, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing OPEN PEER REVIEW DETAILS REVIEWER STATUS Abstract Background This article explored the chemical constituents of water and ethanol extracts from the whole body of Apis mellifera jemenitica honeybee drones and workers, investigating their effects on glucose tolerance test (GTT) curves through feeding experiments conducted on Oryctolagus cuniculus male rabbits. Methods Chemical analysis of the extracts was performed using infrared (IR) spectroscopy alongside the spectral database of organic compounds. Results The water extract revealed a richer diversity of natural products (38 compounds) compared to the ethanol extract (12 compounds). Notably, the water extract comprised various bioactive molecules, including sugars and their derivatives, phenolic compounds, alkaloids, quinones, amino acid derivatives, dipeptides, and organometallic compounds. In contrast, the ethanol extract primarily contained sugar derivatives, phenolic compounds, alkaloids, and pesticides. The water extract decreased the blood glucose level and transformed the GTT curve from a convex to a concave shape. Conclusion The blood glucose-lowering effect of the water extract may be attributed to the anti-diabetic properties of its dipeptides, phenolic compounds and alkaloid contents. READ ALL READ LESS Keywords Chemical composition of honeybees, biological activity, IR spectrometry, spectral database of organic compounds., Biochemistry and chemical ecology, Hive product science Corresponding Author(s) Mohammed Mohammed ( [email protected] ) Close Corresponding author: Mohammed Mohammed Competing interests: No competing interests were disclosed. Grant information: The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University for funding this work through Big groups project under grant number (66/46). Moreover, this research was logistically supported by the Saudi Aramco. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2025 Asseri RMA et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Asseri RMA, Ismail MAA, Ghramh HA et al. Glucose Tolerance Test (GTT) Curves of Water and Ethanol Extracts of Whole Body Apis mellifera jemenitica [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :872 ( https://doi.org/10.12688/f1000research.166622.1 ) First published: 04 Sep 2025, 14 :872 ( https://doi.org/10.12688/f1000research.166622.1 ) Latest published: 10 Jan 2026, 14 :872 ( https://doi.org/10.12688/f1000research.166622.2 )  There is a newer version of this article available. Suppress this message for one day. 1. Introduction Entomotherapy refers to the use of insects and their products for the treatment of various diseases. Insects are known to possess bodies and products that are rich in bioactive compounds, enabling them to act as antimicrobial, antioxidant, anticancer, and immunomodulatory agents ( Guarnieri et al., 2022 ; Khayrova et al., 2022 ; Seabrooks & Hu, 2017 ). This practice is prevalent in several cultures, notably in countries such as China, India, Thailand, and parts of Africa. Traditionally, insects have been used to treat a variety of ailments, including kidney diseases, digestive tract disorders, asthma, chronic cough, liver issues, rheumatoid conditions, and tooth pain ( Siddiqui et al., 2023 ). Honeybee larvae and pupae have been reported to be effective in treating skin wounds, gastrointestinal issues, and mental health distress ( Meda et al., 2004 ). According to Choi (2021) , extracts from the pupae of Apis mellifera L. (ligustica) drones demonstrated a range of biological activities, including antimicrobial, antioxidant, anti-inflammatory, anti-diabetic, anti-obesity benefits, skin whitening effects, prevention of hair loss, and an increase in blood testosterone levels ( Choi, 2021 ). Both the brood (larvae and pupae) and adult honeybees are known to contain various bioactive and nutritious compounds, such as proteins, essential amino acids, saturated and monounsaturated fatty acids, vitamins, minerals, and antioxidants. The nutritional composition of Apis mellifera larvae and pupae is influenced by factors including their diet, health, age, species, climatic conditions, and seasonal variations ( Guiné et al., 2022 ; Ghosh et al., 2016 ). However, it’s also important to note that the whole body of Apis mellifera can contain environmental contaminants like pesticides, heavy metals, and veterinary drugs, which could impact their safety and efficacy ( Traynor et al., 2021 ; Savarino et al., 2020 ; Borkovcová et al., 2022 ). The Glucose Tolerance Test (GTT) is a diagnostic procedure designed to assess a patient’s ability to metabolize glucose effectively. It plays a crucial role in identifying conditions such as diabetes mellitus, insulin resistance, acromegaly (reactive hypoglycemia), and disturbances in carbohydrate metabolism, as well as evaluating pancreatic beta-cell function ( Eyth et al., 2024 ). The Oral Glucose Tolerance Test (OGTT) consists of several key steps: 1) Fasting Preparation: The patient must arrive at the laboratory in a fasting state, ideally in the morning; 2) Initial Blood Sample: A baseline blood sample is collected to measure the fasting blood glucose level; 3) Glucose Administration: The patient is given an oral glucose load, typically 70 grams dissolved in 250 mL of water or 1.75 grams per kilogram of body weight for children; and 4) Subsequent Blood Sampling: Blood samples are taken every 30 minutes for a duration of two to three hours to monitor changes in blood glucose levels. It is essential to collect blood samples in fluoride-containing tubes to prevent a decrease in glucose concentration. The fluoride acts as an inhibitor of glycolysis by blocking aldolase, thereby preserving the glucose levels for accurate measurement ( Eyth et al., 2024 ). This article aimed to: 1) investigate the chemical constituents of water and ethanol extracts of adult Apis mellifera jemenitica honeybees to assess their potential medicinal values; 2) explore the effects of these extracts on the glucose tolerance test (GTT) curves and blood glucose levels using Oryctolagus cuniculus rabbits as experimental subjects; and 3) identify which specific chemical constituents of the extracts may account for their effects on the GTT curve. To our knowledge, this study is unique as it is the first to examine the bioactive compounds, in addition to nutritive biomolecules, in the whole bodies of Apis mellifera jemenitica workers and drones. 2. Material and methods 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. Results To enhance the clarity of this article, the results section is divided into three parts: IR Spectral Analysis, Compound Identification, and GTT Results, highlighting the bioactivity of the water and ethanol extracts. 