Floral and geographical origin of the honey samplesThe microscopic analysis showed that the honey samples were of three floral origins; Acacia, Ziziphus and polyfloral (Fig. 1) and (Table 1).Table 1 The number of the honey samples and their floral origins.The major dominant honeys in Saudi Arabia are the Acacia, Ziziphus, Lavender (lavandula dentate) and Hypoestes forskaolii (Majra)44. The Acacia honey of Saudi Arabia includes Acacia tortilis (Samar), Acacia hamulosa (Gatada), Acacia gerrardii / Acacia origena (Taleh), Acacia ehrenbergiana (Salm), and Acacia asak (Dahiana). The Ziziphus honey is of two types (Ziziphus spina Christi and Ziziphus nummularia)44,45,46.Quality parametersThe results of the honey quality parameters and total sugars are presented in (Table 2) including the mean, standard deviation (SD), minimum and maximum values of each parameter. All the values of the studied quality parameters comply with the values of the Codex and Saudi Food and Drug Authority (SFDA) standards1,47.Table 2 The results of the investigated honey quality parameters.The geographical origin of the honey samples significantly affected the moisture percentage of the Acacia honey and the EC of the polyfloral honey.The geographical origin had significant effect on the moisture percentage and HMF of the Acacia honey and the EC of the polyfloral honey. The floral origin had significant effect on the pH, moisture and HMF of the honey samples from Jazan while it had significant effect on the glucose and the sum of fructose and glucose of Asir honey (Table 2) and (Table 3). Many of the previous studies concluded that the floral and geographical origin had significant effects of the quality parameters of honey48,49,50. In a conference abstract published by the journal of biological chemistry, we reported that the geographical origin had insignificant effect on some studied honey quality parameters including the moisture, acidity and electrical conductivity48. Moreover, the same abstract concluded that the floral origin from one geographical origin (hot climate) had significant effects on the moisture, pH and total sugars51.Table 3 The significant effects of the floral origin on the honey analyzed parameters.Total proteinsThe line equation of the standard curve of the protein assay was Y = 0.0011X + 0.0205 and the R2 was equal to 0.98 (Fig. 2).Fig. 2The standard curves of the studied antioxidant molecules.The mean, SD, minimum and maximum values of the total protein concentration in Acacia, Ziziphus and polyfloral honey samples of Asir region were (667.17, 210.36, 416.0, 1172.0 µg/g), (610.67, 3.06, 608.0, 614.0 µg/g) and (915.48, 198.28, 450.0–1260.0 µg/g), respectively. The protein results of the Acacia, Ziziphus and polyfloral honey samples of Jazan region were (642.06, 186.39, 345.0, 991.0 µg/g), (737.5, 280.72, 539.0, 936.0 µg/g) and (645.8, 49.97, 571.0, 725.00 µg/g), respectively (Fig. 3).Fig. 3The results of the proteins concentration and the catalase activity.The geographical region significantly affected the total protein concentration of Acacia honey (p-value = 0.044) and polyfloral honey (p-value ≤ 0.001). With regard to the effect of the floral origin, the mean total protein concentration of the polyflroal honey of Asir region was significantly more than the protein concentration in the Acacia and Ziziphus honeys (p-value = 0.004 and p-value = 0.011, respectively). The floral origin of Jazan region had insignificant effect on the total protein concentration. The comparison between the mean values of the total protein in the different honey samples showed the significant effects of the geographical and botanical origins on the total protein concentration.The ranges of total proteins in Asir and Jazan honey samples irrespective of their floral origins were (416.20–1260.0 µg/g) and (345.0–991.0 µg/g), respectively. The protein content range of the studied honey samples is different than the range of our pilot study results (345.0–1260.0 µg/g) compared to (222.54–560.5 µg/g)11. Azeredo et al. (2003) measured the protein concentration in honey samples of different botanical origin and a reported high concentration of proteins in two of their honey samples (2236 and 2212 µg/g)35. This study reported significant effects of the geographical and floral origins on the protein concentration of the studied honey samples (Table 3). In our published abstract in the Journal of biological chemistry, we stated that the floral and geographical origins of honey samples had significant effect on the total concentration of honey proteins51. The previous studies proved that the physicochemical properties and chemical composition of honey are affected by the floral and geographical origins of honey43,45,48,49,50,51,52. It has been stated that the protein concentration in honey is impacted by the honeybee species and the honey type53.CatalaseThe standard curve of the catalase is presented in (Fig. 2). There was inverse relationship between the absorbance and the catalase activity and the equation was y = -0.3806x + 0.5837 and R2 = 0.9733.The mean ± SD and range of CAT activity of the Acacia honey of Asir region were 168.70 U/kg ± 76.04 and (52.16–382.89 U/kg), of the Ziziphus honey of Asir were 122.18 U/kg ± 86.07 and (63.33–250.06 U/kg) and the results of Asir polyfloral honey were (162.05 U/kg ± 67.14 and (19.68–238.41 U/kg). The results of the CAT of Asir region honeys were more than those of Jazan region. The results of the CAT activities of the Acacia honey from Jazan were 80.38 U/kg ± 54.84 and (13.24–125.64 U/kg), of the Ziziphus honey from Jazan were 102.68 U/kg ± 2.21 and (101.11–104.24 U/kg) and the results of the Jazan polyfloral honey were 130.66 U/kg ± 36.45 and (65.13–203.66 U/kg) (Fig. 3).The Acacia honey of Asir region had significantly increased mean value of CAT activity compared to the Acacia honey of Jazan (P-value = 0.015). As Asir region has a cold climate, it can