Presence of short and cyclic peptides in Acacia and Ziziphus honeys may potentiate their medicinal values


 Acacia honey is characterized by high nutritional, antioxidant, antibacterial and immuno-modulatory values. This work investigated the presence of short and cyclic peptides in Acacia and Ziziphus honey samples. Acacia honey samples (Acacia tortilis and Acacia hamulosa) and three Ziziphus honeys (Ziziphus spina-christi) were screened for their short and cyclic peptide contents using the LC-MS and the chemical structure databases. Moreover, the total protein content was determined using the Bradford method. The A. tortilis honey contained three short peptides; HWCC, DSST, and ECH, and the A. hamulosa honey sample contained five short peptides and one cyclic peptide. The short peptides of the A. hamulosa honey were Ac-GMGHG-OH (Ac-MGGHG-OH), Boc-R(Aloc)2-C(Pal)-OH, H-C (1)-NEt2·H-C (1)-NEt2, APAP (AAPP), and GAFQ (deamino-2-pyrid-4-yl-glycyl-dl-alanyl-dl-norvalyl-dl-asparagine). The cyclic peptide of the A. hamulosa honey was cyclo[Aad-RGD-d-F] (cyclo[Aad-Arg-Gly-Asp-d-Phe]). The Ziziphus honey was characterized by the presence of either Almiramide B or Auristatin-6-AQ. A. tortilis, A. hamulosa, and Ziziphus honeys are characterized by the presence of short and cyclic peptides which may contribute to their medicinal values.


Introduction
Nutritionally, honey is considered as energy food since it is majorly composed of carbohydrates and it is used in infants and children feeding to boost their growth. Moreover, honey contains some amounts of vitamins, minerals, amino acids, enzymes, and phenolic compounds which qualify it to act as antioxidant and to boost the athletic performance, immune system, and digestion and absorption. Medicinally, honey is used for wound healing due to its high sugar content, low moisture percentage, hydrogen peroxide, gluconic acid, and dicarbonyl molecules including the methylglyoxal. Furthermore, honey is used to treat disorders of hematology and immunity, metabolism and cardiovascular system, oral health, ophthalmology, and gastrointestinal tract beside its usage as anticancer (chemotherapy) and antimicrobial [1,2].
Honey is characterized by proteins such as the enzymes which originate from the honeybees or the plants' nectars and secretions. The protein content of honey is associated with its medical and pharmaceutical value [3,4].
Short or bioactive peptides are composed of small number of amino acids in foods. The bioactive peptides are mostly produced by enzymatic hydrolysis of large proteins from animal and plant origins. Milk and its products, eggs, meat, marine organisms, spinach, soybeans, and cereal grains are the best examples of bioactive peptides containing foods. Some bioactive peptides are chemically synthesized and added to foods for the purpose of increasing their medicinal value. Short peptides affect different body systems including the cardiovascular, endocrine, immune, nervous, and digestive systems. The presence of short peptides in foods prevents their oxidation and degradation by microbes [5][6][7][8].
Cyclic peptides are short peptides with ring structure due to the binding of its amino terminal to its carboxyl terminal by amide bond or other chemical bonds such as the ether, disulfide, and lactone bonds. Cyclic peptides are reported to be found in roasted coffee, cocoa, and malt beside their presence in milk, beverages, chicken, and fermented foods. Biological activities of cyclic peptides include antibacterial, antitumor, immunosuppressive and antioxidant activities. Known functions of cyclic peptides are the cyclo (-Phe-Phe) of chicken essence which inhibits the serotonin transporter and the acetylcholinesterase and the cyclo (-His-Pro) which inhibits rat's food intake and reduces their body weight [5,9]. This article measured the concentration of total proteins and investigated the presence of short and cyclic peptides in Acacia and Ziziphus honey samples after they were authenticated with regard to their floral origin and some quality parameters.

Study design
This study is an observational descriptive study. The disadvantages of descriptive studies include the small number of samples and the difficulty in deriving a general conclusion. However, descriptive studies are useful since they highlight research areas for survey studies [10].

Honey samples and their authentication
Two Acacia and three Ziziphus honey samples were collected directly from the bee farms and their hives. The samples were involved in this study after confirming their floral origin and their conformance to some of the international standards for honey. The two Acacia honeys were collected during the flowering seasons of A. tortilis and A. hamulosa, while the dominant Ziziphus tree in the study area was Z. spina-christi. All the honey samples were collected from Asir region at the southwestern part of Saudi Arabia.
The Ziziphus 3 honey samples were collected from bee farms at sea level altitude, while the A. hamulosa, Ziziphus 1, and Ziziphus 2 samples were harvested from bee farms at 900 m above sea level. A. tortilis was harvested at 2,000 m above sea level. The flowering season of the Z. spina-christi and A. hamulosa ranges from September to November, while the flowering season of A. tortilis is from March to July [11,12].
The floral origin of the honey samples was determined following the method published by Louveaux et al. [13]. The moisture, pH and acidity, conductivity, and diastase activity were determined according to the methods of International honey commission, (2009) [14], while the glucose, fructose, sucrose, and HMF were measured following the methods of Agilent company.