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) C 6 H 12 O 5 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) C 6 H 12 O 5 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 C 8 H 11 Cl 3 O 6 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 C 17 H 25 NO 10 S 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 C 15 H 19 NO 3 S It is used for chemical synthesis ( Chemical book, 2023 ). Water Pubchem: NA SDBS:18269 2,4- dihydroxybenzoic acid C 7 H 6 O 4 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 C 10 H 8 O 2 Antioxidant, antiplatelet aggregation, anti-inflammatory, antimicrobial and anti-protozoa ( Ibrahim and Mohamed, 2016 ). Water Pubchem: 7091 SDBS:1626 6-methyl-2-benzothiazolamine C 8 H 8 N 2 S 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 C 6 H 7 NO 4 S 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 C 9 H 9 N 3 S 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 C 12 H 10 O 2 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 C 7 H 5 F 3 N 2 O 2 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 C 12 H 12 N 2 S HBr NA Anti-angiogenesis ( Bhat et al., 2013 ). Water Pubchem: NA SDBS:26013 O-(N-(dimethylcarbamoylmethyl)acetamido)-N-N-dimethylbenzamide C 15 H 21 N 3 O 3 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 C 22 H 15 NO 2 Anti-tumor, antimicrobial, antioxidant and anti-inflammatory ( Miao et al., 2019 ). Water Pubchem: 275779763 SDBS:30670 2-ethylthio (thiocarbonyl)amino-phenylpropionic acid cyclohexylamine salt C 12 H 15 NO 2 S 2 C 6 H 13 N 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 C 28 H 41 N 3 O 3 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 C 11 H 14 O 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 C 15 H 26 O 2 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 C 12 H 14 CINO 4 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 C 34 H 53 NaO 8 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 C 14 H 16 Cl 2 N 2 O 3 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 C 20 H 12 O 5 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 C 10 H 16 N 2 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 C 10 H 10 N 4 O 2 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) C 12 H 10 ClN 3 O 2 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 C 7 H 10 N 4 O 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 C 19 H 16 N 4 O 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 C 28 H 33 NO 7 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 C 10 H 20 N 2 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 C 18 H 14 N 2 O 2 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 C 14 H 8 BrNO 3 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 C 12 H 18 N 2 O 4 Antimalarial, anti-Alzheimer, antiviral, antifungal, antibacterial and antitumor ( Zhao and Zheng, 2023 ). Water Pubchem: 620240 SDBS:25209 2,5,6-trihydroxy-1,4-naphthoquinone C 10 H 6 O 5 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 C 12 H 16 N 2 O 3 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 C 14 H 20 N 2 O 2 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 C 5 H 11 N 3 O 3 H 2 O 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 C 8 H 11 NO 3 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 C 19 H 22 N 6 O 4 HCl Enhance proteolytic activity of plasmin and trypsin ( Christensen and, Müllertz, 1974 ; Dulay et al., 2005 ). Water Pubchem: NA SDBS:12532 1-nitroguanidine CH 4 N 4 O 2 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 C 2 H 17 Cl 3 CoN 5 O 10 NA Used in synthesis of explosive material ( Ilyushin et al., 2010 ). Water Pubchem: NA SDBS:35464 Potassium diaquabis (malonato) manganite (III) C 6 H 8 KMnO 10 2H 2 O NA synthetic Inorganic compound with magnetic activity ( Delgado et al., 2006 ). Water Pubchem: NA SDBS:26382 alpha-chloralose C 8 H 11 Cl 3 O 6 Pesticide, anesthetic, hypnotics and sedatives ( National Center for Biotechnology Information, 2024 l ). 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 . Table 8. Blood glucose concentration (mg/dL) of the GTT experiments. Time of blood sample GTT Mean SD Minimum Maximum Fasting Control 97.50*$ 17.68 85.00 110.00 Glucose+ water extract 136.50$@ 31.82 114.00 159.00 Glucose + ethanol extract 97.00$ 18.38 84.00 110.00 After 90 minutes Control 114.50#$ 21.92 99.00 130.00 Glucose+ water extract 120.50$ 9.19 114.00 127.00 Glucose+ ethanol extract 90.00$ 4.24 87.00 93.00 After 120 minutes Control 173.00*#$&@ 29.70 152.00 194.00 Glucose+ water extract 123.50$ .71 123.00 124.00 Glucose + ethanol extract 82.50$ 4.95 79.00 86.00 After 150 minutes Control 100.50$& 16.26 89.00 112.00 Glucose+ water extract 115.50$ 14.85 105.00 126.00 Glucose + ethanol extract 124.50 27.58 105.00 144.00 After 180 minutes Control 87.50$@ 10.61 80.00 95.00 Glucose+ water extract 236.00*#&@ 43.84 205.00 267.00 Glucose + ethanol extract 192.00*#&@ 7.07 187.00 197.00 4. Discussion This study can be seen as the first to report the presence of bioactive natural products such as polyphenols, alkaloids, and quinones in the whole body constituents of Apis mellifera jemenitica drones and workers. Previously published articles have primarily focused on the nutritional value of honeybees and their larvae and pupae, emphasizing their content of amino acids, fatty acids, proteins, minerals, and vitamins ( Guiné et al., 2022 ; Ghosh et al., 2016 ). 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 C 6 H 12 O 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 15 H 19 NO 3 S. 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 7 H 6 O 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 10 H 8 O 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 8 H 8 N 2 S ( 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 6 H 7 NO 4 S, 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 9 H 9 N 3 S. 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 12 H 10 O 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 7 H 5 F 3 N 2 O 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 22 H 15 NO 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 28 H 42 ClN 3 O 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 11 H 14 O. 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 34 H 53 NaO 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 ). 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 ). 5. Limitations This study has certain limitations, including the small sample size of experimental rabbits and a somewhat un ideal discussion section (mostly presented as a review) due to the scarcity of published literature on the chemical composition of honeybee bodies 6. Conclusions The water extract of the whole body of Apis mellifera drones and workers is found to be abundant in various compounds, including sugar derivatives, phenolic acids, alkaloids, quinones, amino acid derivatives, and short peptides. When administered, this extract notably altered the glucose tolerance test (GTT) curve from a convex shape to a concave shape, indicating its potential anti-hyperglycemic properties. The observed anti-hyperglycemic effect is likely attributed to the presence of dipeptides, phenolic acids and alkaloids within the water extract. Data availability statement [Figure Share]. [Glucose Tolerance Test (GTT) Curves of Water and Ethanol Extracts of Whole Body Apis mellifera jemenitica]. [ https://doi.org/10.6084/m9.figshare.29339888 ] ( Mohammed et al., 2025 ). This project contains the following underlying data: • Supplement.1. (Shows the searching the SDBS database). • Supplement.2. (Shows the SDBS search results displaying the 39 compounds detected in the water extract of Apis mellifera jementica). • Supplement.3. (Presents the SDBS search results showing the 12 compounds identified in the ethanol extract of Apis mellifera jementica). • Supplement.4. 