be concluded that the catalase activity increases in cold climates. The conclusion is that the geographical origin had significant effect on the CAT activity of the Acacia honey while the floral origin had insignificant effect.It is known that honey catalase is mainly derived from the nectar of the plants and the microorganisms in honey and it is affected by the other components in the nectar of the plants. Similar to our findings one of the literature mentioned that wheat catalase increases in low temperature environment9,54. As a honey component, the catalase is affected by the floral origin, nectar constituents, climate conditions, processing and storage environment9,52,54. Moreover, the results of this study are very low compared to Brazilian honey samples which were with a range of 9970–99070 U/g (9.97–99.07 U/mg)55.Short peptidesShort peptides in the honey samples from Asir regionNine precursor masses from Asir region honey samples were found to be short peptides from Apis mellifera and Apis florea proteins (Table 4). The two honeybee species were present in the study area. According to the mascot search engine, the enzymes that produced the short peptides were trypsin, chymotrypsin, pepsin A, Clostripain (Arg-C) and V8-DE. The trypsin, chymotrypsin and pepsin are produced by the honeybees9 while the Clostripain and V8-DE are of bacterial origin.Table 4 The detected ultra-short peptides in the honey samples from Asir region using the precursor masses and the mascot search engine.Clostripain is a cysteine endopeptidase that cleaves peptide bonds from the carboxyl end of Arginine residues. The clostripain is produced by the Clostridium histolyticum bacterium which is found in the feces of animals56. The V8 is a protease is produced by Staphylococcus aureus and it cleaves peptide bonds from the carboxyl ends of glutamate and aspartate residues57.Presence of short peptides digested by enzymes of bacterial origin in honey suggests presence of these bacteria in the intestinal flora of the honeybees. However, some researchers reported that honey contains some Clostridium species such as the Clostridium botuinum and Clostridium perfringens58,59,60. Presence of short peptides in honey due to the action of clostripain need to be investigated since the Clostridium histolyticum is not present in the flora of the honeybees. Moreover, It is reported by Anjum et al. (2021)58 that staphylococcus species are present in the gut of Apis mellifera.Regarding the honey samples from Asir region, seven short peptides were detected in Acacia honey and two were found in polyfloral honey while no short peptide was detected in the Ziziphus honey samples. The precursor masses that correspond to the short peptides in the Acacia honey were 635.3990, 519.3547, 573.4010, 622.3807, 521.3077, 620.3872 and 393.2230, whereas, the precursor masses corresponding to the short peptides of the polyfloral honeys were 519.3506 and 650.3761 (Table 4).The mascot search showed that the precursor mass of 635.3990 is either a trypsin digest of hunchback protein of Apis mellifera (YCHSL KLHLR; Tyr-Cys-His-Ser-Leu-Lys-Leu-His-Leu-Arg) or a trypsin digest of Tertiapin of Apis mellifera (IIIPHMCWKK; Ile-Ile-Ile-Prl-His-Met-Cys-Trp-Lys-Lys) (Table 4). The hunchback protein is an insect transcription factor that activates of repress gene expression associated with embryo development61. Short peptides containing histidine, tyrosine, phenylalanine, cysteine, lysine and valine are well known by their antioxidant activity63,64. The trypsin digests of the hunchback protein in the honey samples can act as antioxidants. Short peptides containing tyrosine, leucine isoleucine, arginine, phenylalanine, tryptophan, proline and lysine at their carboxyl or amino terminals act as antihypertensive medicine through inhibiting the Angiotensin Converting Enzyme (ACE)65. The two short peptides of the trypsin digest of the hunchback protein contain tyrosine and lysine at their N-terminals and contain leucine and arginine at their C-terminals. i.e. the two short peptides are qualified to inhibit the ACE and act as antihypertensive peptides. The Tertiapin is a protein neurotoxin and is a minor component of the honeybee venom66. The trypsin digest of the Tertiapin (IIIPHMCWKK) has three isoleucine residues at its N-terminal and two lysine residues at its C-terminal which qualifies it to act as antihypertensive medicine65. Moreover, presence of methionine in the structure of the trypsin digest of Tertiapin qualifies it to act as antioxidant beside its content of histidine, cysteine and lysine63,64,67. The presence of proline and tryptophan in the sequence of the trypsin digest of Tertiapin suggests its antimicrobial activity68.The second precursor mass was for chymotrypsin digest with the value of 519.3547 (Table 4). It was a digestion product of Apidermin-2 of Acacia honey samples produced by Apis mellifera. Apidermin-2 is a circular structural protein with antibacterial and antifungal activities69. The Chymotrypsin digest of Apidermin-2 had the sequence of AAVPLAPTIAL (Ala-Ala-Val-Pro-Leu-Ala-Pro-Thr-Ile-Ala-Leu) which can be classified as alanine rich short peptide. Alanine rich peptides are known by their antimicrobial activity70.The third precursor mass (573.4010) was from Acacia honey and it was corresponding to two Clostripain digests for Myosuppressin and orcokinin proteins of Apis mellifera (Table 4). Myosuppressin is an insect neuropeptide famous by its activity as visceral muscle inhibitor such as the muscles of the gastro intestinal tract and heart70. Orcokinin is a neuropeptide in insects and crustaceans that enhance the frequency of gastrointestinal tract muscle contraction72. The clostrpain digests of Myosuppressin and Orcokinin were IRKVCVALSR (Ile-Arg-Lys-Val-Cys-Val-Ala-Ser-Arg) and TAFDNFFKR (Thr-Ala-Phe-Asp-Asn-Phe-Phe-Lys-Arg). The clostripain digest of Myosuppressin can act as antimicrobial (contains repetitive