1. INTRODUCTION:

Recently, many researches have indication on the pro-health and therapeutic effectiveness of phytochemical products, including bee products¹. Bee pollen is used as an antimicrobes, anti-viral, anticancer wound burns healing, , immune stimulator and local analgesic. Some studies have been shown that bee pollen and honey reduce the symptoms of menopausal in female and breast cancer patients, as it relieves the symptoms of menopause in patients who use aromatase inhibitors/ inactivators and tamoxifinn^2,3. Normally, bee pollen is collected by the worker bees and pressed into grains from flowers and trees, then transported to the hive. It is dampened with fragmented and saliva by flightless bees, packed in honeycomb cells, then covered with a thin layer of waxes and honey. The color of bee pollen grain varies from yellow to black^4,5.

There are many factors which contribute to the formation of bee pollen, including climates, plant source and the actions of beekeepers⁶. Pollen is an important source of lipids sacharides, proteins, cures fiber, phenolic compounds, miscellaneous, and minerals vitamins^7-9. Experiments were conducted on the laboratory mouse's that poisoned with ammonium fluoride, trichloroethylene, carbon tetrachloride, and ethionine have confirmed that the bee pollen act to remove the toxicity of organic solvents which destroy the liver cell as well as cause cirrhosis¹⁰. Other studies have confirmed that bee pollen is an anti-inflammatory that effectively inhibits the activity of lipoxygenase and cyclooxygenase, These enzymes are responsible for converting the arachidonic acid into a toxic materials such as leukotrienes and prostaglandin, where it is based on the formation of inflammation in the tissues cells. Because of that it was recommended in the treatment of inflammations, liver diseases and any symptoms of toxicity in this organ¹¹.

Monoamine oxidase (MAO ) as an enzyme (EC 1.4. 3.4) which is highly important enzyme in the metabolism. It is found in all tissues and responsible for the major neurotransmitters that degraded at the central nervous system (CNS). Monoamine oxidase (MAO ) catalyzes the deamination oxidation of amines that corresponding an aldehydes to produce H₂O₂and free amines¹².

In the present work, we studied bee pollen extracts effect on monoamine oxidase activity in human sera samples by using GC-Mass technique.

2. METHODS AND RELATED MATERIALS:

2.1. Bee pollen collection and its extracts preparation:

The pollen grain (bee pollen) was collected from Iraqi beekeepers, during the spring season of 2018. Extracts were prepared using two solvents, where (0.8g) of bee pollen was extracted with (50ml) water, and with (50 ml) absolute ethanol; then, stirred for 24 h at room temperature; after that, filtered in two steps: medicinal lint and filter paper.

2.2. Bee Pollen Extract for GC-Ms:

The extract of bee pollen that analyzed by GC-Ms spectroscopy was prepared by extracting the bee pollen constituents via a mixture of solvents consisted from (10 ml Hexane and 10 ml Ethyl acetate); then (0.5 gm) of bee pollen was added to the mixture. After 2.5 hours, the mixture was filtered. The color of these extracts ranges from deep clear yellow to slightly clear yellow.

2.3. Monoamine Oxidase Activity Assay:

MAO activity assay was implemented, manually according to Cohenand Newen's method¹³. Phosphate buffer solution (0.2 M) was prepared by dissolving (10.999 gm) of NaH₂PO₄ in distill water. Then, volume completed to (500 ml) with water and other solution was prepared by dissolving the (14.08 gm) of Na₂HPO₄ in distill water. After that, the volume was completed to (500 ml) with water. Next, (28 ml) from first solution was added to the (72 ml) of second solution and mixed. Then, the volume was completed to (200 ml) by water to give (pH=7.2). Finally, test and control solutions were prepared as below:

1. Take two test tubes the first is for test add 0.6 ml serum, 0.75ml MAO buffer and benzyl amine 0.15ml but the first is contro add 0.6ml serum and 0.75ml of MAO buffer.

2. Then putting it in a water bath and shaken for (3h) at 37° C. The following chemical solutions were added to both test and control solutions according to the.

3. Add to the test tube of test 0.15ml perchloric acid and 1.5ml of cyclohexane but the test tube of control add 0.15ml benzyl amine, 0.15ml perchloric acid and 1.5ml of cyclohexane.

Then, the mixture was isolated by centrifuged for 8 min. The absorbance was recorded of supernatant at 242 nm. The different volume of bee pollen extracts were (10, 25, 50, 75µl). MAO activity was measured in human serum by replacing (0.75ml) of buffer solution with (50 µl extract + 700 µl buffer). To estimate of Vmax and Km Lineweaver-Burk plot, we used different concentration of substrate (0.008, 0.006, 0.004, 0.0022 M), one time without inhibitor and another time with a constant concentration of inhibitor (50 µl).