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PubMed Abstract | Publisher Full Text Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 04 Sep 2025 ADD YOUR COMMENT Comment Author details Author details 1 Department of Chemical Engineering, King Khalid University, Abha, Aseer Province, Saudi Arabia 2 Department of Biology, King Khalid University, Abha, Aseer Province, Saudi Arabia 3 Honeybees and their Products Research Center, King Khalid University, Abha, Aseer Province, Saudi Arabia 4 Department of Chemistry- Preparatory Year Program, Batterjee Medical College, Abha, Aseer Region, Saudi Arabia 5 Department of Chemistry, King Khalid University, Abha, Aseer Province, Saudi Arabia Raaih Marwae Ahmad Asseri Roles: Investigation, Methodology, Writing – Original Draft Preparation Mohamed Adam Ali Ismail Roles: Conceptualization, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing Hamed A Ghramh Roles: Funding Acquisition, Resources, Writing – Original Draft Preparation, Writing – Review & Editing Wed Mohammed Ali Alarjani Roles: Formal Analysis, Methodology, Resources, Writing – Original Draft Preparation, Writing – Review & Editing Tarik El-Sayed Ali Ismail Roles: Formal Analysis, Methodology, Resources, Validation, Writing – Review & Editing Mogbel A A El-Niweiri Roles: Conceptualization, Formal Analysis, Validation, Writing – Original Draft Preparation, Writing – Review & Editing Mohammed Mohammed Roles: Conceptualization, Formal Analysis, Funding Acquisition, Methodology, Project Administration, Resources, Software, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing Competing interests No competing interests were disclosed. Grant information The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University for funding this work through Big groups project under grant number (66/46). Moreover, this research was logistically supported by the Saudi Aramco. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (2) version 2 Revised Published: 10 Jan 2026, 14:872 https://doi.org/10.12688/f1000research.166622.2 version 1 Published: 04 Sep 2025, 14:872 https://doi.org/10.12688/f1000research.166622.1 Copyright © 2025 Asseri RMA et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Asseri RMA, Ismail MAA, Ghramh HA et al. Glucose Tolerance Test (GTT) Curves of Water and Ethanol Extracts of Whole Body Apis mellifera jemenitica [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :872 ( https://doi.org/10.12688/f1000research.166622.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 1 VERSION 1 PUBLISHED 04 Sep 2025 Views 0 Cite How to cite this report: karlıdağ s. Reviewer Report For: Glucose Tolerance Test (GTT) Curves of Water and Ethanol Extracts of Whole Body Apis mellifera jemenitica [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :872 ( https://doi.org/10.5256/f1000research.183639.r422288 ) The direct URL for this report is: https://f1000research.com/articles/14-872/v1#referee-response-422288 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 10 Dec 2025 semiramis karlıdağ , University of Malatya Turgut Ozal, Malatya, Turkey Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.183639.r422288 In this article, water and ethanol extracts were obtained from the bodies of Apis mellifera jemenitica honeybee drones and workers. The chemical composition of the extracts was investigated. The extracts were also fed to Oryctolagus cuniculus rabbits and their effects ... Continue reading READ ALL In this article, water and ethanol extracts were obtained from the bodies of Apis mellifera jemenitica honeybee drones and workers. The chemical composition of the extracts was investigated. The extracts were also fed to Oryctolagus cuniculus rabbits and their effects on glucose tolerance test (GTT) curves were investigated. This is an original and valuable study that will lay the groundwork for the literature. However, as the authors note, the discussion is weak due to the limited material and the scarcity of published literature on the chemical composition of honeybee bodies. I believe it is a valuable study because it paves the way for future studies of this type. The method section of the article lacks information on how bee samples were collected and when the analyses were conducted. These missing points are noted in the text. Note: My suggestions and corrections are given above the text. It would be more appropriate to use "Chemical composition of honeybee extracts" instead of "Chemical composition of honeybees" in the keywords. I made my corrections and suggestions to the article. please find the attached file via following link : https://f1000research-files.f1000.com/linked/762273.my_corrections_and_suggestions_to_the_article.docx Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: The article can be published by making some changes I suggested. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT karlıdağ s. Reviewer Report For: Glucose Tolerance Test (GTT) Curves of Water and Ethanol Extracts of Whole Body Apis mellifera jemenitica [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :872 ( https://doi.org/10.5256/f1000research.183639.r422288 ) The direct URL for this report is: https://f1000research.com/articles/14-872/v1#referee-response-422288 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: El-Wahed AA. Reviewer Report For: Glucose Tolerance Test (GTT) Curves of Water and Ethanol Extracts of Whole Body Apis mellifera jemenitica [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :872 ( https://doi.org/10.5256/f1000research.183639.r425586 ) The direct URL for this report is: https://f1000research.com/articles/14-872/v1#referee-response-425586 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 03 Nov 2025 Aida Abd El-Wahed , Plant Protection Research Institute, Agricultural Research Centre, Giza, Egypt Not Approved VIEWS 0 https://doi.org/10.5256/f1000research.183639.r425586 Introduction: - The section beginning with “The Oral Glucose Tolerance Test (OGTT) consists of several key steps...” (Eyth et al., 2024) should be omitted; it is unnecessary. The authors should instead discuss the currently used drugs and justify the ... Continue reading READ ALL Introduction: - The section beginning with “The Oral Glucose Tolerance Test (OGTT) consists of several key steps...” (Eyth et al., 2024) should be omitted; it is unnecessary. The authors should instead discuss the currently used drugs and justify the shift toward exploring natural sources. Materials and Methods: The sample size is very small—only four rabbits were used, which were then divided into two groups (n=2). This raises serious concerns about statistical validity. The age of the adult workers and drones used should also be specified. The section describing “The study sample consisted of adult workers and drones …… similar to using dry ice for killing honeybees (Mutinelli, 2021).” is overly detailed and unnecessary. The authors should instead provide a clear and detailed description of the extraction method. The doses derived from adult workers and drones are not mentioned. Furthermore, while the authors state there is limited literature on the chemical composition of honeybee bodies, several studies have already addressed this, such as: (Ghosh, et al., 2016) - ref 1 (Abedelmaksoud, et al., 2024) - ref 2 Analytical Methods: Relying solely on IR spectroscopy for compound identification is insufficient. The study should incorporate additional analytical tools such as mass spectrometry (MS) and nuclear magnetic resonance (NMR) to ensure accurate compound identification. Although a list of various compounds is presented, the authors fail to identify which compounds are responsible for the reported biological activity. The authors present list of compounds with diverse of compounds but they didn’t mentionwhich responsible of the activity Results and discussions: A more comprehensive discussion of the results is needed. The authors should also include clear recommendations and practical implications of the findings. Conclusion Please rewrite the conclusion and include suggestions for future work. The study’s recommendations should be included. Figures: All figures should be revised to achieve higher resolution and publication-quality standards. The graphic abstract recommended. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? No Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required. Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? No References 1. Ghosh S, Jung C, Meyer-Rochow V: Nutritional value and chemical composition of larvae, pupae, and adults of worker honey bee, Apis mellifera ligustica as a sustainable food source. Journal of Asia-Pacific Entomology . 2016; 19 (2): 487-495 Publisher Full Text 2. Abedelmaksoud T, El‐Masarawy M, Altemimi A, Hesarinejad M, et al.: Drones and Workers of HoneybeeApis mellifera L. Dried Powder: Chemical Composition, Antioxidant, and Anticancer Assessment. Food Science & Nutrition . 2024; 12 (12): 10357-10369 Publisher Full Text Competing Interests: No competing interests were disclosed. Reviewer Expertise: Natural Products Chemistry, Isolation, purification, synthesis, structure elucidation of bio-active compounds, Bee products, honeybees I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT El-Wahed AA. Reviewer Report For: Glucose Tolerance Test (GTT) Curves of Water and Ethanol Extracts of Whole Body Apis mellifera jemenitica [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :872 ( https://doi.org/10.5256/f1000research.183639.r425586 ) The direct URL for this report is: https://f1000research.com/articles/14-872/v1#referee-response-425586 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 31 Dec 2025 Mohammed Elimam Ahamed Mohammed , Department of Chemistry, King Khalid University, Abha, Saudi Arabia 31 Dec 2025 Author Response Introduction: Comment: - The section beginning with “The Oral Glucose Tolerance Test (OGTT) consists of several key steps...” (Eyth et al., 2024) should be omitted; it is unnecessary. The authors should instead ... Continue reading Introduction: Comment: - The section beginning with “The Oral Glucose Tolerance Test (OGTT) consists of several key steps...” (Eyth et al., 2024) should be omitted; it is unnecessary. The authors should instead discuss the currently used drugs and justify the shift toward exploring natural sources. Response: Thank you very much for your careful review. As the manuscript focuses specifically on the Glucose Tolerance Test (GTT) and related compounds, detailing the GTT procedure is essential for ensuring the accuracy and reliability of the results. Omitting any step would invalidate the findings. I have addressed your point by changing "patient" to "study subject" to better reflect the research context. Thank you again for your valuable feedback. Materials and Methods: Comment: The sample size is very small—only four rabbits were used, which were then divided into two groups (n=2). This raises serious concerns about statistical validity. Response: We acknowledge that the sample size of experimental animals is limited. This investigation is designed as a pilot study. The decision to use a smaller cohort was guided by ethical considerations, as the potential for serious, life-threatening toxicity from whole-body honeybee extracts in rabbits was previously unknown. Furthermore, this approach aligns with established ethical frameworks for animal research. Several published articles endorse using minimal animal numbers in preliminary investigations, with some citing 3-5 subjects as an acceptable range for pilot studies, and others noting that certain pilot experiments have been conducted with a single animal. Please see the reference below for further context: Michael F. W. Festing and Douglas G. Altman. Guidelines for the Design and Statistical Analysis of Experiments Using Laboratory Animals. ILAR Journal. 2002; 43(4): 244- 258. Comment: The age of the adult workers and drones used should also be specified. Response: Done Comment: The section describing “The study sample consisted of adult workers and drones …… similar to using dry ice for killing honeybees (Mutinelli, 2021).” is overly detailed and unnecessary. The authors should instead provide a clear and detailed description of the extraction method. Response: The extraction method was very simple as mentioned and nothing more. Comment: The doses derived from adult workers and drones are not mentioned. Response: Many thanks. The dose is added to GTT procedure. Comment: Furthermore, while the authors state there is limited literature on the chemical composition of honeybee bodies, several studies have already addressed this, such as: Response: Thank you very much for your suggestion, however, the published articles are concentrating on the nutritional value alone without other natural products such as phenolic compunds and alkaloids. Beside they did not mention the contaminants such as the pesticides. (Ghosh, et al., 2016) - ref 1, This study is already cited. (Abedelmaksoud, et al., 2024) - ref 2 This study is cited in the introduction and the importance of the study is rephrase accordingly. Thanks the reference is of high importance. Analytical Methods: Comment: Relying solely on IR spectroscopy for compound identification is insufficient. The study should incorporate additional analytical tools such as mass spectrometry (MS) and nuclear magnetic resonance (NMR) to ensure accurate compound identification. Response: While mass spectrometry (MS) and NMR are powerful tools, especially when paired with databases or analysis software, our methodology aligns with standard practices for characterizing complex biological samples. A key aspect of our work was utilizing the new IR spectra database from Japan. Additionally, we are pioneering an approach to use IR spectral peaks much like precursor ions are used to query established metabolomics (e.g., MassBank) and protein databases. Comment: Although a list of various compounds is presented, the authors fail to identify which compounds are responsible for the reported biological activity. The authors present list of compounds with diverse of compounds but they didn’t mentionwhich responsible of the activity. Response: Thanks a lot. In the discussion and conclusion sections we have stated some compounds as possibly responsible for the change of the GTT curve. Results and discussions: Comment: A more comprehensive discussion of the results is needed. Response: This was stated as a limitation because the available literature is limited, with existing research predominantly concentrating on nutritional value. Comment: The authors should also include clear recommendations and practical implications of the findings. Response: Statement is added to the conclusion section to meet your comment. Conclusion Comment: Please rewrite the conclusion and include suggestions for future work. The study’s recommendations should be included. Response: Done. Figures: Comment: All figures should be revised to achieve higher resolution and publication-quality standards. Response: Done according to the journal requirements Comment: The graphic abstract recommended. Response: A graphical abstract is created. Introduction: Comment: - The section beginning with “The Oral Glucose Tolerance Test (OGTT) consists of several key steps...” (Eyth et al., 2024) should be omitted; it is unnecessary. The authors should instead