Characterization of Melittin from Honey Bee Venom using Analytical Methods

Abstract

Melittin, the main bioactive component of honey bee venom (HBV), demonstrates significant therapeutic promise, especially as an anticancer agent. This review explores the physicochemical and pharmacological properties of HBV, highlighting melittin's mechanisms of action against cancer cells, such as triggering apoptosis, inhibiting cell proliferation, and preventing metastasis. The effects of traditional collection methods and advanced extraction technologies, including electric shock devices, on venom composition are examined. The study delved deeper into advanced analytical methods such as SDS-PAGE, HPLC-MS, and LC-MS, which are vital to the separation and identification of melittin from venom of honey bees. This study examines these approaches I greater detail. It also looks at delivery systems mediated by nanotechnology as an innovative approach to maximize melittin's therapeutic effectiveness while lowering systemic toxicity. This review highlights melittin's potential as a cancer treatment choice in the future and encourages ongoing advancements in analytical and delivery systems to promote pharmacotherapies based on HBV.

Keywords

Introduction

Different Activity of Melittin has been shown in figure no. 1

The Picture of  Apis Mellifera has been shown in figure no. 2  (23)

• Physical Properties, Chemistry and pharmacology Of Honey Bee Venom

Apitoxin is another name for bee venom, which is produced in bee venom glands as a protective mechanism. Humans have known about its properties since ancient times. Venom production occurs after two of three days of adulthood and decreases with age. Bee venom is a colourless, transparent fluid with a strong odour. The venom of honeybees is a clear liquid that can also turn yellow and it is odourless with an irritating (bitter taste) flavor. The pH of honeybee venom is about 4.5-5.5 (24).

When it arrives into contact with mucous membranes or the eyes, it produces a strong burning sensation and irritation. Because some of its protein have oxidized, the dried form is yellow, while some commercial preparations are brown in colour. A variety of volatile compounds included in honey bee venom are easily lost during the gathering process. The venom of honey bees is believed to be a good source of biogenic amines, peptides and enzymes (25). The bee venom gets rapidly dries and crystallizes into grayish-white crystals, when it comes into touch with air. The venom dissolves in water but does not dissolve in alcohol and ammonium sulphate (20).

The Picture Bee Venom Powder has been shown in figure no.3  (26)

The vivo and in vitro studies are done to examined pharmacology of bee venom (27), (28), (29), (30), (31), (32) Bee venom has a wide range of pharmacological effects, including anti-mutagenic (32), radioprotective (32), anti-hepatotoxic (30), cytoprotective (30), anti-microbial (28), anti-viral (28), anti-inflammatory (28), (30), anti-oxidant (28), (30) anti-arthritic (27), anti-metastatic (29), and anti-tumor (29) effects.

The  Pharmacological Activities of Bee Venom has been shown in figure no. 4

The Procedures for dissecting and separating the sting apparatus's venom reserves has been shown in figure no. 5 :  (A) Use all-purpose fine-tipped tweezers to pull the venom reservoir and sting mechanism out of the bee's body; (B) After the two structures are fully extracted from the bee's body but remain together, use two tweezers (or one pair of micro scissors and one pair of tweezers) to separate them (35).

The Steps for Collection of Bee venom via Electric shock method has been shown in figure no. 6  (37)

Chemical Composition of Melittin has been shown is figure no. 7: A) Molecular structure and B) Amino acid sequences of two melittin isoforms, Apis Mellifera and Apis Cerana. (45)

Diagrammatic representation of melittin’s lytic process has been shown in figure no. 8 (A) Melittin and membrane bilayer, (B) Pore formation, and (C) membrane lysis. (45)

Diagrammatic representation of melittin’s primary anti-cancer modes of action has been shown in figure no. 9 : (23), (12)