Arginine and valine residues)73 and antihypertensive (contains isoleucine at its amino terminal and arginine at its carboxyl terminal)65. The clostrpain digest of Orocokinin can act as antimicrobial since it is rich in phenylalanine (33%)74.The fourth precursor mass was due to the activity of the clostripain with the value of 622.3807 and it is for the pheromone biosynthesis activation neuropeptide (PBAN-type neuropeptide) (Table 4). It is a pheromone with variable functions including pheromone synthesis muscle contraction and diapause induction and termination75. The clostripain digest of the PBAN-type neuropeptide has the sequence of IGFAVFSSFNR (Ile-Gly-Phe-Ala-Val-Phe-Ser-Ser-Phe-Asn-Arg). The clostripain digest can act as anti-hypertensive because it has isoleucine at its amino terminal and Arg at its carboxyl terminal65. Moreover, the digest contains three phenyalanine residues out of eleven amino acids which qualifies it to act as antimicrobial71.650.3761 was the fifth precursor mass and it was pepsin A digest product in a polyfloral honey sample. The mascot search showed that this pepsin A digest is either from Melittin or Homeobox protein H40 (Fragment) of Apis mellifera (Table 4). Melittin is the major bee venom protein that constitutes about 52% of bee venom dry weight. Melittin has promising future as a medicine for the treatment of leukemia76. Generally, Homeobox proteins are transcription factors that play roles in the morphogenesis and differentiation of cells74. The pepsin A digest of melittin had the sequence of ISWIKNKRKQ (Ile-Ser-Trp-Ile-Lys-Asn-Lys-Arg-Lys-Gln) which is lysine rich peptide. Lysine rich peptides containing tryptophan and or aliphatic amino acids act as hypo-cholesterolemic peptides beside their role as antimicrobial and antioxidants63,64,74. The Homeobox 40 digest of the pepsin A was lysine and threonine rich polypeptide (ENKFKTTRYL; Glu-Asn-Lys-Phe-Lys-Thr-Thr-Arg-Tyr-Leu) which is qualified to act as antimicrobial and antioxidant63,64,73,74.The sixth precursor mass was (519.3506) and it was for Apidermin-2 (circular structural protein) of a polyfloral honey sample produced by pepsin A (Table 4). The short peptide was rich in alanine and proline (AAVPLAPTIAL; Ala-Ala-Val-Pro-Leu-Ala-Pro-thr-Ile-Ala-Leu). The alanine and proline rich peptides act as antimicrobials68,70. Proline rich short peptides are reported to act as anti-diabetics78.The seventh precursor mass was (521.3077) obtained from Acacia honey and produced by pepsin A. It was either for a digest from Apamin or Mast cell degranulating peptide of Apis mellifera (Table 4). Apamin is a bee venom neurotoxin protein responsible for the regulation of the gene expression associated with cell development. Pharmacologically, it irreversibly blocks the potassium channels that depend on Ca2+79. Mast cell degranulating peptide is a bee venom peptide strongly de-granulates the mast cells leading to the release of histamine. It acts as anti-inflammatory and blocks potassium channels leading to low blood pressure80. The pepsin A digest of the Apamin and the Mast cell degranulating peptide had the sequence of (SVILITSYF; Ser-Val-Ile-Leu-Ile-Thr-Ser-Tyr-Phe). Many short peptides that contain serine, isoleucine, leucine, valine and threonine possess antioxidant activity64. Furthermore, repetitive amino acids is a character of antimicrobial peptides74.The eights precursor mass (620.3872) was due to the activity pepsin A in Acacia honey sample. The mascot search showed that the mentioned pepsin A digest was either a product of Abaecin or Homeobox protein H17 (Fragment) peptides (Table 4). Abaecin is a proline rich protein secreted by the Apis mellifera in response to bacterial infection81. The Homeobox protein H17 (Fragment) is a transcription factor associated with the morphogenesis and differentiation of insects cell77. The pepsin A digest of Abaecin is an alanine rich peptide (ATICAAFAYVPL; Ala-Thr-Ile-Cys-Ala-Ala-Phe-Ala-Tyr-Val-Pro-Leu) which can act as antimicrobial peptide70. The pepsin A digest of Abaecin can be classified as antioxidant peptide since it contains cysteine, tyrosine, valine, isoleucine, leucine and phenylalanine63,64. The pepsin A digest of the Homeobox H17 was rich in Arginine, Lysine and alanine (QNRRAKAKRL; Gln-Asn-Arg-Arg-Ala-Lys-Ala-Lys-Arg-Leu). Arginine or lysine rich peptides have the ability of penetrating cell membranes which suggests them to be used in drug delivery82,83. Moreover, the pepsin A digest of the Homeobox H17 can act as antimicrobial and hypocholesterolemic peptide because it is alanine and glycine rich, respectively70,74.The ninth precursor mass (393.2230) was produced by the V8 protease of the Staphylococcus spp of an Acacia honey sample. The V8 digest was either for the Brain peptide MVPVPVHHMADELLRNGPDTVI or the Homeobox H90 (Table 4). The brain peptide MVPV is one of the peptide that controls the behavior of bees when foraging for the collection of nectar or pollens84. As a homeobox, the homeobox H90 is a transcription factor involved in the regulation of gene expression associated with the cell differentiation and insect morphogenesis74. The V8 digest of the MVPV brain peptide is (LLRNGPD; Leu-Leu-Arg-Asn-Gly-Pro-Asp) which is rich in leucine. Short peptide with multiple leucine residues regulates the intracellular phosphorylation which is involved in the regulation of many metabolic processes85. The V8 digest of the Homeobox H90 was (LSHCVPE; leu-Ser-Cys-Val-Pro-Glu) which is suitable to act as antioxidant peptide63,64. The short peptides produced by the V8 of the Staphylococcus aureus reflect the usefulness of honey proteomics in detecting the contamination of honey by pathogenic bacteria.Short peptides of the Jazan honey samplesShort peptides in Jazan honey samples were detected in the mascot search engine after choosing the trypsin, chymotrypsin, TrypChymo, pepsin, Arg-C (Clostripain), Lysyl-endopeptidase (Lys-C and