discuss the currently used drugs and justify the shift toward exploring natural sources. Response: Thank you very much for your careful review. As the manuscript focuses specifically on the Glucose Tolerance Test (GTT) and related compounds, detailing the GTT procedure is essential for ensuring the accuracy and reliability of the results. Omitting any step would invalidate the findings. I have addressed your point by changing "patient" to "study subject" to better reflect the research context. Thank you again for your valuable feedback. Materials and Methods: Comment: The sample size is very small—only four rabbits were used, which were then divided into two groups (n=2). This raises serious concerns about statistical validity. Response: We acknowledge that the sample size of experimental animals is limited. This investigation is designed as a pilot study. The decision to use a smaller cohort was guided by ethical considerations, as the potential for serious, life-threatening toxicity from whole-body honeybee extracts in rabbits was previously unknown. Furthermore, this approach aligns with established ethical frameworks for animal research. Several published articles endorse using minimal animal numbers in preliminary investigations, with some citing 3-5 subjects as an acceptable range for pilot studies, and others noting that certain pilot experiments have been conducted with a single animal. Please see the reference below for further context: Michael F. W. Festing and Douglas G. Altman. Guidelines for the Design and Statistical Analysis of Experiments Using Laboratory Animals. ILAR Journal. 2002; 43(4): 244- 258. Comment: The age of the adult workers and drones used should also be specified. Response: Done Comment: The section describing “The study sample consisted of adult workers and drones …… similar to using dry ice for killing honeybees (Mutinelli, 2021).” is overly detailed and unnecessary. The authors should instead provide a clear and detailed description of the extraction method. Response: The extraction method was very simple as mentioned and nothing more. Comment: The doses derived from adult workers and drones are not mentioned. Response: Many thanks. The dose is added to GTT procedure. Comment: Furthermore, while the authors state there is limited literature on the chemical composition of honeybee bodies, several studies have already addressed this, such as: Response: Thank you very much for your suggestion, however, the published articles are concentrating on the nutritional value alone without other natural products such as phenolic compunds and alkaloids. Beside they did not mention the contaminants such as the pesticides. (Ghosh, et al., 2016) - ref 1, This study is already cited. (Abedelmaksoud, et al., 2024) - ref 2 This study is cited in the introduction and the importance of the study is rephrase accordingly. Thanks the reference is of high importance. Analytical Methods: Comment: Relying solely on IR spectroscopy for compound identification is insufficient. The study should incorporate additional analytical tools such as mass spectrometry (MS) and nuclear magnetic resonance (NMR) to ensure accurate compound identification. Response: While mass spectrometry (MS) and NMR are powerful tools, especially when paired with databases or analysis software, our methodology aligns with standard practices for characterizing complex biological samples. A key aspect of our work was utilizing the new IR spectra database from Japan. Additionally, we are pioneering an approach to use IR spectral peaks much like precursor ions are used to query established metabolomics (e.g., MassBank) and protein databases. Comment: Although a list of various compounds is presented, the authors fail to identify which compounds are responsible for the reported biological activity. The authors present list of compounds with diverse of compounds but they didn’t mentionwhich responsible of the activity. Response: Thanks a lot. In the discussion and conclusion sections we have stated some compounds as possibly responsible for the change of the GTT curve. Results and discussions: Comment: A more comprehensive discussion of the results is needed. Response: This was stated as a limitation because the available literature is limited, with existing research predominantly concentrating on nutritional value. Comment: The authors should also include clear recommendations and practical implications of the findings. Response: Statement is added to the conclusion section to meet your comment. Conclusion Comment: Please rewrite the conclusion and include suggestions for future work. The study’s recommendations should be included. Response: Done. Figures: Comment: All figures should be revised to achieve higher resolution and publication-quality standards. Response: Done according to the journal requirements Comment: The graphic abstract recommended. Response: A graphical abstract is created. Competing Interests: NA Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 31 Dec 2025 Mohammed Elimam Ahamed Mohammed , Department of Chemistry, King Khalid University, Abha, Saudi Arabia 31 Dec 2025 Author Response Introduction: Comment: - The section beginning with “The Oral Glucose Tolerance Test (OGTT) consists of several key steps...” (Eyth et al., 2024) should be omitted; it is unnecessary. The authors should instead ... Continue reading Introduction: Comment: - The section beginning with “The Oral Glucose Tolerance Test (OGTT) consists of several key steps...” (Eyth et al., 2024) should be omitted; it is unnecessary. The authors should instead discuss the currently used drugs and justify the shift toward exploring natural sources. Response: Thank you very much for your careful review. As the manuscript focuses specifically on the Glucose Tolerance Test (GTT) and related compounds, detailing the GTT procedure is essential for ensuring the accuracy and reliability of the results. Omitting any step would invalidate the findings. I have addressed your point by changing "patient" to "study subject" to better reflect the research context. Thank you again for your valuable feedback. Materials and Methods: Comment: The sample size is very small—only four rabbits were used, which were then divided into two groups (n=2). This raises serious concerns about statistical validity. Response: We acknowledge that the sample size of experimental animals is limited. This investigation is designed as a pilot study. The decision to use a smaller cohort was guided by ethical considerations, as the potential for serious, life-threatening toxicity from whole-body honeybee extracts in rabbits was previously unknown. Furthermore, this approach aligns with established ethical frameworks for animal research. Several published articles endorse using minimal animal numbers in preliminary investigations, with some citing 3-5 subjects as an acceptable range for pilot studies, and others noting that certain pilot experiments have been conducted with a single animal. Please see the reference below for further context: Michael F. W. Festing and Douglas G. Altman. Guidelines for the Design and Statistical Analysis of Experiments Using Laboratory Animals. ILAR Journal. 