Diagram illustrating the suggested anti-cancer modes of action of melittin and bee venom has been shown in figure no. 10 :   Examples of cytochrome C (Cyt C), nuclear factor-κB include reactive oxygen species (ROS), deoxyribonucleic acid (DNA), protein 53 (p53), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated Xprotein (Bax), Bcl-2 homologues antagonist/killer (Bak), and death receptor (DR);  MMP2/9, matrix metaloproteinase 2 and 9; cMyc, c-Myelocytomatosis; Bcl-xL, B-cell lymphoma extra-large; AIF, apoptosis-induced factor; EndoG, endonuclease G; TRAILR, TNF-related apoptosis-inducing ligand receptor; FasR, fas receptor; VEGF, vascular endothelial growth factor; VCAM1 vascular cell adhesion molecule 1; ICAM1, intercellular adhesion molecule 1;  (53)

• Characterisation of Melittin from Honey Bee Venom

There are several methods for characterisation of Melittin form BV which consist of separation methods, chemical strategies based on common protein reactions, ad biological testing. Several aanalytical methods are employed to accelerate the eventual standardization of HBV. The primary means of separating, purifying and characterizing HBV peptides were achieved through gel chromatography and thin-layer chromatography. The identification of melittin may also be accomplished using capillary electrophoresis. In spite of this, HPLC remains the primary chromatographic method for peptides and proteins, whether for fingerprinting, product standardization, or quality control.  Melittin, apamin, and tertiapin structural analyses, which include peptides in solution at various temperatures, have been the primary applications of spectroscopic methods (54).

To study the complex substances modern pharmaceutical analysis requires advanced analytical methods. Thus, researcher use “hyphenated techniques” which combine multiple analytical methods for better sensitivity, resolution and specificity (55). When two separate analytical methods are combined or coupled with the aid of a suitable interface, it is referred to as a hyphenated technique (56).  By combining spectroscopic techniques like FTIR, PDA, MS and NMR with chromatographic methods like HPLC, GC, CE .Thus the hyphenated techniques integrate separation technologies with spectroscopic methods to enable both quantitative and qualitative analysis of complex samples (57).

The amount of melittin in bee venom may be measured using a variety of analytical methods, including reversed-phase high-performance liquid chromatography (RP-HPLC). Following RP-HPLC, the detection methods are diode array detector (DAD) (58), ultraviolet (UV) detector (59) , photodiode array detector (PDA), and tandem mass spectrometry (MS/MS) (60,61).   Also, ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QqTOF-MS) is used for the quantitative measurement of melittin in Asian honeybee venom (Apis cerana) (62).

ANALYTICAL TECHNIQUES TO ACCESS MELITTIN FROM HBV HAS BEEN SHOWN IN TABLE NO. 1 (54).

HPLC-MS and LC-MS is one of the hyphenated technique used for characterisation of melittin. Spectroscopic techniques are typically paired with chromatographic procedures. Chemical components in a combination that were pure or almost pure were separated using chromatography, and spectroscopy generates specific data for identification through the use of library or standard spectra. The term "hyphenated techniques" refers to a variety of methods that combine separation and strategies for separation, separation-identification, and identification-identification (56).

MATERIAL AND METHOD

• Chemicals

HPLC-grade acetonitrile and water, and phosphoric acid (85%) were from Merck (Darmstadt, Germany). Melittin (91.8% purity) was from Sigma (M2272, 119K4001) (63). 

• Sample preparation

One mg of powdered venom of honey bee was transferred into a screw capped tube and dissolved in 10 mL of pure water by mixing with a vortex mixer for 3 min. following 5 min a centrifuging at 4000 rpm, 3 mL of supernatant was transferred to a 5 mL vial. This solution was filtered using a syringe filter with a 0.45 µm membrane for the HPLC analysis. 1gram of cream with one mg of honey bee venom and 10mL of 0.4%  Phosphoric acid in water (elute A) were added in a screw-capped tube, heated for 5 minutes at 70^oC and then mixed. The solution was centrifuged at 4000 rpm for 10 min at ambient temperature. After being filtered, the supernatant was combined with one milliliter of hexane to clarify it. For HPLC analysis, the aqueous solution was filtered using a 0.45 µm membrane 63).