The inhibition percentage of both enzymes were calculated according to the equation:

activity with extract

(%Inhibition = 100 - --------------------------------------------× 100) [18].

activity without extract

2.5. Gas Chromatography-Mass Spectrophotometry (GC-MS) analysis technique,

GC-Ms identification was implemented by Shimadzu GCMS-QP2010Ultra, and column (5Ms), under conditions, that temperature programmed (60, 180, 300)^°C at rate (0, 15, 10) with temperature rogrammed respectively, and column oven temp 60 ^°C. Injection mode splitless with total flow 20.7 ml/min and pressure 100 Kpa in hold time 1 min. GC was carried out under with high syringe speed conditions and injection mode splitless with pumping time 5 at injection temperature 150^° C with purge flow 30ml/min.

3. RESULTS AND DISCUSSION:

Recently studies about Bee pollen fixed information on its ingredients, that containing on materials could interact and reacted with many chemical compounds and act as anti-oxidants, therefore the experimented their effects on some enzymes that take part in oxidation reduction reactions. The results in the present study demonstrated the different volume on MAO activity, which the effects were clear and significant as inhibitors for two extracts (ethanolic and aqueous) on this enzyme. The maximum volume were used from two extracts from stock solution which equal to (75µl), which gave maximum inhibition percentage and shown in figure (1, 2) and table (1), that equal to (57%) for MAO and when using aqueous extract, and (79%) for MAO and when using ethanolic extract. The kinetic parameters also were calculated according to the Line-weaver burk plot in table (2) from figure (3, 4), and the results in the presence of inhibitors that recorded in the present study equal to (km =0.011M) (v_max =66) IU by using aqueous extracts, and (km =0.018 M), (v_max =28) IU by using ethanolic extract for MAO activity.

Table (1): Effect of different volumes of aqueous and alcoholic Bee pollen extracts on activity of MAO in human sera.

Aqueous of extract volume (µl)	MAO activity (IU/ml)	Inhibition %
0.00	38.16	0.00
10	36.68	3.87
25	31.44	17.61
50	23.73	37.81
75	16.28	57.33
Alcoholic extract volume (µl)	MAO activity (IU/ml)	Inhibition %
0.00	38.16	0.00
10	27.15	28.85
25	19.11	49.92
50	11.30	70.38
75	7.64	79.97

Figure (1): MAO inhibition percentages with different volume of aqueous extract.

Figure (2): MAO inhibition percentages with different volume of ethanolic extract.

Table (2): Kinetic parameter; Km, V max, and type of inhibition for aqueous and alcoholic bee pollen extracts on MAO activity.

Enzyme	Km (M)	*Vmax (mol/ml/min)*	Type of inhibition
MAO aq.	0.011	66.7	Competitive
MAO alc.	0.018	28.5	Mixed inhibition

Figure (3):Lineweaver – Burk plot at the presence and abcence of bee pollen with aqueous extract for MAO.

Figure (4): Lineweaver – Burk plot at the presence and abcence of bee pollen alcoholic extract for MAO.

Our results showed that alcoholic extract have a higher activity than an aqueous extract on the enzyme because alcoholic extract as an organic solvent have been able to extract more compounds that may be cause the enzymes inhibition.

People who had been take antidepressants drugs may undergoing from crisis of hypertensive, and it may be fatal¹⁴. Due to of bee pollen have phenolic compounds, it have anti-Parkinson effects¹⁵. Also, bee pollen is not just useful as a dietary supplementation but as a functional one, therefore we recommend to carry out more studies about bee pollen extract and using it to treat some disease such as inflammation, cancer and depression because its inhibition of MAO and where MAO regulates serotonin and catecholamin in nervous system ^[16]. Bee pollen is rich in polyphenols¹⁷; which may be responsible inhibitory effect for their extracts. Due to the hydrophobic interaction between phenolic compounds and non polar groups in the active site of MAO, that consists of 420A^° hydrophobic substrate cavity intercorrected to an entrance cavity of 290A^° and the obvious site for the amino group of substrate is a tyrosine aromatic cage¹⁸. The important role of polyphenol in chemical reaction behavior is its ability to form a complex with metal ion¹⁹. interaction of polyphenols with iron ion that incorporated with chemical structure of enzymes, therefore these polyphenols prevent active reactions of enzymatic reaction and finally inhibition of enzyme²⁰.

On the other demonstration, the results in the present study demonstrated the Bee pollen ingredients compounds that may be founded in comparison with library; which containing on 40 compounds, as shown in table (3) that depended on chromatogram in figure (5). GC-Ms library comprised very large approximately two hundred thousand. These peaks refer to the identity of these constituents that have been fixed in other studies, which 95% of total mass identified by this technique²¹. Because this library allows impermanent identification of many basic compounds in Bee pollen components. The temperature programmed that used in the present study may be yield a different compound than other studies. Because low temperature program was giving isothermal retention is not over 1% terpenoids²². Also the obtained results of components in extracts differs from other studies belonged to the using other solvent to extraction. Which sugar compounds equal to 54% and poly unsaturated fatty acids were a little amount 1.1% when using ethanol as a solvent²³.

Figure (5): TIC chromatogram for Bee pollen, extracted via (1:1 Hexane and Ethyl acetate). Which the numbers indicated to these compounds in table (5) that obtained by a GC–MS analysis.

Table (3): Bee pollen constituents with data that obtained identified by GC-Ms analysis, which extracted via (1:1 Hexane and Ethyl acetate).

