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

Abstract

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 µg 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.

1. 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.

2. Materials and Methods

2.1. 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.

2.2. 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.

2.3. 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.

2.3.2. 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.

2.3.3. 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.

2.4. Qualitative Phytochemical Analysis

The different extracts of propolis were subjected to qualitative analysis to identify its constituents, including alkaloids, saponins, tannins, flavonoids, terpenoids, phlobatanins, proteins, and carbohydrates, by following standard protocols [36,37,38,39].

2.5. 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.

2.6. 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.

2.7. Bacterial Strains

The five bacterial strains used in this study, viz. Pseudomonas aeruginosa (MTCC1688), Escherichia coli (MTCC443), Klebsiella pneumoniae (MTCC19), Cutibacterium acnes (MTCC843), and Staphylococcus aureus (MTCC96) were obtained from the Institute of Microbial Technology (IMTECH), Chandigarh, India. As a positive control, streptomycin (10 µg mL⁻¹) was used.

3. Results

3.1. 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.

3.2. GC-MS Analysis

Since EEKP produced the maximum number of phytochemicals when compared to MEKP and AqEKP, GC-MS analysis was performed to determine EEKP’s chemical profile. The GC-MS chromatogram of C. indica propolis extract shown in Figure 2 shows a total of 68 peaks corresponding to bioactive compounds identified by comparing their mass spectral fragmentation patterns to those of known compounds described in the NIST library. The chemical constituents identified in Table 2 are listed by retention time, peak area (%), and molecular weight. The most common phyto-constituents found in propolis extract are alpha-D-Mannopyranoside, methyl, cyclic 2,3:4,6-bis-ethyl boronate (21.17%), hexadecanoic acid, methyl ester (9.91%), nona-2,3-dienoic acid, ethyl ester (4.75%), bacteriochlorophyll-c-stearyl (4.41%), 10-methyldodecan-4-olide (3.89%), and nickel, cyclopentadienyl-1,2,3-trimethylallyl (3.12%).

3.3. 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.

4. 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].

5. 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, anti-inflammatory, 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.

Author Contributions

Conceptualization, I.M. and F.S.; methodology, I.M., T.R.K., M.I.Z. and P.A.; software, S.A. and F.S.; validation, S.U.D.W., F.S., S.A. and W.A.M.; formal analysis, S.U.D.W.; investigation, T.R.K. and M.I.Z.; resources, W.A.M. and S.A.; data curation, P.A.; writing—original draft preparation, I.M.; writing—review and editing, F.S., S.A. and W.A.M.; visualization, W.A.M.; supervision, I.M.; project administration, I.M.; funding acquisition, W.A.M. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

Authors are thankful to the Researchers Supporting Project number (RSP2022R516), King Saud University, Riyadh, Saudi Arabia, for supporting this work.

Conflicts of Interest

The authors declare no conflict of interest.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures and Tables

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.

Figure 2. Gas chromatography–mass spectrometry (GC-MS) chromatogram of significant propolis compounds.

Figure 3. The zone of inhibition (mm) of different extracts of C. indica propolis against the selected bacterial strains. (A) EEKP vs. P. aeruginosa. (B) EEKP vs. S. aureus. (C) EEKP vs. K. pneumoniae. (D) MEKP vs. K. pneumoniae. (E) MEKP vs. P. aeruginosa. (F) AqEKP vs. S. aureus. EEKP: ethanolic extract of Kashmiri propolis; MEKP: methanolic extract of Kashmiri propolis; AqEKP: Aqueous extract of Kashmiri propolis.

Table 1

Phytochemical screening of propolis extracts.

Phytoconstituents	EEKP	MEKP	AqEKP
Alkaloids	++	++	+
Tannins	++	+	+
Saponins	++	++	+
Terpenoids	+	+	-
Flavonoids	++	++	+
Phlobatanins	++	-	-
Anthraquinones	+	+	+
Carbohydrates	++	+	+
Resins	++	+	-
Coumarins	+	+	+
Quinones	++	-	-
Proteins	+	+	-

EEKP: ethanolic extract of Kashmiri propolis; MEKP: methanolic extract of Kashmiri propolis; AqEKP: aqueous extract of Kashmiri propolis; ++: abundant, +: moderate, -: absent.