Lys-N), flavastacin (Asp-N). As mentioned above the trypsin, chymotrypsin and pepsin are secreted by the honeybees while the clostripain is normally isolated from the Clostridium histolyticum. The lysyl enopeptidases are produced by the lysobacter strains which are reported to be present in honey86. The flavastacin cleaves peptide bonds from the amino terminal of Aspartate and it is isolated from Flavobacterium meningosepticum bacterium87. Presence of short peptides in honey due to flavastacin action may be due to contamination of honey by the water borne Flavobacterium meningosepticum. However, the microorganisms are considered as a major source of enzymes in honey9.This study reported that twenty seven precursor masses from the Jazan honeys are for enzymes digests of variable proteins. The first precursor mass was (416.1897) in a polyfloral honey produced by the trypsin enzyme. The mascot search showed that the mentioned trypsin digest is for the diuretic hormone class 2 of Apis mellifera (Table 5). The diuretic hormones of insects induce diuresis in response to energy mobilization due to crop draining into the gut or due to environmental stresses88. The sequence of the obtained trypsin digest of the Diuretic hormone class-2 was (GLDLGLSR; Gly-leu-Asp-Leu-Ser-Arg) (Table 5). Leucine rich short peptides act as regulatory molecules for metabolism and presence of the amino acids of this short peptide is an indicator of its antimicrobial activity85,89. Decker et al. (2022) stated that antimicrobial peptides contain frequent amino acids (Alanine, Cysteine, Glycine, Histidine, Isoleucine, Lysine, Leucine, Proline, Arginine, and Valine) and occasionally (Aspartate, Glutamate, Phenylalanine, Serine, Threonine, Tryptophan, and Tyrosine) while three amino acids are not found in the structure of the antimicrobial peptides (Asparagine, glutamine and Methionine)89.Table 5 The detected ultra-short peptides in the honey samples from Jazan region using the precursor masses and the mascot search engine.The second precursor mass (280.0965) was from polyfloral honey for clostripain (Arg-C) action. The mascot search showed that this clostripain was for either Melittin of Apis dorsata with the sequence of (KRQE; Lys-Arg-Gln-Glu) or two homeobox proteins E30 or E60 with one sequence (RVKR; Arg-Val-Lys-Arg) (Table 5). The clostripain digest of the Apis dorsata melittin contains arginine and lysine which are character of the peptides that can penetrate cell membranes and act as drug delivery vehicle82,83. The clostripain digest of the homeoboxes (E30 or E60) contains 2 Arg, one Lys and one Val which propose it to act as a drug delivery molecule and as anti ACE because it contains Arg at its N-terminal and Arg at its C-terminal65,82,83.The third precursor mass was from poyfloral honey sample produced by Apis florea with the value of (458.2721) and it a product of chymotrypsin activity. The chymotrypsin digest was for the melittin according to the mascot search result with sequence of (IKNKRKQ; Ile-Lys-Asn-Lys-Arg-Lys-Gln) (Table 5). The chymotrypsin digest of the melittin is rich in Lysine and contains arginine which a feature of cell penetrating peptides according to Ma et al. (2023)82. Chymotrypsin digest of Melittin can act as antioxidant because it contains isoleucine and Lysine63,64. Moreover, Lysine or arginine rich peptides are proved to have antimicrobial activity without mentioning that if they contain glutamine, asparagine or methionine or not89,90.The fourth short peptide had the precursor mass of (510.2713) and it was a clostripain product of a polyfloral honey sample produced by Apis mellifera. The short peptide was digested from Pro-corazonin protein. Corazonin is a neuropeptide hormone of insects which has effects on visceral muscles and regulates coloration91. The clostripain digest of the Pro-corazonin had the sequence of (QPAPTNNNY; Gln-Pro-Ala-Pro-Thr-Asn-Asn-Asn-Tyr) (Table 5). Short peptides containing sequences of QP, PT, PA and AP are reported to have anti-diabetic activity92. Furthermore, glutamine/ asparagine rich peptides play role in the control of epigenetic (staple and inheritable changes at chromosomal level)93.The fifth precursor mass (427.2311) was for trypsin digest either for Larval-specific very high density lipoprotein, diuretic hormone class-2 or chymotrypsin inhibitor in a polyfloral honey sample with sequences of (VAPFPHGK; Val-Ala-Pro-Phe-Pro-His-Gly-Lys), (GFSGSQAAK; Gly-Phe-Ser-Gly-Ser-Gln-Ala-Ala-Lys) and (ICTMQCR; Ile-Cys-Thr-Met-Gln-Cys-Arg), respectively (Table 5). Larval-specific very high density lipoprotein is responsible for the transport and storage of lipids through the hemolymph during metamorphosis. Metamorphosis is the active holometabolism during the development through the four stages of honeybees (egg, larva, pupa and adult)94. The chymotrypsin inhibitor is a 56 amino acid protein which inhibits both chymotrypsin and cathepsin G95. The trypsin digest of the Larval-specific very high density lipoprotein is proline rich peptide containing valine and alanine which is a character of anti-diabetic peptides beside its antioxidant activity because it contains valine, pheylalanine, histidine and lysine63,64,92. The trypsin digest of the diuretic hormone class-2 (GFSGSQAAK; Gly-Phe-Ser-Gly-Ser-Gln-Ala-Ala-Lys) is glycine, serine and alanine rich peptide which is proposed to act as neuroprotective and suggested for the treatment of Alzheimer disease96. The trypsin digest of the chymotrypsin inhibitor can act as anti-hypertension since it contains isoleucine at its N-terminal and arginine at its C-terminal65. Furthermore, it can act as antioxidant because of its content of isoleucine, cysteine, methionine and threonine63,64,67.The sixth precursor mass (608.3546) was Apisimin digest of a chymotrypsin in a polyforal honey sample. Apisimin is a royal jelly protein with 54 amino acids that do not contain sulfur containing amino