GC-MS Analysis, Phytochemical Screening, and Antibacterial Activity of Cerana indica Propolis from Kashmir Region

: Propolis is a resinous compound produced by honey bees. It contains bioactive molecules that possess a wide range of biological functions. The chemical composition of propolis is affected by various variables, including the vegetation, the season, and the area from which the sample was collected. The aim of this study was to analyze the chemical composition and assess Cerana indica propoli’s antibacterial efﬁcacy from the Kashmir region. Gas chromatography-mass spectrometry (GC-MS) analysis was used to determine the chemical composition of Kashmiri propolis. A range of bacterial strains was tested for antimicrobial activity using different extracts of propolis by agar well diffusion technique. Propolis was found to be rich in alkaloids, saponins, tannins, and resins. The chemical characterization revealed the presence of 68 distinct phytocompounds using GC-MS, and the most predominant compounds were alpha-D-mannopyranoside, methyl, cyclic 2,3:4,6-bis-ethyl boronate (21.17%), followed by hexadecanoic acid, methyl ester (9.91%), and bacteriochlorophyll-c-stearyl (4.41%). The different extracts of propolis showed speciﬁc antibacterial efﬁcacy against multidrug-resistant (MDR) strains viz., Pseudomonas aeruginosa (MTCC1688), Escherichiacoli (MTCC443), Klebsiella pneumonia (MTCC19), Cutibacterium acnes (MTCC843), and Staphylococcus aureus (MTCC96). The EEKP showed the highest zone of inhibition against S. aureus (17.33) at 400 µ g mL − 1 . According to the ﬁndings of this study, bee propolis contains a variety of secondary metabolites with various pharmacological activities. Furthermore, because of its broad spectrum of positive pharmacological actions and the fact that it is a promising antibacterial agent, more research on propolis is warranted.


Introduction
Propolis (also known as bee glue) derives its name from the Greek word pro, which means "in front of" or "at the entrance to," and polis, which means "community" or "city".This natural substance is a unique mixture of various natural components with distinct properties and aids in the protection of the beehive [1,2].It is made up of a resinous substance that honeybees collect from the buds and exudates of certain trees and plants, which they then combine with beeswax and the enzyme-rich secretion of bee salivary glands, including β-glycosidase [3][4][5].
Honeybee colonies frequently employ propolis for hive repairs as a sealant to plug the cracks and restrict the openings, limiting the entry of intruders and maintaining the temperature inside the hive at the ideal level for bees, about 35 • C [6].In addition, they embalm dead invaders with bee glue to prevent decomposition, protect the bee larvae, store honey, and remove potential sources of microbial infestations [7].Propolis becomes sticky when above room temperature, but it is hard and brittle at low temperatures [8].It has a very distinctive and pleasant aroma.Depending on its origin and age, its coloration varies from yellow to red to dark brown.Even the existence of transparent propolis has been reported [9,10].Propolis has been revealed to have antimicrobial properties [11,12], and it has been used for centuries in traditional medicine and remedies.
The complex chemical content of propolis is constantly changing due to various geographical conditions; generally, raw propolis is composed of plant resin (45-55%), wax (25-35%), essential (5-10%), and aromatic oils (5%), pollen, and other natural components (5%).[13].Depending on the species of bee, the plant's origin, the habitat, and the storage conditions, thechemical composition of propolis varies [14,15].So far, over 300 chemical compounds have been isolated and identified from the propolis, including sugars, polyols, hydroxy acids, fatty acids, cardanols, flavan derivatives, triterpenes, prenylated flavanones, anacardic acids, aromatic acids, and their esters, and chalcones, [6,16].Additionally, due to its complex chemical composition, it has been shown that propolis possesses antimicrobial [17,18], antioxidant [19][20][21], anti-inflammatory [22,23], and antifungal properties [24].The bioactivity of propolis has been implicated in its flavonoid, phenolic, diterpenic acid, and aromatic acid contents [25].Globally, propolis formulations are effective antibacterial agents [26][27][28].Propolis is effective against a wide range of bacterial strains, though it is most effective against Gram-positive bacteria and less effective against Gram-negative bacteria [4,29].The findings of numerous studies have revealed that the antibacterial action is based on obstruction of bacterial movement and the activity of various bacterial enzymes, as well as a weakening of the cytoplasmic membrane [29].Propolis from several countries, including Spain, Romania, and China, was found to contain fructose, glucose, galactose, stachyose, and sucrose [30].Propolis contains essential amino acids, such as proline and arginine, which are needed for cell regeneration and can vary depending on the flora and environment around bee colonies [31].Terpenoids are the volatile components of propolis that are responsible for the odor or scent that propolis emits.It also significantly contributes to propolis essential oil extract's biological effects, such as antibacterial and anti-inflammatory properties [32].According to Sturm and Ulrih (2020), terpenoids were isolated in propolis for the first time in 2011, and 133 terpenes have been reported in propolis so far.Only 16 alkaloids have been isolated from Brazil and Algeria to date [33].Hexadecanoic acid, methyl ester, is a fatty acid with antioxidant, hepatoprotective, anticancer, anti-inflammatory, anticoronary, anti-arthritic, antieczemic, antihistaminic, and antiandrogenic properties [34].
Therefore, the current study aims to evaluate the antibacterial activities, physicochemical characterization, phytochemical screening, and gas chromatography-mass spectrometry (GC-MS) analysis of various propolis extracts from the chosen area.