Determination of total protein concentration
The total protein concentration was determined in the studied samples using the spectrophotometric method of Bradford (1976) [3]. The protein in the samples (50% W/V; 100 µL) was reacted with coomassie brilliant blue (5 mL) and the absorbance was measured at a wavelength of 595 nm. Albumin was used in preparation of standard curve (0-500 µg/mL).

Liquid chromatography-mass spectrometry (LC-MS)
The chemical constituents and the presence of short peptides in honey samples were investigated using the LC-MS. Hunter software was used to analyze the mass. Tuning and optimization are carried out before any run and on each single day as recommended by the manufacturer.

Chemical structure databases search
The molecular formulas obtained from the LC-MS were searched in the PubChem, ChemSpider, and Molbase databases to investigate the possible isomers. However, one short peptide is published by the University of Dortmund-Germany.

Statistical analysis
The agglomerative hierarchical Cluster analysis of the Statistical Package for Social Sciences (SPSS) was used to group the honey samples according to the values of the physicochemical parameters and the total proteins.

Results
The honey microscopic pollen analysis showed that all the honey samples were mono-floral with dominance of one pollen type by more than 60% ( Figure 1).
The results of the measured quality parameters were within their ranges in the Codex Alimentarius standards of honey [15] (Table 1). However, some honeys were with marginal diastase activity compared to the standards which may be due to the geographical and climatic conditions.

Total protein concentration
The R 2 was 0.976, while the equation of the standard curve line was Y = 0.00142X + 0.042. The A. hamulosa and the Ziziphus 2 honey samples had the highest concentration of proteins ( Table 1).

The agglomerative hierarchical clustering
The honey samples were divided into three groups (levels).
Level one was composed of A. hamulosa, Ziziphus 2, and

LC-MS and the search in chemical structure databases
The LC-MS showed that the Ziziphus honey samples had one short peptide. Three short peptides were found in the A. tortilis honey, while five short peptides and one cyclic peptide were reported for the A. hamulosa honey.

The short peptides of the A. tortilis honey
The three short peptides of the A. tortilis honey were: (1) HWCC (His-Trp-Cys-Cys) with the molecular formula of C 23 H 29 N 7 O 5 S 2 which is retrieved from the ChemSpider database ( Table 2 and Figure 3) [16].

The short and cyclic peptides of the A. hamulosa honey
The A. hamulosa honey was characterized by five short peptides and one cyclic peptide.   (I) The five short peptides and their databases were: 1. The PubChem search showed that the molecular formula C 19 H 29 N 7 O 7 S is similar to the molecular formula of two short peptides Ac-GMGHG-OH (Ac-Gly-Met-Gly-His-Gly-OH) and Ac-MGGHG-OH (Ac-Met-Gly-Gly-His-Gly-OH) ( Table 2 and Figure 4) [20].    Table 2 and Figure 4) [25,26]. Moreover, the C 19 H 27 N 5 O 6 is the molecular formula of Alfuzosin hydroxy acid. However, the Alfuzosin and its metabolites are routine drugs for Benign Prostatic Hyperplasia (BPH) [27].

The short peptide of the Ziziphus honey samples
The Ziziphus 2 honey sample contained a short peptide with the molecular formula of C 40 H 64 N 6 O 6 . In the PubChem, the short peptide is registered as Auristatin-6-AQ, a Figure 3: The LC-MS spectra of the short peptides in the Acacia tortilis honey sample. The three short peptides were HWCC, DSST, and ECH (CHE). The molecular structure of the HWCC were retrieved from the ChemSpider, Molbase, and PubChem databases [16][17][18][19].
dipeptide containing two methylated valine residues [29]. The ChemSpider and PubChem search showed that the molecular formula is for Almiramide B, a short peptide with phenylalaninamide, three methylated valine residues and one methyl alanine ( Table 2 and Figure 5) [30,31].

Discussion
All the studied quality parameters of the honey samples were within the ranges of the Codex Alimentarius standards except the diastase activity of two Ziziphus honey samples which was in the lower margin. The marginal activity of the diastase enzyme may be due to the climatic conditions of Tehama (Geographical region). The Ziziphus honey had no short peptides, while the two Acacia honeys contained short and cyclic peptides.
The first short peptide of the A. tortilis honey is the (His-Trp-Cys-Cys) which is available in the ChemSpider database [16]. It proposed that presence of cross links between His-Cys and Trp-Cys play a regulatory role of host enzymes [32]. Dipeptides that possess tryptophan, tyrosine, cysteine, or methionine have antioxidant activities [33].
The L-aspartic acid-L-seryl-L-seryl-L-threonyl (DSST) (CAS No.: 748808-93-7) of the A. tortilis honey sample has the molecular formula of C 14 H 24 N and is registered in the Molbase database [17]. Proteins and short peptides containing Asp, Ser, and threonine are reported to act as binders and transporters of cations and their related substrates [34,35]. Thus, the presence of the DSST short peptide in the A. tortilis honey may be an indication for its mineral content.
The A. tortilis honey contained a third short peptide identified as Gln-Cys-His (ECH) [18] or Cys-His-Gln (CHE) [19]. No published article containing the three amino acids was retrieved. However, Cys-His are known to be responsible for acetyl transfer reactions in enzyme mimics [36].
Concerning the short peptides of the A. hamulosa, the N-acetyl-L-methionyl-glycyl-glycyl-L-histidyl-glycine is the first identified short peptide in the PubChem of the national center for biotechnology information [20]. Peptides that contain the sequence of Gly-Gly-His are known to act as copper binding proteins [37][38][39]. Moreover, it is reported that presence of tyrosine, tryptophan, methionine, lysine, cysteine, and histidine in a short peptide increases its antioxidant activity [40]. Nα-tert-Butyloxycarbonyl-(Nδ,ω-diallyloxycarbonyl)-L-arginyl-(S-palmitoyl)-L-cysteine (Boc-Arg(Aloc)2-Cys(Pal)-OH) was the second short peptide of the A. hamulosa honey. A search of the literature showed that this short peptide is synthesized for the purpose of identifying the function of viral proteins and their modified forms [21]. Cysteine residues of short peptides and their position are well-known to possess an antioxidant activity because of the direct interaction between the SH and the radicals [41]. Some Arginine containing short peptides in royal jelly samples have been proved to possess antioxidant activities [42]. Since this short peptide contains both arginine and cysteine, it may qualify the A. hamulosa honey to act as antioxidant diet.
The third short peptide in the A. hamulosa was the L-cysteine diethylamide (1 → 1′)-disulfide compound with L-cysteine diethylamide (H-C (1)-NEt2·H-C (1)-NEt2) [22], with score percentage of 98.6%. This dipeptide was produced and tested as inhibitor of L-cystine crystallization and a possible treatment for cystinuria and prevention of cystine renal stones [22]. The A. hamulosa honey may be a possible inhibitor of the formation of cystine renal stones. The fourth supposed short peptide in the A. hamulosa honey have sequence of Ala-Ala-Pro-Pro in the PubChem [23] and the sequence of Ala-Pro-Ala-Pro in the Chem-Spider database [24]. Short peptides rich in proline are known to possess an antioxidant activity [43,44]. Strong capacity of hydroxyl radical scavenging was reported for the Alanine containing dipeptides [45].
The A. hamulosa fifth short peptide was reported as deamino-2-pyrid-4-yl-glycyl-DL-alanyl-DL-norvalyl-DL-asparagine (deamino-G(4-pyridyl)DL-A-DL-NV-DL-N) in the PubChem [25] and gly-ala-phe-gln in the ChemSpider [26]. Presence of hydrophobic amino acids such as alanine, glycine, and valine in the sequence of short peptides facilitates the binding of these short peptides to fatty acids leading to the inhibition of lipid oxidation [39]. Presence of phenylalanine in a short peptide indicates its radical scavenging capability [40], while short peptides containing Asparagine or glutamine in the mid exhibited antihypertensive activity [46,47].
The cyclic peptide of the A. hamulosa honey was the cyclo[L-alpha-homoglutamyl-L-arginyl-glycyl-L-alpha-aspartyl-D-phenylalanyl] (cyclo[Aad-RGD-D-F]) which is found in the PubChem database [28]. The cyclic peptide is used in bioassay for integrin receptor [28]. According to its cyclic nature and amino acid content, this peptide could be used as antioxidant, antimicrobial, antitumor and can also be used as weight loss inducing peptide [5,9].
The Z. spina-christi honey contained Almiramide B short peptide which acts as anti-Leishmania donovani and anti-Trypanosoma brucei brucei [48,49]. Auristatin-6-AQ is potently active against human cancer cell lines [50]. This study is limited due to the small number of honey samples. Future studies should be designed to involve more Acacia honey samples and the short peptides should be extracted or synthesized so as to investigate their biological and medicinal activities.

Conclusion
The A. tortilis honey contained three short peptides, while five short peptides and one cyclic peptide were found in the A. hamulosa honey. The Ziziphus spina-christi honey can be used as anticancer, anti-Leishmania donovani, and anti-Trypanosoma brucei brucei. The presence of the short and cyclic peptides in the Acacia honey qualifies it to act as a natural medicine such as the Manuka honey.
Acknowledgements: The authors extend their appreciation for the Poison Control and Medical Forensic Chemistry Centre, Ministry of Health, Asir region, Saudi Arabia for permitting them to perform the LC-MS analysis. Informed consent (if applicable): The bee keepers did not agree to publish the latitude and longitude of their bee farms, because they believe that if their location is published, many beekeepers will bring their bee farms to the location. The geographical location of the samples is mentioned as wide areas such as Bisha and Tehama.
Ethical approval: The conducted research is not related to either human or animal use.
Data availability statement: The research data are available upon request.