• Standard solution

The stock solution was made by dissolving the melittin in pure water to achieve the concentration of 1000 µg/mL. The stock solution was diluted with pure water and this diluted solution stored at 4°C in the dark before analysis to produce a 200 µg/mL working standard solution. The analyte level anticipated in the samples determined the concentration of the standard solutions. Melittin was calibrated using five concentrations (63).

• Chromatogephic conditions

The high-performance liquid chromatography (HPLC) was performed using the Knauer UHPC/PLC PLATINE system (Knauer, Berlin, Germany), which included a PDA detector, confined column chamber, and two pumps. The samples were separated at 25°C and chilled at a rate of 1mL/min using a Europa Protein 300 C18 column (250 x 4 mm, 5 m). The mobile phase consisted of acetonitrile (eluent A) and 0.4%   phosphoric acid in water (eluent B), with a gradient elution that began at 5% to 52% A over 15 minutes, then rose to 80% A in 5 minutes, and ultimately reached 100% A in another 5 minutes. Every sample was examined three times after being injected with a volume of 20 μL. The PDA detector covered the wavelength range of 200 to 380 nm, with the detection wavelength set at 220 nm. By comparing the retention times and UV spectra of the analytes with known substances and published data, the analytes were identified (63).

• HPLC (High performance liquid chromatography) (35)

Most components of honey bee venom can be separated from one another and identified using a technique called high performance liquid chromatography (HPLC). Melittin, tertiapin, MCDP, apamin and several enzymes (phospholipase A2 (PLA2), hyaluronidase) are the most significant substances found in the protein/peptide fraction. It is quite challenging to validate the results because there aren't any reference materials with certified values of the bee venom's constituent parts. For the purpose of removing the matrix effects and enhancing accuracy and precision, applying an internal standard is a definite requirement.

The following is the procedure:

1. Dilute all samples with a 25 ug/ml cytochrome c solution (internal standard) in deionized water.
2. Make the solutions for honey bee venom by diluting 3 mg of the product in 10 milliliters of internal standard solution.
3. Dissolve the melittin in an internal standard solution to create the standard solutions.  The solutions will be applied to the design of calibration curves.  The following standard solution concentrations (at least six dilutions) are advised: melittin: 30–300 mg.  After five minutes of sonicating all prepared solutions, filter through 0.45-mm membrane filters.
4. Analyze produced standard solutions and solutions containing honey bee venom using HPLC.  Use the column with the same specifications, such as the SynChropack C8 6.5 mm, 4.6 x 100 mm column. Eluents A and B contain 0.1% TFA in water and acetonitrile: water (80:20) respectively,

Separation conditions are as follows: a linear gradient of 5% B to 80% B at 30 minutes; flow rate ¼ 1 ml/min; injection volume ¼ 40 ml; separation temperature ¼ 25 -C; and k ¼ 220 nm.

1. Use their retention times to determine melittin.
2. Using comparable relative peak areas (peak area of an analyte divided by peak area of internal standard), create the melittin calibration curves.
3. Using the usual calibration curve calculations, determine the concentrations of the honey bee venom that was examined.

•      Mass spectrometry (35)

High performance liquid chromatography-mass spectroscopy (HPLC-MS) or liquid chromatography-mass spectrometry (LC-MS) is the preferred technique for a thorough qualitative examination of the protein composition of bee venom. The wide variations in the concentrations of different proteins in the venom extracts, however, undermine the technique. In particular, tryptic digests of bee venom samples exhibit an overrepresentation of peptides derived from melittin and the different isoforms of phospholipase C. A number of techniques for pre-fractionating proteins before analysis are discussed in order to increase the dynamic range of proteomic analysis.  Highly abundant proteins are usually removed using affinity reagents, such as cocktail sets of antibodies. We present the use of a combinatorial peptide library that is commercially accessible and serves as a "equalizer." Hexapeptides attached to beads make up this peptide library. During the washing processes, saturation causes excess, abundant protein to be removed.