Chemicals and Apparatus
All of the chemicals and reagents used were of analytical grade purity.Methanol, ethanol, nutrient agar, Mueller-Hinton agar (MHA), and dimethyl sulfoxide (DMSO) were purchased from Himedia (Mumbai, India).Other chemicals used in this experiment were of the highest quality and commercially available on the market.Streptomycin was also procured from Himedia (Mumbai, India).Throughout the media preparations and measurements, ultrapure water was used.

Collection of Propolis Sample
Crude propolis samples produced by Ceranaindica bees were obtained from honeybee colonies in Chunt-Waliwar, Ganderbal, Jammu, and Kashmir, India (Figure 1a,b).By scraping the surfaces of the walls, frames, entrances, and coverings, propolis was recovered from the beehive [35].Prior to analysis, the propolis sample was stored at −20 • C.

Extraction Procedure 2.3.1. Ethanolic Extract (EEKP)
The 30 g of powdered propolis was extracted using a maceration process with 100 mL of ethanol for at least 3 days.The extract was filtered by using Whatman No.1 filter paper.The extract was dried under pressure using a rotatory evaporator and kept at −20 • C prior to further analysis.

Methanolic Extract (MEKP)
The 30 g of powdered propolis was extracted using a maceration process with 100 mL of methanol for at least 3 days.The extract was filtered using Whatman No. 1 filter paper.The extract was dried under pressure using a rotatory evaporator and kept at −20 • C prior to further analysis.

Aqueous Extract (AqEKP)
About 30 g of Propolis was chopped into pieces and extracted with 100 mL of distilled water.The samples were heated for 5 min on a hot plate with constant agitation at 60 • C before being left at room temperature for a night before being filtered.The resulting mixture was filtered through Whatman filter paper No.1 and concentrated at low temperatures using the freeze-drying method.The extracted samples were kept at −20 • C before the analysis.

Identification and Quantification of Bioactive Compounds Using GC-MS
GC-MS analysis was performed by using GC-MS/MS-7000D Agilent (Agilent technologies, Santa Clara, CA, USA), equipped with Agilent J&W, GC-MS column HP-5Ms (15 m × 250 mm × 0.25 µm).The carrier gas was helium at a flow rate of 1 mL/min, and the injector temperature was 280 • C in split-less mode.The oven temperature was initially held at 60 • C for 4 min before being increased to 150 • C at a rate of 10 • C/min for 15 min.The following parameters were used to optimize the mass spectra: the source temperature is 280 • C, and the transfer temperature is 150 • C. The solvent delay time was 2 min, and the scan range was 35-500 Da.The temperature was finally raised to 310 • C. The GC's total run time was 40.5 min.By comparing their mass spectra to data from the National Institute of Standards and Technology (NIST) library, the compounds were identified.

Antibacterial Screening
The agar well diffusion method was used to assess the antibacterial activity of various propolis extracts [40].After being sterilized at 121 • C for 25 min, all the selected bacterial colonies were first sub-cultured in Nutrient Agar Media 2 (NAM) with a pH of 7.About 4 mL of the NAM was added to test tubes to produce the agar slants, which were then incubated at 37 • C overnight.Tubes containing 15 mL of MHA were inoculated with freshly prepared bacterial inoculums using a sterile loop to ensure uniform distribution of inoculums.The pre-inoculated medium was poured into Petri plates and allowed to solidify before cutting 8 mm wells with a sterile cork borer.The wells were filled with 100 µL of extract at various concentrations (100, 200, and 400 µg mL −1 ) and an equivalent volume of DMSO, which served as a negative control.After standing for 30 min to allow for pre-diffusion of the extract into the medium, the plates were incubated at 37 • C for 16-20 h.The inhibition zones on the plates were ascertained, and the findings were compared to the positive control containing streptomycin (10 µg mL −1 ).All assays were carried out in triplicate, and mean values were obtained.