2002; 43(4): 244- 258. Comment: The age of the adult workers and drones used should also be specified. Response: Done Comment: The section describing “The study sample consisted of adult workers and drones …… similar to using dry ice for killing honeybees (Mutinelli, 2021).” is overly detailed and unnecessary. The authors should instead provide a clear and detailed description of the extraction method. Response: The extraction method was very simple as mentioned and nothing more. Comment: The doses derived from adult workers and drones are not mentioned. Response: Many thanks. The dose is added to GTT procedure. Comment: Furthermore, while the authors state there is limited literature on the chemical composition of honeybee bodies, several studies have already addressed this, such as: Response: Thank you very much for your suggestion, however, the published articles are concentrating on the nutritional value alone without other natural products such as phenolic compunds and alkaloids. Beside they did not mention the contaminants such as the pesticides. (Ghosh, et al., 2016) - ref 1, This study is already cited. (Abedelmaksoud, et al., 2024) - ref 2 This study is cited in the introduction and the importance of the study is rephrase accordingly. Thanks the reference is of high importance. Analytical Methods: Comment: Relying solely on IR spectroscopy for compound identification is insufficient. The study should incorporate additional analytical tools such as mass spectrometry (MS) and nuclear magnetic resonance (NMR) to ensure accurate compound identification. Response: While mass spectrometry (MS) and NMR are powerful tools, especially when paired with databases or analysis software, our methodology aligns with standard practices for characterizing complex biological samples. A key aspect of our work was utilizing the new IR spectra database from Japan. Additionally, we are pioneering an approach to use IR spectral peaks much like precursor ions are used to query established metabolomics (e.g., MassBank) and protein databases. Comment: Although a list of various compounds is presented, the authors fail to identify which compounds are responsible for the reported biological activity. The authors present list of compounds with diverse of compounds but they didn’t mentionwhich responsible of the activity. Response: Thanks a lot. In the discussion and conclusion sections we have stated some compounds as possibly responsible for the change of the GTT curve. Results and discussions: Comment: A more comprehensive discussion of the results is needed. Response: This was stated as a limitation because the available literature is limited, with existing research predominantly concentrating on nutritional value. Comment: The authors should also include clear recommendations and practical implications of the findings. Response: Statement is added to the conclusion section to meet your comment. Conclusion Comment: Please rewrite the conclusion and include suggestions for future work. The study’s recommendations should be included. Response: Done. Figures: Comment: All figures should be revised to achieve higher resolution and publication-quality standards. Response: Done according to the journal requirements Comment: The graphic abstract recommended. Response: A graphical abstract is created. Introduction: Comment: - The section beginning with “The Oral Glucose Tolerance Test (OGTT) consists of several key steps...” (Eyth et al., 2024) should be omitted; it is unnecessary. The authors should instead discuss the currently used drugs and justify the shift toward exploring natural sources. Response: Thank you very much for your careful review. As the manuscript focuses specifically on the Glucose Tolerance Test (GTT) and related compounds, detailing the GTT procedure is essential for ensuring the accuracy and reliability of the results. Omitting any step would invalidate the findings. I have addressed your point by changing "patient" to "study subject" to better reflect the research context. Thank you again for your valuable feedback. Materials and Methods: Comment: The sample size is very small—only four rabbits were used, which were then divided into two groups (n=2). This raises serious concerns about statistical validity. Response: We acknowledge that the sample size of experimental animals is limited. This investigation is designed as a pilot study. The decision to use a smaller cohort was guided by ethical considerations, as the potential for serious, life-threatening toxicity from whole-body honeybee extracts in rabbits was previously unknown. Furthermore, this approach aligns with established ethical frameworks for animal research. Several published articles endorse using minimal animal numbers in preliminary investigations, with some citing 3-5 subjects as an acceptable range for pilot studies, and others noting that certain pilot experiments have been conducted with a single animal. Please see the reference below for further context: Michael F. W. Festing and Douglas G. Altman. Guidelines for the Design and Statistical Analysis of Experiments Using Laboratory Animals. ILAR Journal. 2002; 43(4): 244- 258. Comment: The age of the adult workers and drones used should also be specified. Response: Done Comment: The section describing “The study sample consisted of adult workers and drones …… similar to using dry ice for killing honeybees (Mutinelli, 2021).” is overly detailed and unnecessary. The authors should instead provide a clear and detailed description of the extraction method. Response: The extraction method was very simple as mentioned and nothing more. Comment: The doses derived from adult workers and drones are not mentioned. Response: Many thanks. The dose is added to GTT procedure. Comment: Furthermore, while the authors state there is limited literature on the chemical composition of honeybee bodies, several studies have already addressed this, such as: Response: Thank you very much for your suggestion, however, the published articles are concentrating on the nutritional value alone without other natural products such as phenolic compunds and alkaloids. Beside they did not mention the contaminants such as the pesticides. (Ghosh, et al., 2016) - ref 1, This study is already cited. (Abedelmaksoud, et al., 2024) - ref 2 This study is cited in the introduction and the importance of the study is rephrase accordingly. Thanks the reference is of high importance. Analytical Methods: Comment: Relying solely on IR spectroscopy for compound identification is insufficient. The study should incorporate additional analytical tools such as mass spectrometry (MS) and nuclear magnetic resonance (NMR) to ensure accurate compound identification. Response: While mass spectrometry (MS) and NMR are powerful tools, especially when paired with databases or analysis software, our methodology aligns with standard practices for characterizing complex biological samples. A key aspect of our work was utilizing the new IR spectra database from Japan. Additionally, we are pioneering an approach to use IR spectral peaks much like precursor ions are used to query established metabolomics (e.g., MassBank) and protein databases. Comment: Although a list of various compounds is presented, the authors fail to identify which compounds are responsible for the reported biological activity. The authors present list of compounds with diverse of compounds but they didn’t mentionwhich responsible of the activity. Response: Thanks a lot. In the discussion and conclusion sections we have stated some compounds as possibly responsible for the change of the GTT curve. Results and discussions: Comment: A more comprehensive discussion of the results is needed. Response: This was stated as a limitation because the