Proteins recovered from the beads are separated by SDS-PAGE, cut into peptides and then analyzed via LC-MS or HPLC-MS. The peptide analysis can be performed using any high-resolution mass spectrometer (Orbitrap, QTOF) for proteomic analysis, but the use of FT-ICR-MS mass meters will be discussed later. It should be noted that quantitative data on variations in protein abundance are lost when employing the peptide library technique.

• SDS-page (35)

We explain how to utilize SDSPAGE on a 10% SDS-PAGE in this approach. For small proteins (less than 10 kDa), a Tris-tricine-SDS-PAGE (Sch€agger & VON Jagow, 1987) is the preferable method.

• SDS-PAGE gel casting:

A commercial casting stand (small gel format) and frame can be used to cast a SDS-PAGE. Put two glass plates in the holder, spaced apart by a spacer, after assembling the casting stand. Make use of deionized water to check for leaks.

1. Prepare separating gel solution  by mixing 3.8 mL of deionized water with 3.4 mL of acrylamide/            bisacrylamide (30% / 0.8% w/v), 2.6 mL of 1.5M Tris (pH 8), and 0.1 ml of 10 % SDS. Mix this solution.
2. Add 100 10% (w/v) ammonium persulfate.
3. Gently shake the mixture.
4. Administer 10 TEMED.
5. Again gently shake the mixture.
6. Pipette the solution among the glass plates down to about 2 cm from the edge of the front plate.
7. Fill the space with isopropanol until it overflows.
8. Let the gel to polymerize for 20–30 min.
9. In the meantime, mix 3.975 ml of deionized water with 0.67 ml of acrylamide/bisacrylamide (30% / 0.8% w/v), 1.6 ml 0.5 M Tris-HCl (pH 6.8) and 0.05 ml 10% SDS to create the stacking gel solution.
10. Throw away the isopropanol.
11. Add 0.5 ml of 10% ammonium persulfate to the stacking gel solution.
12. Give a little shake.
13. Add 0.005 ml TEMED.
14. Again shake it gently.
15. Add the solution on the top of the separation gel.
16. Present the well- forming comb, being careful not to let air get caught behind the teeth.
17. Wait for at least 20–30 min for gelation.
18. Allow the gel to stand on room temperature for 2-3 hours to let the polymerization finish. Another option is to keep the gel overnight at 4⁰ C.

•     Preparation for gel electrophoresis.

1. First, remove the comb.
2. Remove the gel-containing glass plates from the casting frame and place them in the cell buffer tank.
3. Fill the inner chamber with running buffer (25 mM Tris-HCL, 200 mM glycine, 0.1% SDS), and then continue after overflowing until the buffer in the outer chamber reaches the necessary level.
4. Prepare a 1mL sample buffer solution in 0.2M Tris-HCL at pH 6 by adding 10% (w/v) SDS, 10mM dithiothreitol, 20% (9 w/v) glycerol, and 0.05% (w/v) bromophenol blue. This buffer should be diluted five times before use since it is five times more concentrated. 
5. Combine 50 lg of eluted proteins from the protein enrichment step with recommended quantity of sample buffer to get a volume of 10.

•      Running of the gel electrophoresis.

1. Fill a well with this sample; ideally the third or fourth lane. Don’t let it overflow.
2. Fill the first lane with the protein marker solution.
3. Attach electrodes and cover the top. 
4. Assign 100V as the voltage. Until the blue bromophenol front hits the bottom of the gels, and continue electrophoresizing.
5. After removing the gel cassette, place the gel in a box for the cleaning procedures.

•      Liquid chromatography-mass spectrometry (35)

1. Dissolve the dried peptide extract in 15ug of 20% acetonitrile /0.1% formic acid.
2. Vortex
3. Pipette 5 ug in a vial containing an HPLC sample. .
4. Utilize an LC-MS gradient, analyze the material for 1–3 hours. Through a central facility or specialist laboratory, the majority of laboratories will have success to such a system. Readers who are interested are directed to specialized literature.