Phytochemical Analysis
The propolis extracts were phytochemically analyzed and found to contain bioactive components, such as terpenoids, flavonoids, alkaloids, phenols, tannins, and saponins.The efficacy of the three solvents in extracting various components of propolis is compared in Table 1.

Antibacterial Activity
Table 3 and Figure 3 show the antibacterial properties of different extracts of Kashmiri propolis.EEKP, MEKP, and AqEKP had nearly identical antimicrobial activities.Small differences were observed in the majority of cases (1-3 mm).Only in the cases of E. coli and C. acnes did MEKP and AqEKP show no activity at 100 µg mL −1 .

Discussion
The propolis extracts used in the current study showed the existence of a range of bioactive components, including carbohydrates, aldehydes, flavonoids, alkaloids, terpenoids, alcohols, cardiac glycosides, tannins, coumarins, amino acids, phytobatanins, saponins, etc.These phytochemicals could be responsible for the pharmacological properties of the propolis extracts.Propolis is commercialized globally and is considered a significant source of phytochemicals that have pharmacological effects [41].The presence of 68 compounds from various groups was revealed by the GC-MS analysis, including flavonoids, flavonoid derivatives, terpenes, aromatic acids, and their related esters.
The extracts of propolis have a strong effect against bacteria, such as Enterococcus spp., Escherichia coli, and Staphylococcus aureus [42,43].In the present studies, the antibacterial activity of various extracts of propolis was tested against an array of bacterial strains.The EEKP was found to possess comparatively stronger antimicrobial activity than MEKP and AqEKP.The highest microbial activity in the EEKP may be due to the highest concentration of flavonoid and phenolic compounds, which may inhibit microorganisms [44].Another factor is that the majority of naturally occurring secondary metabolites are highly soluble in organic solvents; as a result, the ethanolic extract has the highest microbial activity, followed by MEKP and AqEKP [45].The propolis extracts had significant antibacterial activity against Staphylococcus aureus but no activity against E. coli, according to studies from China and Canada [46,47].The propolis ethanolic extracts have shown the highest levels of antimicrobial activity against a variety of microorganisms in Brazil [48].In Vietnam, propolis crude extract demonstrated significant antibacterial activity against S. aureus and inhibited E. coli at lower doses [49].This is because propolis contains flavones and flavonols that have been isolated from it [50], as well as large levels of terphenyl esters and hydroxybenzoic acid, both of which have antibacterial and antifungal properties [51].Components of propolis, such as Pinocembrin, exhibit antibacterial action against Streptococcus species, whereas p-Coumaric acid, artepillin,3-phenyl-4-di hydrocinnamylocinnamic acid inhibits H. pylori, and Apigenin substantially inhibits bacterial glycosyltransferase [52].

Conclusions
The present work is the first approach to identify numerous bioactive components by GC-MS analysis and to assess the antibacterial potency of various extracts of C. indica propolis from the Kashmir region.The GC-MS analysis of propolis has revealed the presence of 68 bioactive compounds that have a wide range of pharmacological potential, including antibacterial, antifungal, anti-protozoal, antioxidant, hepatoprotective, antiinflammatory, anticancer, and so on.The presence of these bioactive compounds in propolis is responsible for various therapeutic and pharmacologic properties in traditional medicine.More research is needed to isolate a specific substance that results in a positive result in a biological assay, and appropriate methodologies for in-depth studies should be developed.

Figure 1 .
Figure 1.(a) The image of map showing the location from where the C. indica propolis was collected.(b) The image of crude propolis sample produced by C. indica honeybees.

Table 3 .
Antimicrobial capacity of various extracts of propolis against tested bacterial strains using agar well diffusion method.

µg/mL) Zone of Inhibition Diameter (mm)
Results in the table are expressed in millimeters (mm) and each value is in triplicate represented as mean ± S.E.M.; -represents no zone of inhibition.EEKP: ethanolic extract of Kashmiri propolis; MEKP: methanolic extract of Kashmiri propolis; AqEKP: Aqueous extract of Kashmiri propolis.