available literature is limited, with existing research predominantly concentrating on nutritional value. Comment: The authors should also include clear recommendations and practical implications of the findings. Response: Statement is added to the conclusion section to meet your comment. Conclusion Comment: Please rewrite the conclusion and include suggestions for future work. The study’s recommendations should be included. Response: Done. Figures: Comment: All figures should be revised to achieve higher resolution and publication-quality standards. Response: Done according to the journal requirements Comment: The graphic abstract recommended. Response: A graphical abstract is created. Competing Interests: NA Close Report a concern COMMENT ON THIS REPORT Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 04 Sep 2025 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 Version 2 (revision) 10 Jan 26 read read Version 1 04 Sep 25 read read Aida Abd El-Wahed , Plant Protection Research Institute, Agricultural Research Centre, Giza, Egypt semiramis karlıdağ , University of Malatya Turgut Ozal, Malatya, Turkey Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2026 karlıdağ s. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 22 Jan 2026 | for Version 2 semiramis karlıdağ , University of Malatya Turgut Ozal, Malatya, Turkey 0 Views copyright © 2026 karlıdağ s. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The authors have satisfactorily addressed all of the comments and suggestions raised during the review process. The revisions have improved the clarity and scientific quality of the manuscript. In its current form, the manuscript is suitable for indexing. Competing Interests No competing interests were disclosed. Reviewer Expertise The article can be published by making some changes I suggested. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) karlıdağ s. Peer Review Report For: Glucose Tolerance Test (GTT) Curves of Water and Ethanol Extracts of Whole Body Apis mellifera jemenitica [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :872 ( https://doi.org/10.5256/f1000research.193925.r449132) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-872/v2#referee-response-449132 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2026 El-Wahed A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 13 Jan 2026 | for Version 2 Aida Abd El-Wahed , Plant Protection Research Institute, Agricultural Research Centre, Giza, Egypt 0 Views copyright © 2026 El-Wahed A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The manuscript recommends acceptance in the current form Competing Interests No competing interests were disclosed. Reviewer Expertise Natural Products Chemistry, Isolation, purification, synthesis, structure elucidation of bio-active compounds, Bee products, honeybees I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) El-Wahed AA. Peer Review Report For: Glucose Tolerance Test (GTT) Curves of Water and Ethanol Extracts of Whole Body Apis mellifera jemenitica [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :872 ( https://doi.org/10.5256/f1000research.193925.r449133) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-872/v2#referee-response-449133 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 karlıdağ s. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 10 Dec 2025 | for Version 1 semiramis karlıdağ , University of Malatya Turgut Ozal, Malatya, Turkey 0 Views copyright © 2025 karlıdağ s. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions In this article, water and ethanol extracts were obtained from the bodies of Apis mellifera jemenitica honeybee drones and workers. The chemical composition of the extracts was investigated. The extracts were also fed to Oryctolagus cuniculus rabbits and their effects on glucose tolerance test (GTT) curves were investigated. This is an original and valuable study that will lay the groundwork for the literature. However, as the authors note, the discussion is weak due to the limited material and the scarcity of published literature on the chemical composition of honeybee bodies. I believe it is a valuable study because it paves the way for future studies of this type. The method section of the article lacks information on how bee samples were collected and when the analyses were conducted. These missing points are noted in the text. Note: My suggestions and corrections are given above the text. It would be more appropriate to use "Chemical composition of honeybee extracts" instead of "Chemical composition of honeybees" in the keywords. I made my corrections and suggestions to the article. please find the attached file via following link : https://f1000research-files.f1000.com/linked/762273.my_corrections_and_suggestions_to_the_article.docx Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise The article can be published by making some changes I suggested. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (0) karlıdağ s. Peer Review Report For: Glucose Tolerance Test (GTT) Curves of Water and Ethanol Extracts of Whole Body Apis mellifera jemenitica [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :872 ( https://doi.org/10.5256/f1000research.183639.r422288) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-872/v1#referee-response-422288 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 El-Wahed A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 03 Nov 2025 | for Version 1 Aida Abd El-Wahed , Plant Protection Research Institute, Agricultural Research Centre, Giza, Egypt 0 Views copyright © 2025 El-Wahed A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Not Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Introduction: - The section beginning with “The Oral Glucose Tolerance Test (OGTT) consists of several key steps...” (Eyth et al., 2024) should be omitted; it is unnecessary. The authors should instead discuss the currently used drugs and justify the shift toward exploring natural sources. Materials and Methods: The sample size is very small—only four rabbits were used, which were then divided into two groups (n=2). This raises serious concerns about statistical validity. The age of the adult workers and drones used should also be specified. The section describing “The study sample consisted of adult workers and drones …… similar to using dry ice for killing honeybees (Mutinelli, 2021).” is overly detailed and unnecessary. The authors should instead provide a clear and detailed description of the extraction method. The doses derived from adult workers and drones are not mentioned. Furthermore, while the authors state there is limited literature on the chemical composition of honeybee bodies, several studies have already addressed this, such as: (Ghosh, et al., 2016) - ref 1 (Abedelmaksoud, et al., 2024) - ref 2 Analytical Methods: Relying solely on IR spectroscopy for compound identification is insufficient. The study should incorporate additional analytical tools such as mass spectrometry (MS) and nuclear magnetic resonance (NMR) to ensure accurate compound identification. Although a list of various compounds is presented, the authors fail to identify which compounds are responsible for the reported biological activity. The authors present list of compounds with diverse of compounds but they didn’t mentionwhich responsible of the activity Results and discussions: A more comprehensive discussion of the results is needed. The authors should also include clear recommendations and practical implications of the findings. Conclusion Please rewrite the conclusion and include suggestions for future work. The study’s recommendations should be included. Figures: All figures should be revised to achieve higher resolution and publication-quality standards. The graphic abstract recommended. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? No Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required. Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? No References 1. Ghosh S, Jung C, Meyer-Rochow V: Nutritional value and chemical composition of larvae, pupae, and adults of worker honey bee, Apis mellifera ligustica as a sustainable food source. Journal of Asia-Pacific Entomology . 2016; 19 (2): 487-495 Publisher Full Text 2. Abedelmaksoud T, El‐Masarawy M, Altemimi A, Hesarinejad M, et al.: Drones and Workers of HoneybeeApis mellifera L. Dried Powder: Chemical Composition, Antioxidant, and Anticancer Assessment. Food Science & Nutrition . 2024; 12 (12): 10357-10369 Publisher Full Text Competing Interests No competing interests were disclosed. Reviewer Expertise Natural Products Chemistry, Isolation, purification, synthesis, structure elucidation of bio-active compounds, Bee products, honeybees I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. reply Respond to this report Responses (1) Author Response 31 Dec 2025 Mohammed Elimam Ahamed Mohammed, Department of Chemistry, King Khalid University, Abha, Saudi Arabia Introduction: Comment: - The section beginning with “The Oral Glucose Tolerance Test (OGTT) consists of several key steps...” (Eyth et al., 2024) should be omitted; it is unnecessary. The authors should instead discuss the currently used drugs and justify the shift toward exploring natural sources. Response: Thank you very much for your careful review. As the manuscript focuses specifically on the Glucose Tolerance Test (GTT) and related compounds, detailing the GTT procedure is essential for ensuring the accuracy and reliability of the results. Omitting any step would invalidate the findings. I have addressed your point by changing "patient" to "study subject" to better reflect the research context. Thank you again for your valuable feedback. Materials and Methods: Comment: The sample size is very small—only four rabbits were used, which were then divided into two groups (n=2). This raises serious concerns about statistical validity. Response: We acknowledge that the sample size of experimental animals is limited. This investigation is designed as a pilot study. The decision to use a smaller cohort was guided by ethical considerations, as the potential for serious, life-threatening toxicity from whole-body honeybee extracts in rabbits was previously unknown. Furthermore, this approach aligns with established ethical frameworks for animal research. Several published articles endorse using minimal animal numbers in preliminary investigations, with some citing 3-5 subjects as an acceptable range for pilot studies, and others noting that certain pilot experiments have been conducted with a single animal. Please see the reference below for further context: Michael F. W. Festing and Douglas G. Altman. Guidelines for the Design and Statistical Analysis of Experiments Using Laboratory Animals. ILAR Journal. 2002; 43(4): 244- 258. Comment: The age of the adult workers and drones used should also be specified. Response: Done Comment: The section describing “The study sample consisted of adult workers and drones …… similar to using dry ice for killing honeybees (Mutinelli, 2021).” is overly detailed and unnecessary. The authors should instead provide a clear and detailed description of the extraction method. Response: The extraction method was very simple as mentioned and nothing more. Comment: The doses derived from adult workers and drones are not mentioned. Response: Many thanks. The dose is added to GTT procedure. Comment: Furthermore, while the authors state there is limited literature on the chemical composition of honeybee bodies, several studies have already addressed this, such as: Response: Thank you very much for your suggestion, however, the published articles are concentrating on the nutritional value alone without other natural products such as phenolic compunds and alkaloids. Beside they did not mention the contaminants such as the pesticides. (Ghosh, et al., 2016) - ref 1, This study is already cited. (Abedelmaksoud, et al., 2024) - ref 2 This study is cited in the introduction and the importance of the study is rephrase accordingly. Thanks the reference is of high importance. Analytical Methods: Comment: Relying solely on IR spectroscopy for compound identification is insufficient. The study should incorporate additional analytical tools such as mass spectrometry (MS) and nuclear magnetic resonance (NMR) to ensure accurate compound identification. Response: While mass spectrometry (MS) and NMR are powerful tools, especially when paired with databases or analysis software, our methodology aligns with standard practices for characterizing complex biological samples. A key aspect of our work was utilizing the new IR spectra database from Japan. Additionally, we are pioneering an approach to use IR spectral peaks much like precursor ions are used to query established metabolomics (e.g., MassBank) and protein databases. Comment: Although a list of various compounds is presented, the authors fail to identify which compounds are responsible for the reported biological activity. The authors present list of compounds with diverse of compounds but they didn’t mentionwhich responsible of the activity. Response: Thanks a lot. In the discussion and conclusion sections we have stated some compounds as possibly responsible for the change of the GTT curve. Results and discussions: Comment: A more comprehensive discussion of the results is needed. Response: This was stated as a limitation because the available literature is limited, with existing research predominantly concentrating on nutritional value. Comment: The authors should also include clear recommendations and practical implications of the findings. Response: Statement is added to the conclusion section to meet your comment. Conclusion Comment: Please rewrite the conclusion and include suggestions for future work. The study’s recommendations should be included. Response: Done. Figures: Comment: All figures should be revised to achieve higher resolution and publication-quality standards. Response: Done according to the journal requirements Comment: The graphic abstract recommended. Response: A graphical abstract is created. View more View less Competing Interests NA reply Respond Report a concern El-Wahed AA. Peer Review Report For: Glucose Tolerance Test (GTT) Curves of Water and Ethanol Extracts of Whole Body Apis mellifera jemenitica [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2025, 14 :872 ( https://doi.org/10.5256/f1000research.183639.r425586) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-872/v1#referee-response-425586 Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions Adjust parameters to alter display View on desktop for interactive features Includes Interactive Elements View on desktop for interactive features Competing Interests Policy Provide sufficient details of any financial or non-financial competing interests to enable users to assess whether your comments might lead a reasonable person to question your impartiality. 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