acids, arginine, histidine, tyrosine, tryptphan and proline97. The sequence of the amino acids of the chymotrypsin Apisimin digest was (SKIVAVVVLAAF; Ser-Lys-Ile-Val-Ala-Val-Val-Val-Leu-Ala-Ala-Phe) (Table 5). Alanine, arginine and valine rich peptides are reported to act as antimicrobial molecules70,73.The seventh precursor mass (658.4379) was a digest product of the bacterial enzyme lysylyl endopeptidase (Lys-C) with the sequence of (AKRLQEAEIEK; Ala-Lys-Arg-Leu-Gln-Glu-Ala-Glu-Ile-Gu-Lys). The mascot search showed that this sequence is for homeobox H 17 protein (Table 5). Due to its repetitive sequence of alanine and glutamate, it may act as antioxidant as stated by Chen et al. (2015)92. Chen research group found that short peptides form walnut proteins containing repetitive sequences of Alanine and glutamate (ADIYTEEAGR) has antioxidant capacity98.The eights precursor mass (520.3415) was obtained from a polyfloral honey sample by the action of the Lys-C bacterial endopeptidase. The mascot search showed that the precursor mass was for a short peptide from the venom phospholipase A2 of Apis dorsata. The Lys-C digest of the phospholipase A2 is (LEHPVTGCGK; leu-Glu-His-Pro-Val-Thr-Gly-Cys-Gly-Lys). As beekeeping in Saudi Arabia does not involve Apis dorsata, we can conclude that this sample is either imported from outside Saudi Arabia or mixed with honey from abroad. The digest of the phospholipase A2 is expected to have antioxidant activity because it contains leucine, histidine, valine, threonine, cysteine and lysine63,64. Furthermore, the phospholipase A2 can act as inhibitor of ACE (anti-hypertension) because it contains leucine at its N-terminal and lysine at its C-terminal65.The ninth precursor mass (493.3322) was for the brain peptide MVPV due to the activity of the bacteria flavastacin enzyme. The sequence of the flavastacin digest was (VPVPVHHMA; Val-Pro-Val-Pro-Val-His-His-Met-Ala) (Table 5). Because of its amino acid content and sequence, the flavastacin digest of the brain peptide is expected to act as anti-diabetic (VPVPV), antioxidant due to its content of valine, histidine and methionine and antimicrobial because of the poly valine residues63,64,73,92.The tenth precursor mass (357.2246) was a Clostripain digest for the histone 4 of Apis mellifera. It has the sequence of (TLYGFGG; Thr-Lys-Tyr-Gly-Phe-Gly-Gly) (Table 5). Histone 4 (H4) is one of the histone proteins (H2A, H2B, H3 and H4) that form nucleosome with DNA strands. Histone proteins are major regualtors of gene expression (replication, transcription, translation)99. The Clostripain digest of H4 is qualified to act as antioxidant due its content of threonine, lysine, tyrosine, phenylalanine63,64 and antimicrobial because it is a glycine rich short peptide100.The eleventh precursor mass (328.2059) was a Clostripain digest of definsin-1 of Apis mellifera carnica or Apis mellifera with the sequence of (ADRHR; Ala-Asp-Arg-His-Arg) (Table 5). Definsin-1 is an antimicrobial peptide that is secreted by the salivary gland and it is associated with social immunity101. The Clostripain digest of Definsin-1 can penetrate the cell membrane because it is Arginine rich and it can act as antimicrobial73,82.The twelfth precursor mass (389.2388) was obtained from a polyfloral honey due to the action of the bacterial lysylyl peptidase (Lys-N) on the Abaecin antibacterial peptide (Table 5). The Lys-N digest sequence is (KWPQGY; Lys-Trp-Pro-Gln-Gly-Tyr) which can act as antioxidant due its content of lysine and tyrosine63 and antimicrobial because of the tryptophan and proline68.The flavastacin was the endopeptidase that produced the thirteenth precursor mass (519.3292) in a polyfloral honey sample. The flavastacin digest was from the definsin-1 of Apis mellfiera and it has the sequence of (DLWDKRFG; Asp-Leu-Trp-Asp-Lys-Arg-Phe-Gly) (Table 5). Presence of leucine, lysine, and phenylalanine may increase the antioxidant activity of this flavastacin digest63,64. The flavastacin digest short peptides may act as antimicrobial because its sequence is composed of amino acids well known to be in the structure of antimicrobial peptides89,102. Moreover, this flavastacin digest contains leucine, arginine and lysine which is a character of the short peptides that modulate the immune system activity92,103.The fourteenth precursor mass (622.3818) was for a short peptide (IGFAVFSSFNR; Ile-Gly-Phe-Ala-Val-Phe-Ser-Ser-Phe-Asn-Arg) produced by the action of the clostripain on the PBAN-type neuropeptide of Apis mellifera (Table 5). Phenylalanine rich peptides are well known by their antimicrobial activity70,104. Furthermore, the short peptide of the PBAN-type neuropeptide contains isoleucine at its N-terminal and arginine at it C-terminal which may qualify it to act as anti-hypertension beside its activity as antioxidant because of its content of isoleucine, phenylalanine, valine and serine63,64,65.The fifteenth precursor mass (517.3110) was for a short peptide (QILIDANVF;Gln-Ile-Leu-Ile-Asp-Ala-Asn-Val-Phe) produced by the action of the chymotrypsin on the Apisimin (royal jelly protein of Apis mellifera) (Table 5). The chymotrypsin digest of the Apisimin contains all the essential branched chain amino acids (leucine, isoleucine and valine) which reduces the risk of branched chain amino acids related metabolic syndromes105. Because the chymotrypsin digest of Apisimin contains multiple iselucine, leucine, valine and phenylalanine, it can act as antioxidant and antimicrobial63,64,89.The precursor mass (367.1107) was the sixteenth one which is a product of the lysylyl-endopeptidse (Lys-N) in a polyfloral honey sample. The Lys-N digest was for ATP synthase protein 8 of the Apis mellifera ligustica with the sequence (PQMMPM; Pro-Gln-Met-Met-Pro-Met). The Apis mellifera ligustica is not within the beekeeping species in Saudi Arabia and presence of its protein in a local honey may reflect that the honey was mixed with an imported honey (Table 