Table 2

GC-MS analysis used to identify bioactive compounds in propolis extract (mean ± SD, n = 3.0).

S. N.	Phytocompounds	Molecular Formula	Molecular Weight (g/mol)	Retention Time (min)	CAS Number	Peak Area ± SD (%)
1	2-Azido-2,4,4,6,6-pentamethylheptane	C12H25N3	211.35	3.874	1000293-29-0	1.17 ± 0.01
2	2-Azido-2,4,4,6,6,8,8hepta methylnonane	C16H33N3	267.45	3.974	1000293-29-1	0.44 ± 0.00
3	Methyl 9,10-octadecadienoate	C19H34O2	294.5	4.097	1000336-45-7	0.36 ± 0.00
4	Methyl 8,9-octadecadienoate	C19H34O2	294.5	4.349	1000336-45-0	1.84 ± 0.02
5	Cyclohexane, 1,3-butadienylidene	C10H14	134.22	4.620	53864-08-7	0.75 ± 0.01
6	Methyl 10,11-octadecadienoate	C19H34O2	294.5	5.292	1000336-45-3	1.14 ± 0.01
7	1-Propanimine, N-(2-ethylcyclohexyl-, N-oxide	C11H21NO	183.29	12.163	1000187-25-8	0.18 ± 0.00
8	Tridecanoic acid, 12-methyl-, methyl ester	C15H30O2	242.4	14.146	5129-58-8	1.55 ± 0.01
9	Decanoic acid, silver(1+) salt	C10H19AgO2	279.12	14.944	13126-67-5	0.98 ± 0.00
10	Methyl 13, 14- octadecadienoate or 13,14-18:2	C19H34O2	294.5	15.961	1000336-46-2	1.42 ± 0.01
11	i-propyl 12-methyl-trideca	C17H34O2	270.5	16.136	1000336-60-4	0.45 ± 0.00
12	4-n-Hexylthiane, s,s- dioxide	C11H22O2S	218.36	16.746	70928-52-8	0.83 ± 0.00
13	Cyclopentyl-methyl-phosphinic acid,2-isopropyl-5-methyl-cyclohexyl ester	C16H31O2P	286.39	17.079	1000194-56-2	0.97 ± 0.00
14	4(axial)-Ethenyl-1,2(equatorial)-dimethyl-trans-decahydroquinol-4-ol,N-oxide	C13H23NO2	225.33	17.734	62299-73-4	0.14 ± 0.00
15	Hexadecanoic acid, methyl ester	C17H34O2	270.5	18.080	112-39-0	9.91 ± 0.12
16	.alpha,-D-Mannopyranoside, methyl, cyclic 2,3:4,6-bis(ethyl boronate)	C11H20B2O6	269.9	18.900	61553-44-4	21.17 ± 0.20
17	Cis-2-methyl-4-n-butylthiane, s,s-dioxide	C10H20O2S	218.36	19.362	1000215-67-7	2.21 ± 0.02
18	Borinic acid, diethyl-,1-cyclododecen-1-yl ester	C16H31BO	250.2	20.467	61142-73-2	1.11 ± 0.01
19	Trihexadecyl borate	C48H99BO3	735.1	20.751	2665-11-4	0.17 ± 0.00
20	Nona-2,3-dienoic acid, ethyl ester	C11H18O2	182.26	21.323	1000187-19-2	4.75 ± 0.04
21	Methyl 10,11-tetradecadienoate	C15H26O2	238.37	21.710	1000336-31-8	1.95 ± 0.01
22	1-Hexadecanaminium, N,N,N-trimethyl-,octadecanoate	C37H77NO2	568	21.775	124-23-2	0.70 ± 0.00
23	Methyl 8-methyl-decanoate	C12H24O2	200.32	21.943	1000336-49-1	0.21 ± 0.00
24	Tropine N-oxide	C8 H15NO2	157.21	23.019	35722-43-1	1.12 ± 0.01
25	Ethyl trans-4-decenoate	C12H22O2	198.3	23.112	76649-16-6	1.15 ± 0.01
26	1,2-Oxathiane, 6-dodecyl-, 2,2-dioxide	C16H32O3S	304.5	24.395	15224-88-1	0.10 ± 0.00
27	Ethyl hydrogen dimethylamidophosphate, sodium salt	C7H20NO3PSi	175.1	24.750	1000445-96-4	1.30 ± 0.01
28	9-Borabicyclo[3.3.1]nonane, 9(3-methoxycyclohexyl)oxy-	C15H27BO2	250.19	24.902	1000150-57-9	0.50 ± 0.00
29	1,2:5,6-Di-O-ethylborandiyl-D-glucohexodialdose	C10H16B2O6	253.9	25.545	74143-57-0	0.23 ± 0.00
30	11-Dodecen-1-ol, 2,4,6-trimethyl-, (R,R,R)-	C15H30O	226.4	25.890	27829-54-5	0.11 ± 0.00
31	9-Tetradecenal, (Z)-	C14H26O	210.36	26.840	53939-27-8	1.73 ± 0.01
32	10-Methyldodecan-4-olide	C13H24O2	212.33	26.937	1000370-40-6	3.89 ± 0.03
33	Boroxin, tripropyl	C9 H21B3O3	209.7	27.535	7325-08-8	0.19 ± 0.00
34	Methyl 3,4-tetradecadienoate	C15 H26O2	238.37	27.573	1000336-28-1	0.35 ± 0.00