•    Nanotechnology approach for bee venom and Melittin 

MEL is considered an anticancer chemotherapeutic drug, but its ability to break down quickly and exhibit non-specific cellularlytic behavior poses significant challenges. The toxic effects of intravenous MEL therapy, such as hemolysis, (64) hinder its widespread adoption in cancer treatment. It has recently been clear that MEL and its conjugates may be utilized to treat certain cancer types by conjugating with hormone receptors, gene therapy, and nanoparticles (45). The toxicity, non-specificity, breakdown, low bioavailability, and hemolysis are all potential drawbacks of using melittin for cancer treatment (64). These are being overcome by means of methods such as nanotechnology, gene therapy, and immunoconjugation to improve efficacy in both in vitro and animal models (65).

Bee venom therapy can be done through electrophoresis, topical forms, injections, or direct stings. Traditional sting therapy is painful, has an inconsistent dosage, and needs to be administered repeatedly because of melittin's short half-life (66).  Researchers are creating alternatives like polymer and (NPs) nanoparticle-based delivery methods in order to improve stability and control because of the short plasma half-life and dosage difficulties of bee venom (53).

Effect of Bee Venom Loaded on Nanoparticles (NPs) and Polymers

A sustained-release system is necessary to produce long-term therapeutic benefits because of the disadvantages of honeybee stings and venom injections. Recently, nanoparticles (NPs) that are effective carriers of bioactive chemicals like bee venom have been created using biodegradable polymers such as poly-D., L. and Glyc acid (PLGA), alginate, and chitosan. The delivery and release profile of bee venom is improved by loading it onto these polymer-based NPs, which lowers the frequency of administration and increases patient adherence. By modifying the polymer type, molecular weight, or nanoparticle architecture, it is possible to accurately change the breakdown rate of these nanostructures from days to years. This regulated release ensures that bee venom remains effective for a long period of time (66).  It is also found that nano-fungal chitosan loaded with bee venom greatly enhanced anti-cancer action against cervical carcinoma (HeLa) cells, causing substantial apoptosis in a time- and dose-dependent manner (67).

Recent research has shown that delivery of nanovaccines targeting the LN leads to both humoral and cellular immune responses, which can help treat tumor (68). Due to the limited number and variable immunogenicity of tumor-associated antigens (TAAs), it is challenging to manufacture nano vaccines. Although neoantigens have potential, they need sophisticated prediction methods. On the other hand, whole-cell tumor antigens offer a wide range of antigens, which lowers the possibility of tumor escape and recurrence. Although the manufacturing procedure is time-consuming, Recently created chitosan nanoparticles packed with entire cell lysates to target dendritic cells in the lymph node (LN) (69). Methods for releasing antigens in situ, such as radiotherapy or oncolytic viruses, produce only little immune responses and require immunomodulators. As a result, there is a critical need for LN-targeted nanovaccines that can effectively activate antigen-presenting cells (APCs) and release whole-cell antigens (70). Melittin's lytic action is primarily attributed to its capacity to integrate into phospholipid bilayers and disrupt the integrity of cell membranes; however, when administered via nanoparticles, it may travel to intracellular membranes, activate the intrinsic pathway, and result in apoptosis (65).

Researchers developed α-peptide-NPs, a high-density lipoprotein-mimicking scaffold, and successfully loaded melittin to create ultrasmall α-melittin-NPs (10–20 nm) (71). These nanoparticles reduce melittin’s cytotoxicity to red blood cells while retaining its ability to induce tumor cell death and release whole-tumor antigens in situ. Their optimal size allows efficient lymph node (LN) targeting, enhancing melittin’s immunomodulatory effects. In vivo studies show that α-melittin-NPs rapidly drain to LNs, activate antigen-presenting cells (APCs), and trigger systemic immune responses. In a B16F10 tumor model, they eliminated 70% of primary and 50% of distant tumors, demonstrating strong potential as LN-targeted whole-cell nanovaccines for cancer immunotherapy.  α-Melittin-NPs are LN-targeted whole-cell nanovaccines that efficiently slow tumor progression and elicit long-lasting, targeted, and strong systemic antitumor immunity. Most significantly, the necessary dose of melittin peptide is significantly reduced by the use of nanotechnology and intratumoral injection, resulting in additional cost savings (70).