5). The Lys-N digest is a methionine rich peptide that is known to act as an antioxidant67. Furthermore, the Lys-N contains sequences of PQ and MP which is reported to act as anti-diabetic92.The seventeenth precursor mass (380.2157) was for the short peptide (GRENQR; Gly-Arg-Glu-Asn-Gln-Arg) which was produced by the action of Clostripain on the Orcokinin peptide (neuropeptide associated with visceral muscle contraction) of the Apis mellifera (Table 5). The Clostripain digest of the orcokinin is an arginine rich peptide which is expected to be capable of penetrating cell membranes and act as antimicrobial73,82.The mascot search showed that the eighteenth precursor mass (302.0782) was for the short peptide (TKINK; Thr-Lys-Ile-Asn-Lys) that is produced by the trypsin from the ATP synthase protein 8 of Apis mellifera (Table 5). The trypsin digest of the ATP synthase is a lysine rich peptide that is capable of penetrating the cell membranes, act as antimicrobial and antioxidant63,64,89. Moreover, the trypsin digest of the ATP synthase may act as cholesterol lowering peptide because it is rich in lysine and contains isoleucine73.The nineteenth short peptide (VAPFPHG; Val-Ala-Pro-Phe-Pro-His-Gly) had the precursor mass of (363.1751). Lys-N was the endopeptidase that produced the short peptide from the Larval-specific very high density lipoprotein of the Apis mellifera (Table 5). Due to its content of phenylalanine, valine and histidine, the Lys-N digest may act as antioxidant and because it is rich in proline it is suggested to have antimicrobial activity63,64,68.The twentieth short peptide (DRHRRVTC; Asp-Arg-His-Arg-Arg-Val-Thr-Cys) was obtained from definsin-1 by the action of the flavastacin endopeptidase (Asp-N). The precursor mass corresponding to the Asp-N digest was (521.3077) (Table 5). The Asp-N digest of the definsin-1 may act as antimicrobial and has the capability of invading the cell membranes because it is as arginine rich peptide73,74,82. Also, this short peptide may act as antioxidant due to its content of histidine, valine, threonine and cysteine63,64.The mascot search showed that the twenty one precursor mass (455.3233) was for the short peptide (KKWNWF; Lys-Lys-Trp-Asn-Trp-Phe) which was produced by the action of pepsin from the ATP synthase protein 8 of the Apis mellifera ligustica (Table 5). The pepsin digest of the ATP synthase is lysine and tryptophan rich peptide which may qualify it to have antimicrobial activity with the ability of penetrating the cell membranes81,105. Also, this pepsin digest can act as hypocholesterolemic peptide because it is rich in lysine and contains tryptophan74.The mascot search for the precursor mass (620.3872) showed that it is a pepsin digest for either Abaecin or homeobox protein H17 of Apis mellifera. The short peptide of abaecin is (ATICAAFAYVPL; Ala-Thr-Ile-Cys-Ala-Ala-Phe-Ala-Tyr-Val-Pro-Leu) while that of homeobox H17 is (QNRRAKAKRL; Gln-Asn-Arg-Arg-Ala-Lys-Ala-Lys-Arg-Leu) (Table 5). The pepsin digest of abaecin is alanine rich which may qualify it act as antimicrobial70, and it may be with antioxidant activity because it contains threonine, isoleucine, cysteine, phenalalnine, tyrosine, valine and leucine63,64. The pepsin digest of the homeobox H17 is rich in alanine, arginine and lysine which qualifies it to act as antimicrobial and anti-hypercholesterolemia70,73,74,89.The short peptide number 23 has the sequence of (VSSIVSGANVSAVLL; Val-Ser-Ser-Ile-Val-Ser-Gly-Ala-Asn-Val-Ser-Ala-Val-Leu-Leu) with the precursor mass of (708.4044) and it is produced as a result of the pepsin action on the royal jelly protein (Apisimin) of Apis mellifera (Table 5). The pepsin digest of apisimin is rich in valine (4), serine (4), alanine (2) and leucine (2) which is a character of antimicrobial peptides89. Leucine rich short peptides are reported to regulate metabolic pathways85.The twenty fourth precursor mass (446.2507) was for the trypsin digest of abaecin (bee venom protein) with sequence of (IKWPQGY; Ile-Lys-Trp-Pro-Gln-Gly-Tyr) (Table 5). Short peptides containing tryptophan and proline are reported to have antimicrobial activity and short peptides that contains a sequence of proline and glutamine have anti-diabetic activity68,92. Gua et al. (2009) stated that short peptides from royal jelly protein containing tyrosine at their C-terminal have potent antioxidant activity107.The twenty fifth precursor mass (587.3875) was for a sequence of amino acids (DHHLKTHMR; Asp-His-His-Leu-Lys-Thr-His-Met-Arg) produced from the Protein krueppel of the Apis mellifera by the action of the trypsin (Table 5). The Krueppel protein is a juvenile hormone responsible for the regulation of metamorphosis in insects. Metamorphosis is the transformation from bee animal stages egg, larva, pupa and adult108. Histidine rich peptides are reported to have antimicrobial and cell penetrating activities109. The trypsin digest of the Krueppel protein may be with antioxidant activity because it contains histidine, leucine, threonine and methionine63,64,67.The mascot search for the twenty six precursor mass (597.3459) showed it is for the Lys-C digest of the Hymenoptaecin of the Apis mellifera with the sequence of (SRPSLDIDYK; Ser-Arg-Pro-Ser-Leu-Asp-Ile-Asp-Tyr-Lys) (Table 5). The Hymenoptaecin is an antibacterial peptide secreted by the honeybees in response to infection110. The Lys-C digest of the hymenoptaecin may act as antioxidant because it contains serine, leucine, isoleucine, tyrosine and lysine63,64. Short peptides rich in acidic amino acids such as aspartic and glutamic acids are stated to be with antimicrobial activities100.The last precursor mass (383.1745) was for a clostripain digest of the definsin-2 with the sequence of (NGVCICR; Asn-Gly-Val-Cys-Ile-Cys-Arg) (Table 5). Definsin-2 is an antibacterial peptide secreted form the lymph for the individual immunity101. The clostripain digest of definsin-2 is expected to have antioxidant activity due to its content of valine, cysteine and isoleucine64. Moreover, cysteine rich peptides are proved to have antimicrobial activity111.Total phenolic acidsThe equation of the standard curve of the total phenolic acids was Y = 53.947X- 0.0061 and the R2 = 0.9997 (Fig. 2).The high concentration of phenolic acids was reported for the Jazan honey samples as follows: Acacia Jazan (504.1 ± 195.41 mg GA/100g), Ziziphus Jazan (658.35 ± 159.45 mg GA/100g) and polfloral Jazan (476.49 ± 111.99 mg GA/100g) compared to Acacia Asir (198.20 ± 279.16 mg GA/100g), Ziziphus Asir (96.91 ± 11.00 mg GA/100g) and polyfloral Asir (179.24 ± 72.58 mg GA/100g) (Fig. 4). The honeys form Jazan region had significantly high concentration of phenolic acids compared to those from Asir region with p-values ≤ 0.005 (Table 3). The floral origin had insignificant effect on the concentration of the total phenolic acids either in Jazan or Asir region.Fig. 4The results of the total phenolic acids an flavonoids in the studied honey samples.Suleiman et al. (2020) reported that Acacia and Ziziphus honey samples from different altitudes in Asir region had mean concentrations of total phenolic acids of (67.18 ± 2.39 and 91.83 ± 1.47mg GA/100g) and (81.37 ± 2 and 96.99 ± 1.59 GA/100g), respectively112. Mohammed et al. (2020)113 found mean concentrations of total phenolic acids in Acacia honeys from Asir region were (81.47 ± 1.25 and 91.33 ± 0.96 mg GA/100g). However, this study found that the mean values of total phenolic acids in Acacia and Zziphus honey samples from Asir region were comparable to the previous studies with the mean values of 198.20 ± 279.16 mg GA/100g and 96.91 ± 11.00 mg GA/100g, respectively. Vazquez et al. (2021) reported a range of (48–203 mg GA/100g) for total phenolic acids in variable honey samples of different floral origins (chestnut, black berry, eucalyptus and heather) from Galicia in the Northwest region of Spain114. This study reported a wider range of total phenolic acids concentration in the studied honey samples (16.6–1890.4 mg GA/100g). The wider range of total phenolic acids may be due to the different geographical and floral origins. Thirty Croatian Acacia honey samples were reported to be with mean and range concentration of total phenolic acids of (43.66 ± 6.45 mg GA/kg) and (31.72–80.11 mg GA/Kg), respectively115. A Malaysian study reported that the concentration of total phenolic acids in Acacia honey depends on the harvest season and that the range of total phenolic acids was from 129.16 to 341.67 mg GA/Kg (12.916–34.167 mg GA/100g)116. The mean and range of total phenolic acids of the Croatian and Malaysian studies in the Acacia honey were very low compared to the results of this study which may be because of the different sub species of the Acacia plant and the different geographical origin52.Acacia tortilis honey samples from Oman were reported to be with a mean value of total phenolic acids of 2236 mg GA/kg (223.6mg GA/100g)117, which is high compared to the mean value Acacia honey of Asir (198.20 ± 279.16 mg GA/100g) and lower than the mean value of Acacia honey from Jazan region (504.1 ± 195.41 mg GA/100g). The same study117, registered an average concentration of phenolic acids (1279mg GA/Kg = 127 mg/100g) in Omani Ziziphus honey. This study reported high mean concentration of phenolic acids in the Ziziphus honey from Jazan region (658.35 ± 159.45 mg GA/100g) compared to the Omani Ziziphus honey and comparable mean value of the Ziziphus honey from Asir region (96.91 ± 11.00 mg GA/100g). The Omani study found that the mean value of the phenolic acids in multifloral honeys was (1066 mg GA/Kg = 106.6 mg GA/100g)117. The mean values of the multifloral honeys from Oman were low compared to the mean value of the phenolic acids of the Jazan polyfloral honeys (476.49 ± 111.99 mg GA/100g) and Asir polyfloral honeys (179.24 ± 72.58 mg GA/100g).FlavonoidsThe standard curve equation for the flavonoids was (Y = 38.417 + 0.1102) while the R2 was 0.964 (Fig. 2). The results of the flavonoids concentration in the honey samples from Asir and Jazan regions are comparable with values (mg QE/ 100g) of Acacia Asir (5.03 ± 5.81), Acacia Jazan (3.99 ± 3.20), Ziziphus Asir (1.27 ± 0.05), Ziziphus Jazan (1.85 ± 0.78), Polyfloral Asir (4.07 ± 2.63) and Polyfloral Jazan (5.00 ± 2.12) (Fig. 4). The floral and geographical origins of the honey samples had insignificant effect on the concentration of flavonoids.A Previous study on Acacia and Ziziphus honey samples from Asir region showed that our results are lower than the flavonoid mean concentration in Acacia and Ziziphus honey samples from variable altitudes in Asir region112. Suleiman et al. (2020)112 reported mean flavonoid values of (2.02 ± 0.87 and 8.24 ± 0.49 mg QE/100g) for Acacia honey and (9.15 ± 0.56 and 5.43 ± 0.49 mg QE/100g) for Ziziphus honey. Moreover, Mohammed et al. (2020)112 reported high level of flavonoids in Acacia honey from Asir region with the mean values of 8.78 ± 0.23 and 10.63 ± 0.53 mg QE/100g.Studies from Romania and Italy reported decreased ranges of flavonoids in honey compared to the results of this study. The ranges of flavonoids were as follows; Romania (0.91 to 2.42 mg QE/100g)118 and Italy (0.45 to 1.01 mg QE/100 g)119.Al-Farsi et al. (2018)117 reported very high concentration of flavonoids in Omani honey compared to the findings of this study as follows; Acacia tortilis (2143 mg QE/Kg = 214.3 mg QE/ 100g), Ziziphus (848 mg QE/Kg = 84.8 mg QE/ 100g) and multifloral (925 mg QE/Kg = 92.5 mg QE/ 100g).DPPHThe ability of the honey samples from Asir and Jazan regions to scavenge the DPPH are comparable. The reduction percentage (%) were as follows: Acacia Asir (31.23 ± 16.43), Acacia Jazan (35.03 ± 16.95), Ziziphus Asir (59.03 ± 15.88), Ziziphus Jazan (38.95 ± 32.6), Polyfloral Asir (49.45 ± 26.0) and Polyfloral Jazan (53.4 ± 49.66) (Fig. 5). the geographical origin had insignificant effect on