35	Cyclohexanecarboxylic acid, 2-hydroxy-, monoannhydride with 1-butaneboranic acid, cyclic ester, trans-	C11H19BO3	210.1	27.922	24372-06-3	1.16 ± 0.01
36	3,9-Dispiro[5,5]undecane, 3,9-diethyl-, 2,4,8,10-tetraoxa-3,9-dibora-	C9H18B2O4	211.9	27.987	58163-67-0	0.82 ± 0.00
37	Methyl myristoleate	C15H28O2	240.38	28.132	56219-06-8	0.38 ± 0.00
38	Bacteriochlorophyll-c-stearyl	C52H72MgN4O4	841.5	28.429	1000164-49-7	4.41 ± 0.06
39	alpha,-D-Xylofuranose, cyclic 1,2,:3,5-bis(butylboronate)	C13H24B2O5	282	28.559	52572-01-7	0.16 ± 0.00
40	Z-11-Tetradecenoic acid	C14H26O2	226.35	29.615	1000130-83-3	1.63 ± 0.01
41	Chloroacetic acid, dodecyl ester	C14H27ClO2	262.81	30.061	6316-04-7	0.33 ± 0.00
42	Ethyl 1-(8-amino-1-naphthyl)-1,2,3-triazole-4-carboxylate	C15H14N4O2	282.3	30.539	116114-01-3	1.78 ± 0.01
43	Nickel, 2,6,10-dodecatriendi-1,12-diyl-	C12H18Ni	220.97	30.901	39330-67-1	0.12 ± 0.00
44	Iron, tricarbonyl-(1,4,5,6,-eta,-4,5-dimethyl-4-hexane-1,6-diyl-1-carboxylic acid, ethyl ester), (endo)-	C14H18FeO5	322.13	30.917	1000149-58-0	0.86 ± 0.00
45	7-Hexadecyn-1-ol	C16H30O	238.41	31.179	822-21-9	1.59 ± 0.01
46	Nickel, cyclopentadienyl-1,2,3-trimethylallyl-	C11H16Ni	206.94	31.392	77932-64-0	3.12 ± 0.02
47	11,13-Dimethyl-12-tetradecen-1-ol acetate	C18H34O2	282.5	31.608	1000130-81-0	0.73 ± 0.00
48	3-Methylpyrazolobis(diethylboryl) hydroxide	C12H26B2N2O	236.0	31.715	1000159-71-9	0.37 ± 0.00
49	1-[3-(2,6,6-trimethyl-cyclonex-2-enyl)-4,5-dihydro-3H-pyrazol-4-yl]-ethanone	C14H22N2O	234.34	31.964	1000185-64-0	2.13 ± 0.02
50	Pinane-2,3-diol, 2,3-o-ethaneboronate-	C12H21BO2	208.11	32.077	1000158-69-2	0.21 ± 0.00
51	1H-1,2,3,4-Tetrazole, 5-[(2-methoxyphenoxy)methyl]-	C9H10N4O2	206.2	32.102	1000337-41-2	1.24 ± 0.01
52	1,1-Bicyclohexyl, 4-propoxy-4-propyl-	C18H34O	266.5	32.290	98321-58-5	1.60 ± 0.01
53	Methyl 10,11-tetradecadienoate	C15H26O2	238.37	32.484	1000336-31-8	0.20 ± 0.00
54	Tris(tert-butyldimethylsilyloxy)arsane	C18 H45AsO3Si3	468.7	33.414	1000366-57-5	1.00 ± 0.01
55	9-Borabicyclo[3.3.1]nonane, 9-[3-(dimethylamino)propyl]-	C13H26BN	207.2	33.718	1000160-15-2	2.51 ± 0.03
56	Methyl 12,13-tetradecadienoate	C15H26O2	238.37	33.792	1000336-33-7	0.27 ± 0.00
57	Methyl 8,9-octadecadienoate	C19H34O2	294.5	34.063	1000336-45-0	0.98 ± 0.00
58	Methyl 4,5-tetradecadienoate	C15H26O2	238.37	34.125	1000336-28-7	0.10 ± 0.00
59	.beta,-D-Mannofuranoside, 2,3:5,6-di-ethylboranediyl-cis-nerolidyl	C25H42B2O6	460.2	34.528	1000155-68-3	0.93 ± 0.00
60	Undec-10-ynoic acid,octyl ester	C19H34O2	294.5	34.599	1000406-16-1	1.88 ± 0.01
61	Methyl 3-cis,9-cis,12-cis-octadecatrienoate	C19H32O2	292.5	35.019	1000336-38-4	0.28 ± 0.00
62	Z,E-2-methyl-3,13-octadecadien-1-ol	C19H36O	280.5	35.074	1000131-10-5	0.29 ± 0.00
63	2,2-Dimethyl-6-methylene-1-[3,5-dihydroxy-1-pentenyl]cyclohexan-1-perhydrol	C14H24O4	256.339	35.436	1000212-02-6	0.25 ± 0.00
64	9,12-octadecadienoyl chloride, (z,z)-	C18H31ClO	298.9	35.633	7459-33-8	0.34 ± 0.00
65	1-(4-Amino-furazan-3-yl)-5-methoxymethyl-1H-[1,2,3]triazole-4-carboxylic acid hydrazide	C7H10N8O3	254.21	36.005	1000300-77-2	0.22 ± 0.00
66	Iron, tris, (eta,3-2-propenyl)-	C9H15Fe	179.06	37.255	94139-77-2	1.13 ± 0.01
67	E,E,Z-1,3,12-Nonadecatriene-5,14-diol	C19H34O2	294.5	37.342	1000131-11-4	0.26 ± 0.00
68	Phenylboronic acid, 2TMS derivative	C12H23BOSi2	266.29	39.981	7560-51-2	0.30 ± 0.00