Theoretically, Melittin (MLT) has a strong anticancer potential because of its ability to kill tumor cells (72), (73)   It can control gene expression and alter immunity without causing drug resistance. However, its widespread cytolytic and hemolytic toxicity restricts its clinical application. Improvements in nanotechnology have made it possible to target MLT delivery using nanoparticles, lowering toxicity and increasing therapeutic effectiveness (74).  Despite the availability of MLT-based therapies, more research is still needed on its antitumor processes and delivery techniques. Nanoparticles have benefits such as biocompatibility, adjustable pore size, and a large surface area. Researchers have enhanced functionalization strategies, such as bionic modifications, stimuli-responsive systems, and active targeting, by utilizing receptor variances and site-specific stimuli, resulting in increased bioavailability, safety, and delivery efficiency of melittin in vivo (75). The report summarizes recent advances and examines potential avenues for future study (75).

Melittin (MLT) was delivered effectively in vivo by perfluorocarbon nanocarriers with a PFOB core, resulting in tumor accumulation and considerable tumor growth retardation without toxicity (45).  MLT also increases the bioavailability of orally administered drugs by crossing intestinal barriers, and targeted delivery via hemifusion reached endothelial and cancer cells(47).  For stomach cancer, a legumain-activable MLT-decorated nanovehicle improved sorafenib delivery (76). Additionally, plasmid DNA encoding MLT is used in nanotech-based gene therapy, which has the potential for targeted therapy without any resource issues (75).

Research on cancer prevention and treatment has shown a great deal of interest in medication creation techniques mediated by nanotechnology. The unusual physicochemical characteristics of nanoparticles, such as nanometer size, high surface area-to-mass ratio, and effective interaction with cells, suggest that this technology has a bright future in the treatment of cancer. Numerous nanotechnology-mediated MEL conjugates have been successfully produced and evaluate in preclinical models for a wide variety of human cancers. In vitro tumor cell selectivity of sterically immunoliposomal peptides has been created and evaluated in BV (45).

CONCLUSION AND FUTURE PROSPECTS

Since both BV and its major peptide MEL are promising candidates for cancer treatment. The cumulative evidence thus far points towards the possibility of using BV and all of its components, especially MEL, as a cancer treatment. It's crucial to keep researching and developing MEL's potential as a cancer therapy option due to its anticancer properties. In areas where access to standard treatments is restricted, bee venom (BV) and melittin (MEL), its main peptide, demonstrate significant promise as inexpensive cancer cures. The effects of standard chemotherapeutic treatments are mirrored by the way that MEL causes apoptosis, impairs mitochondrial activity, and prevents angiogenesis and metastasis. Its membrane-lytic character and nonspecific toxicity, however, restrict its clinical application. By increasing targeted delivery, reducing hemolysis, and boosting treatment effectiveness, new strategies, especially nanotechnology, provide a viable solution.

By increasing the accessibility and efficacy of novel treatments, ongoing innovation in MEL-based nanocarriers has the potential to revolutionize cancer care. Due to its strong anticancer, anti-inflammatory, and antimicrobial characteristics, melittin, the major bioactive peptide in honey bee venom, is a viable option for therapeutic uses. The use of sophisticated analytical methods like SDS-PAGE, HPLC-MS, and LC-MS has allowed for accurate characterization and quantification of melittin, while nanotechnology-based delivery methods have greatly enhanced its stability, bioavailability, and targeted effectiveness. Despite its therapeutic promise, its clinical use is restricted by difficulties such hemolytic toxicity and a brief half-life in the plasma. To establish the safety and efficacy of melittin, future research should concentrate on improving nanocarrier systems, investigating gene therapy and immunoconjugates, and performing rigorous clinical trials. The use of melittin in personalized medicine and combination therapies may transform cancer treatment and expand its pharmacological applications.

We believe that a variety of further changes are still needed in order to increase the efficacy of BV and MEL in treating cancer. Although BV or MEL have not yet been tested as cancer therapies, we hope that ongoing research on this topic will eventually enable the assessment of these substances as potential anticancer treatments in the future. Thus, melittin should be characterized by use of hyphenated techniques to increase interest in research concerning the Bee venom and its potential cancer treatment.

ABBREVIATIONS

• HBV: Honey Bee Venom
• SDS-PAGE: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
• HPLC-MC: High Performance Liquid Chromatography-Mass Spectrometry
• LC-MS: Liquid Chromatography-Mass Spectrometry
• IgE: Immunoglobulin E
• RT: Radiotherapy
• MEL: Melittin
• 12VDC: 12 Volt Direct Current
• MCDP: Mast Cell-Degranulating Peptide
• SKOV-3: Susan Komen Ovarian-3
• HER2: Human Epidermal Growth Factor Receptor 2
• EGFR: Epidermal Growth Factor Receptor
• DR: Death Receptor
• JAK2/STAT3: Janus Kinase 2/ Signal Transducer and Activator of Transcription 3 Pathway
• ERK: Extracellular Signal Regulated kinase
• T & NK cell: T and Natural Killer cell
• IFN: Interferon
• PBMCs: Peripheral Blood Mononuclear Cells
• ICAM-1: Intercellular Adhesion Molecule-1
• JNK: C- Jun N-terminal kinase
• Nf-xB: Nuclear factor kappa B
• Bcl2: B-cell lymphoma 2
• iNOS: Inducible nitric oxide synthase
• COX2: Cyclooxygenase-2
• cPLA2: Phospholipase A2
• MeCP2: Methyl - CpG binding protein 2
• HIF-1α Protein: Hypoxia inducible factor 1α Protein
• VEGF: Vascular Endothelial Growth Factor
• Bax, Bak: Bcl-2-associated Xprotein, Bcl-2 homologues antagonist/killer
• AIF: Apoptosis Inducing Factor
• EndoG: Endonuclease G
• Cyt-C: Cytochrome C
• ROS: Reactive Oxygen Species
• DNA: Deoxyribonucleic Acid
• MMP2/9: Matrix Metaloproteinase 2 And 9
• CMyc: c-Myelocytomatosis
• Bcl-xL: B-cell lymphoma extra-large
• TNA: Triple Negative Adenocarcinoma
• FasR: fas receptor
• VCAM I: Vascular Cell Adhesion Molecule -1
• CE: Capillary Electrophoresis
• FTIR: Fourier Transform Infrared Sprectroscopy 
• PDA: Photodiode array detector
• NMR: Nuclear Magnetic Resonance Spectroscopy
• RP-HPLC : Reverse Phase –High Performance Liquid Chromatography
• DAD: Diode Array Detector 
• UPLC-QqTOF-MS: Ultra Performance Liquid Chromatography –Quadrupole Time –Of- Flight Mass Sprectrometry
• FT-ICR-MS: Fourier Transform Ion Cyclotron Resonance Mass Sprectroscopy
• TEMED:  N,N,N,N-tetramethylethylenediamine
• HCL: Hydrogen Chloride
• Nps: Nanoparticles
• LN: Lymph node
• APCs: Antigen Presenting Cells

DECLARATIONS

Ethics approval and consent to participate

(Not Applicable) This work did not require formal ethics approval as it did not involve the use of honey bees practically.

Consent for publication

All authors have reviewed and agreed for publication.

Availability of data

No datasets are generated by any own assumption. Data was collected from different review and research papers.

Competing interests

The authors have no potential conflicts of interest regarding this publication. 

Funding

There was no funding for this project.

Author’s Contributions

• NT wrote the main manuscript text.
• PS has guided for preparation of main manuscript text.
• PW has reviewed the manuscript.
• SV has reviewed the manuscript. 
• All authors read and approved for the final manuscript text.

ACKNOWLEDGEMENTS

Not Applicable

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