the reduction percentage of the studied honey samples while the floral origin of Asir regin had significant effect (Acacia Asir Vs Polyfloral Asir) (Table 3).Fig. 5The DPPH reduction percentage of the studied honey samples.Alshammari et al. (2022) found that Saudi Acacia gerrardii Benth honey and Ziziphus spina Christi had the highest DPPH reduction percentage of 66.4% and 32.6%, respectively119. The DPPH scavenging activity of Jazan Ziziphus honey samples of this study and that of Alshammari et al. (2022) are comparable but the result of Asir Ziziphus honey is almost double that of Alshammari120. The mean DPPH scavenging activity of Asir Acacia honey of this study is very low compared to the scavenging activity of the Acacia gerrardii Benth.Moniruzzaman et al. (2013) studied Malaysian honey samples produced by three different honeybee species and three floral origins and concluded that the highest DPPH scavenging activity was reported for the Tualang honey with a reduction percentage of (59.89%) and the Acacia honey was characterized by the lowest reduction percentage121. Serbian polyfloral honeys were stated to be with radical scavenging activity ranging from 1.31% up to 25.61%122, which is very low compared to result of the polyfloral honeys of this study. Ethiopian honeys from different geographical origins were with DPPH reduction range of (23.74–40.11%)43.FRAPThe FRAP standard curve had the equation of (Y = -0.1403 + 1.3557) and the R2 of 0.9869 (Fig. 2). The results of the FRAP assay of the studied Asir and Jazan honey samples were comparable except for the Ziziphus honey which significantly increased in the Asir honey compared to Jazan honey (p-value ≤ 0.001). The floral origin of Asir honey had significant effect on the FRAP assay (Table 3). The results of the FRAP assay (mg of Fe3+/100g) for the studied honeys were Acacia Asir (205.77 ± 113.29), Acacia Jazan (240.92 ± 75.17), Ziziphus Asir (580.20 ± 112.28), Ziziphus Jazan (142.20 ± 83.16), Polyfloral Asir (233.04 + 163.13) and Polyfloral Jazan (304.26 ± 157.47) (Fig. 6).Fig. 6The results of the FRAP and AEAC antioxidant activities.Al-Hindi et al. (2011)123 studied the antioxidant and antibacterial activities of Saudi unifloral (including Acacia and Ziziphus) and multifloral honey samples and found that the FRAP activity of Acacia honey was 0.4833 ± 0.011 mg of Fe3+/2g of honey (24.165 mg of Fe3+/100g) while the FRAP activity of the two Ziziphus honeys were 0.5958 ± 0.01 and 0.7837 ± 0.009 mg of Fe3+/2g of honey (29.79 and 39.185 mg of Fe3+/100g). A Malaysian Acacia honey was the FRAP activity of 82.386 ± 5.930 mg TE/100g of honey which is very low compared to the findings of this study which may be due to the different geographical origin and the Acacia subspecies124. The antioxidant activities of honey samples depend on thier geographical and floral origins51,52,125.AEACThe straight line equation of the AEAC assay was (Y = -458.98X + 1.3234) and the R2 was 0.9922 (Fig. 2). The mean and the Standards deviation of the AEAC (mg of VitC/ 100g) were as follows: Acacia Asir (308.519 ± 172.47), Acacia Jazan (323.5369 ± 82.24), Ziziphus Asir (363.20 ± 340.18), Ziziphus Jazan (135.42 ± 99.53), Polyfloral Asir (485.97 ± 312.14) and Polyfloral Jazan (330.703 ± 88.31) (Fig. 6). The level of vitamin C equivalent antioxidant capacity was significantly increased in the Polyfloral honey of Asir region compared to the Acacia honey of Asir region and to the Polyfloral honey of Jazan region (P-value ≤ 0.05) (Table 3).Honey samples from different geographical origins and an unidentified floral origins in Ethiopia were with Ascorbic acid equivalent capacity ranging from 16.23 to 26.59 mg VitC/ 100g43. Acacia and multifloral honey samples from Burkina Faso were stated to have an AEAC of 17.50 ± 0.05 mg/100g and (from 10.20 ± 0.59 to 37.87 ± 0.26 mg/100g), respectively126. Similar to our findings that polyfloral honey samples were with the highest AEAC, Islam et al. (2012)127 stated that multifloral honey samples from Bangladesh had the highest AEAC compared to monofloral honeys (34.1 ± 1.4mg/100 g). German Acacia honey and Pakistani Ziziphus jujube scored AEAC of 31.09 ± 0.008 and 26.66 ± 0.71 mg/100g, respectively128.Antioxidant Short peptides of the honey samples and their antioxidant activityHoney samples containing short peptides with possible antioxidant activity because of their amino acid content and sequence were with DPPH radical scavenging, FRAP and AEAC. The antioxidant power increased with the increased number of the antioxidant short peptides (Fig. 7). The antioxidant activity of the short peptides is referred to their content and sequence of amino acids and their mechanism is through proton electron donation, metal chelating and regulation of the activity of antioxidant enzymes129,130. The antioxidant activity of the short peptides is studied by many previous studies63,64,67.Fig. 7The antioxidant activity of honey samples increased with the increase of the number of the antioxidant short peptides.Pearson’s correlationsThe Pearson’s correlation showed that there were significant correlations between the DPPH radical scavenging and the FRAP, AEAC and the total protein. The AEAC was significantly correlated to the DPPH and total proteins. The FRAP was significantly correlated to the DPPH and total phenolic acids (Table 6). The results showed the capability of the proteins to act as radical scavenger and as detoxifier through proton donation. Many previous studies proved the role of proteins as antioxidant molecules131,132,133,134.Table 6 The correlations between the studied parameters and their significance.Limitations of the studyThe limitations of this study include the : 1) small number of samples and 2) other factors known to affect the chemical composition and biological activities of honey are not considered such as honeybee species, nutrition and health, bee keeping practices and soil composition.