Table 3

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

Quantity of Extract (µg/mL)	Zone of Inhibition Diameter (mm)
	Bacterial Strains
	S. aureus	P. aeruginosa	K. pneumonae	E. coli	C. acnes
EEKP
100	12.33 ± 0.33	11.00 ± 0.58	11.33 ± 0.33	09.33 ± 0.33	09.67 ± 0.67
200	14.67 ± 0.67	13.33 ± 0.33	13.67 ± 0.67	11.67 ± 0.33	11.67 ± 0.33
400	17.33 ± 0.67	16.33 ± 0.67	15.67 ± 0.33	13.67 ± 1.20	14.00 ± 0.58
MEKP
100	11.67 ± 0.88	10.33 ± 0.33	10.00 ± 0.58	-	-
200	14.00 ± 0.58	12.67 ± 0.33	12.00 ± 1.15	11.00 ± 0.58	10.67 ± 0.33
400	16.00 ± 0.58	15.00 ± 0.58	14.33 ± 0.33	13.00 ± 0.58	13.33 ± 0.33
AqEKP
100	10.67 ± 0.88	07.67 ± 3.84	07.00 ± 3.51	-	-
200	13.00 ± 0.58	11.67 ± 0.33	11.67 ± 0.33	6.33 ± 3.18	10.33 ± 0.33
400	14.67 ± 0.33	13.67 ± 0.67	13.33 ± 0.33	10.67 ± 0.88	12.33 ± 0.33
Streptomycin (10 µg/mL)	19.33 ± 0.88	19.67 ± 0.33	21.00 ± 0.58	18.33 ± 0.33	19.33 ± 0.67

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.

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