Peak	R. Time	Area	Area%	Height	Height %	Name
1	3.06	6227124	54.89	875674	34.21	E,E,Z-1,3,12- Nonadecatriene-5,14—diol
2	3.85	101108	0.89	48624	1.90	Acetic acid, butyl ester
3	4.16	461633	4.07	241418	9.43	2-Hexanol, 2-methyl-
4	4.57	88607	0.78	30915	1.21	p-Xylene
5	5.75	50183	0.44	6597	0.26	Heptadecane, 7-methyl-
6	6.08	89517	0.79	53130	2.08	Decane
7	7.28	65753	0.58	26721	1.04	Nonane,4,5-dimethyl-
8	7.55	60233	0.53	12227	0.48	4,6- Dinitro-2-(4-trifluromethoxy-phenyl)-2H-indazole
9	7.58	53537	0.47	25410	0.99	Silane, dimethyl(undec-2-enyloxy)heptadecyloxy
10	8.32	94246	0.83	36758	1.44	Methyl 4,6-decadieny ether
11	8.42	63772	0.56	20011	0.78	4-Hydroxy-4-methylhex-5-enoic acid, tert. -butyl ester
12	8.60	60951	0.54	38508	1.50	4,6-Methanocyclobuta[cd]cyclopropa[3,4]benzo[1,2-a]pentalene-5(1H)-one, la,2-bis(acetyloxy)-6-
13	8.75	47346	0.42	6428	0.25	1,3-Dihydro-5-(2-nitrophenyl)-2H-1,4-benzodiazepin-2-one
14	10.57	106755	0.94	52734	2.06	l-Tetradecene
15	12.02	215416	1.90	97344	3.80	Phenol, 2,4-bis(1,1-dimethylethyl)-
16	12.95	257126	2.27	72502	2.83	n-Pentadecanol
17	14.73	50602	0.45	9723	0.38	l-Hecanethiol, 2-(9-borabicyclo[3.3.1]non-9-yloxy)-
18	15.30	109964	0.97	46631	1.82	n-Nonadecanol-1
19	17.05	46462	0.41	9382	0.37	Methyl 2-hudroxy-pentadecanpate
20	17.22	64466	0.57	15407	0.60	1,2-benzenedicarboxylic acid, ditridecyl ester
21	17.49	69664	0.61	25498	1.00	9-Tricosene,(Z)-
22	18.42	54439	0.48	19677	0.77	3-Chloropropionic acid, heptadecyl ester
23	18.62	50546	0.45	7388	0.29	Silanamine, 1,1, l-trimethyl-N-(phenylmethyl)-
24	19.49	91490	0.81	37650	1.47	n-Nonadecanol-1
25	20.47	90113	0.79	35239	1.38	Eucosane
26	20.63	852078	7.51	160188	6.26	2,6,10,14,18,22-Tetracoshexaene,2,6,10,15,19,23-hexamethyl-, (all-E)
27	21.36	250050	2.20	62366	2.44	Eicosane
28	21.47	55317	0.49	11789	0.46	l-Cyano-7methyl-4,6,7,1 lb-tetrahydro-5H-spiro[isoxazolidino[3,2-a]benz-2-azepin-5-cyclohexane
29	21.68	54449	0.48	13261	0.52	1-[2,4Bis(trimethulsiloxy)phenyl]-2-[(4-trimethylsiloxyphenyl] propan-l-one
30	21.81	118987	1.05	28609	1.12	Octadecane, 9-ethyl-9-heptyl-
31	21.92	121231	1.07	25268	0.99	6-methoxy-9H-purine tbdms
32	22.03	117963	1.04	31817	1.24	2,4,6-Cycloheptatrien-l-one, 3,5-bis-trimethylsilyl
33	22.06	46019	0.41	31662	1.24	Benzoicacid, 2,3-bis[(trimethylsilyl)oxy]-, trimethylsilyl ester
34	22.11	81399	0.72	32901	1.29	2’,4’-Dihydroxyacetophinone,bis(trimethylsilyol ester
35	22.2	615159	5.42	186903	7.30	Eicosane
36	22.35	75517	0.67	29985	1.17	Silicie acid, diethyl bis(trimethysilyl) ester
37	22.39	47435	0.42	28865	1.13	1,2-Bis(trimethylsilyl)benzene
38	22.47	128062	1.13	26176	1.02	Pentasiloxane. Dodecamethyl-
39	22.54	60965	0.54	21369	0.83	Cyelotrisilocane, hecamethyl
40	22.59	48745	0.43	16890	0.66	Cyelotrisiloxane, hecamethyl-
		1134429	100.00	2559651	100.0

4. CONCLUSION:

Bee pollen extract can be used to treat depression because it has an effect on inhibiting of AMO and, inflammation and cancer because it is act as aromatase inhibitors.

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Received on 02.02.2021 Modified on 19.12.2021

Research J. Pharm. and Tech. 2022; 15(8):3731-3735.

DOI: 10.52711/0974-360X.2022.00625

Effect of Aqueous and Alcoholic Bee Pollen Extracts on Monoamine Oxidase Activity

2.1. Bee pollen collection and its extracts preparation: