Keywords

Mushroom, Bioactive components, Extraction methods, Solvents, extracts

Introduction

Different plants and fungal materials have been used in traditional medicine to solve varieties of health problems. The effectiveness of various combinations of herbs and other ingredients used by traditional medical practitioners depends on the constituents of the selected plant or fungi materials. The availability of the constituents is a factor of the adopted extraction method. The extraction of a given compound from a substrate depends on both the solubility of the compound and the polarity of the solvent. Modern extraction techniques such as accelerated solvent extraction, supercritical fluid extraction, microwave-assisted extraction and ultrasound-assisted extraction are presently explored in nutraceuticals extractions from plants. These modern techniques are mainly targeted at decreasing extraction time, while increasing yield and enhancing extract’s quality with a reduced solvent consumption. Dadi et al. [1] improve the total phenolic content, total flavonoid content, and antioxidant activity assessed from Moringa stenopetala leaves by optimizing ultrasonic-assisted extraction using response surface methodology. They recorded an extraction time of 26 min, 68 % ethanol concentration, and solvent-to-sample ratio of 42 mL/g.

Oil extraction is mainly a diffusion process in which the solvent penetrates into the lipid containing cells of a raw sample material, thereby forming a solution of the oil in the solvent. Though some of these plants and fungal materials are still very relevant in traditional medicine for treatment of diseases mainly of microbial related infections [2], most of their isolates have shown potential anti-inflammatory and antidiabetic properties in preliminary studies [3].

Pleurotus tuberregium is a notable saprotroph that produces a food storage sclerotium upon its consumption of decaying wood [4]. It is one mushroom that both its basidiocarp and sclerotium are of economic importance due to their nutritive and medicinal properties [4]. The extract of this notable fungus has been used in traditional combinations for the treatment and management of diseases ranging from skin infections, inflammation, childhood malnutrition, headache, stomach problem, cold, asthma, fever, high blood pressure, diabetes and small pox [5]. Isikhuehmen and LeBauer [6] reported the use of this mushroom and its extract in Eastern Nigeria as a thicker and flavoring agent for soups and for the treatment of heart related ailments, while it is used for the treatment and management of asthma, cough and obesity in the Southern part of Nigeria [6]. Aside Nigeria and Africa, P. tuber-regium also grows in Asia and Australia [7].

Other therapeutic activities such as antitumour, immunomodulatory, antioxidant, anti-inflammatory, hypocholesterolaemic, antihypertensive, antihyperglycaemic, antimicrobial and antiviral activities Pleurotus spp. has also been reported [8]. Though these therapeutic activities are exhibited by the extracts or isolated compounds, fermentation broth, mycelia and fruiting bodies of Pleurotus spp. [9], the biochemical mechanisms are still elusive due poor characterization and identification of the bioactive components. The aim of this present study is to extract the bioactive components in the sclerotum of P. tuber-regium using different solvents, to analyze the extracts obtained for identification of the bioactive components and to ascertain the solvent that gives a better yield.

Materials and Methods

Sample collection, preparation and extraction

A quantity of 10.0 kg of fresh sclerotia of Pleurotus tuberregium purchased at Zarama Market in Southern Nigeria was washed, peeled and the white inner parts were sliced using a sterilized knife. The sliced samples were allowed to dry at room temperature in a dust free environment for a period of fourteen days. The samples were ground in a warring blender into a fine powder. Using an analytical weighing balance, 10 g of the ground sample was weighed into three well stopper bottles. A volume of 20 mL of a specific extraction solvent (methanol, hexane and dichloromethane) was added to each stopper bottle. The mixtures were vigorously agitated and were left to stand for 5 days, while that of soxleth extraction was done in a soxleth apparatus, using ethanol as the solvent. The crude extract from each process was collected by filtering into a quartz beaker in a fume hood. The process was repeated twice and the combined aliquot collected from each extraction solvent were separately concentrated on a steam berth to 5.0 mL and purified by passing through a pasture pipette packed with silica gel and anhydrous sodium sulphate. The purified samples were air dried to 2.0 mL for gas chromatographic analysis.

GC-MS analysis of extracts

The extracts were analysed using a combined gas chromatograph model HP 6890 and mass spectrometer model 5973 (Agilent Tech.) fitted with a capillary column HP-5 MS (5% phenylmethylsiloxane) 30.0 m × 250 μm × 0.25 μm, using Helium as a carrier gas at initial column temperature 120°C for 5 minutes. Thereafter, the column temperature was increased at 5°C per minutes to 320°C and held for 5 minutes. Electron impact ionization for mass spectroscopy was done at ionization energy of 70 eV. The oil was diluted with 98% hexane and 2 μL of the diluted sample was automatically injected into Agilent Tech. model 5973 mass spectrometer. The constituent compounds were identified using the Chem-Office software attached tothe MS library. The names and structures of the component oils were confirmed using the database of National Institute of Standard and Technology (NIST).

Results

The Chromatogram of bioactive components of methanol, hexane, dichloromethane and soxhlet extracts of the sclerotia of P. tuberregium are shown in Figures 1-4, respectively. The highest peak for the methanol extract was observe at 32.644 min. Hexane extract has its highest peak at 31.459 min., while dichloromethane and soxhlet extracts have their highest peaks at 14.254 min. and 18.060 min. respectively.

Figure 1: Chromatogram of bioactive components of methanol extracts of the sclerotia of P. tuberregium.

Figure 2: Chromatogram of bioactive components of hexane extract of the sclerotia of P. tuberregium.

Figure 3: Chromatogram of bioactive components of dichloromathane extract of the sclerotia of P. tuberregium.

Figure 4: Chromatogram of bioactive components of sohxlet extract of the sclerotia of P. tuberregium.

The retention time, percentage concentration, molecular formula, molecular weight and structures of the bioactive components of methanol, hexane, dichloromethane and soxhlet extracts of the sclerotia of P. tuberregium are shown in Tables 1-4. (3aR, 4R, 7R)-1,4,9,9-Tetramethyl-3, 4,5,6,7,8-hexahydro-2H-3a,7-methane was the highest bioactive component in the methanol extract with a value of 62.856 % followed by Urs-12-en-28-oic acid, 3-hydroxy-,methyl ester, (3.beta.)- With a value of 23.151 %. Hexane extract has it highest bioactive component as Hexasiloxane, tetradecamethyl- with a value of 29.170 % followed by Hexacosane, 13-dodecyl- with a value of 28.339 %, while Bis(2-ethylhexyl) phthalate and Eicosane were the highest bioactive components observed in dichloromethane extract with values of 5.092% and 4.721% respectively. The highest bioactive component observed in the sohxlet extract was Phthalic acid, 3-chlorobenzyl butyl ester with a value of 25.490 % followed by 1,4-Naphthalenedione, 5,8-dihydroxy- 2,7-dimethoxy- with a value of 23.513 %.

S/N	Compound	Retention Time (min)	Percentage of the total	Molecular formula	Molecular weight	Structure
1	2H-Cyclopropa[a]naphthalen-2-one	32.366	13.992	C15H24	204.3511
2	(3aR,4R,7R)-1,4,9,9-Tetramethyl-3, 4,5,6,7,8-hexahydro-2H-3a,7-methane	32.644	62.856	C8H15N3	153.22
3	Urs-12-en-28-oic acid, 3-hydroxy-, methyl ester, (3.beta.)-	33.498	23.151	C30H48O3	456.700

Table 1: Bioactive components of methanol extract of sclerotia of P. tuberregium.

S/N	Compound	Retention Time (min)	Percentage of the total	Molecular formula	Molecular weight	Structure
1	Cyclododecane	11.964	0.300	C12H2	168.3190
2	Nonane, 2-methyl-	12.537	0.401	C10H22	142.2817
3	Undecane	13.133	0.363	C11H24	156.3083
4	Dodecane, 2,6,10-trimethyl-	13.196	1.234	C15H32	212.4146
5	Octadecane, 1-chloro-	14.246	0.435	C18H37Cl	288.939
6	Tetratetracontane	14.351	0.764	C44H90	619.1854
7	1,1,1,5,7,7,7-Heptamethyl-3,3-bis(trimet hylsiloxy)tetrasiloxane	14.641	0.549	C13H40O5Si6	444.967
8	Hexadecanoic acid, methyl ester	15.784	0.688	C17H34O2	270.4507
9	n-Hexadecanoic acid	16.501	7.223	C16H32O2	256.4241
10	Cycloheptasiloxane, tetradeca methyl-	16.562	1.909	C14H42O7Si7	519.0776
11	Hexadecanoic acid, ethyl ester	16.667	1.913	C18H36O2	284.4772
12	9,12-Octadecadienoic acid,methyl ester, (E,E)-	18.031	0.765	C19H34O2	294.4721
13	9-Octadecenoic acid, methyl ester (E)-	18.116	0.643	C19H36O2	296.4879
14	Methyl stearate	18.501	0.600	C19H38O2	298.511
15	Heptasiloxane, hexadeca methyl-	18.672	1.149	C16H48O6Si7	533.1472
16	Linoelaidic acid	18.877	8.730	C18H32O2	280.4455
17	9,17-Octadecadienal, (Z)-	18.955	3.233	C18H32O	264.4461
18	Linoleic acid ethyl ester	18.981	4.337	C20H36O2	308.4986
19	9,12-Octadecadienoic acid (Z,Z)-	19.081	3.668	C18H32O2	280.4455
20	Octadecanoic acid	19.234	1.839	C18H36O2	284.4772
21	Hexadecanoic acid, 2-methyl propyl ester	19.346	1.622	C20H40O2	312.5304
22	Cycloeicosane	19.522	2.099	C20H40	280.5316
23	Cyclononasiloxane, octadeca methyl-	20.865	1.227	C18H54O9Si9	667.3855
24	Tricosane	21.009	2.206	C23H48	324.6272
25	Tetracosane	22.515	4.205	C24H50	338.6538
26	3-Isopropoxy-1,1,1,5,7,7,7-Heptamethyl-3,3-bis(trimet hylsiloxy)tetrasiloxane	23.052	1.645	C18H52O7Si7	577.200
27	Pentacosane	24.016	5.347	C25H52	352.6804
28	2,5-Dihydroxy benzoic acid, 3TMS derivative	25.115	2.085	C16H30O4Si3	370.6635
29	Hexacosane	25.342	4.261	C26H54	366.7070
30	Heptacosane	26.463	2.728	C27H56	380.7335
31	Cyclononasiloxane, octadeca methyl-	26.720	2.297	C18H54O9Si9	667.3855
32	Docosane, 7-hexyl-	27.448	1.261	C28H58	394.7601
33	2,6,10-Trimethyltridecane	27.700	1.004	C16H26	218.3776
34	Cyclononasiloxane, octadeca methyl-	28.042	0.904	C18H54O9Si9	667.3855
35	Hexacosane, 13-dodecyl-	0.737	28.339	C38H78	535.0259
36	Ergost-5-en-3-ol, acetate, (3.beta.,24R)-	29.096	0.718	C30H50O2	442.7168
37	Hexasiloxane, tetradecamethyl-	1.095	29.170	C14H42O5Si6	458.9933
38	(R)-6-Methoxy-2,8-dimethyl-2-((4R, 8R)-4,8, 12-trimethyltri decyl)chroman	29.594	1.316	C28H48O2	416.6795
39	Ergosta-4,7,22- trien-3-one	29.652	0.885	C28H42O	394.633
40	Stigmastan-3,5-diene	29.825	3.742	C29H48	396.6914
41	Vitamin E	30.196	4.094	C29H50O2	430.71
42	Ergost-5-en-3-ol, (3.beta.)-	30.823	1.070	C28H48O	400.6801
43	Stigmasterol	31.058	1.514	C29H48O	412.6908
44	(1-Cyclohexyl methyl-3-methylbut-2- enylthio) benzene	31.396	0.374	C13H18	174.282
45	gamma.-Sitosterol	31.459	5.677	C29H50O	414.7067
46	2(1H)Naphthalenone, 3,5,6,7, 8,8a-hexa hydro-4,8a-dimethyl-6-(1-methyl ethenyl)-	31.958	2.504	C15H22O	218.3346
47	3-(2-Thienyl)-4,5,dihydro-5-isoxazo lemethanol	35.955	0.295	C8H9NO2S	183.2276
48	(3aR,4R,7R)-1,4,9,9-Tetra methyl-3, 4,5,6, 7,8-hexahydro -2H-3a,7-meth anoazulen-2-one	32.620	1.010	C8H15N3	153.22
49	Tert-Butyl dimethylsilyl 2,3-dimethyl	33.447	0.675	C14H22O2Si	250.4088

Table 2: Bioactive components of hexane extract of sclerotia of P. tuberregium

S/N	Compound	Retention Time (min)	Percentage of the total	Molecular formula	Molecular weight	Structure
1.	Carbonic acid, hexadecyl prop-1-en -2-yl ester	8.085	0.173	C9H10O3	166.1739
2.	Carbonic acid, nonyl vinyl ester	9.440	0.792	C12H22O3	214.3013
3.	5-Acetoxy methyl-2-furaldehyde	10.634	0.982	C8H8O4	168.1467
4.	Hexadecane	11.962	1.672	C16H34	226.4412
5.	[1,2,3,4]Tetrazolo[1,5-b][1,2, 4]triazine, 5,6, 7,8-tetrahydro-	13.482	0.663	C3H6N6	126.12
6.	1-Nonadecene	14.173	1.811	C19H38	266.5050
7.	Octadecane	14.254	4.162	C18H38	254.4943
8.	Bicyclo[6.1.0]non-1-ene	14.431	0.793	C9H14	122.211
9.	1-Methylene-2-vinylcyclo pentane	15.193	0.875	C8H12	108.181
10.	Nonadecane	15.423	0.774	C19H40	268.5209
11.	Hexadecanoic acid, methyl ester	15.780	0.950	C17H34O2	270.4507
12.	Dibutyl phthalate	16.283	3.100	C16H22O4	278.3435
13.	n-Hexadecanoic acid	16.425	3.167	C16H32O2	256.4241
14.	1-Docosene	16.625	3.364	C22H44	308.5848
15.	Eicosane	16.717	4.721	C20H42	282.5475
16	9,12-Octadeca dienoic acid (Z,Z)-, methyl ester	18.029	0.689	C19H34O2	294.4721
17	Heneicosane	18.070	1.175	C21H44	296.5741
18	9,12-Octadeca dienoic acid (Z,Z)-	18.808	2.689	C18H32O2	280.4455
19	1-Docosene	19.438	3.145	C22H44	308.5848
20	Docosane	19.533	4.304	C22H46	310.6006
21	Methoxyacetic acid, 2-tridecyl est er	20.995	1.043	C16H32O3	272.429
22	Heptadecyl trifluoroacetate	22.424	3.008	C19H35F3O2	352.4752
23	Tricosane, 2-methyl-	22.509	3.745	C24H50	338.6538
24	Pentacosane	23.987	1.344	C25H52	352.6804
25	Bis(2-ethylhexyl) phthalate	24.674	5.092	C24H38O4	390.5561
26	Trichloroacetic acid, pentade cyl ester	25.275	2.827	C17H31Cl3O2	373.786
27	Heptadecane, 2-methyl-	25.340	3.866	C18H38	254.4943
28	Heptacosane	26.460	2.475	C27H56	380.7335
29	17-Pentatria contene	27.417	2.249	C35H72	492.9462
30	Octacosane	27.458	3.463	C28H58	394.7601
31	Squalene	27.711	0.905	C30H50	410.718
32	Tricosane	28.348	4.378	C24H50	338.6538
33	Docosane, 9-octyl-	29.161	4.520	C30H62	422.8133
34	Stigmastan-6, 22-dien, 3,5-de dihydro-	29.629	0.770	C28H46O	398.68
35	1-Octadecane sulphonyl chloride	29.923	1.161	C18H37ClO2S	353.003
36	26-Nor-5-chole sten-3.beta. -ol-25-one	30.017	1.215	C29H48O2	428.6902
37	Tetracontane, 3,5,24-trimethyl-	30.639	2.995	C43H88	605.1770
38	Ergost-5-en-3-ol, (3.beta.)-	30.829	1.637	C28H48O	400.691
39	Stigmasterol	31.061	4.407	C29H48O	412.6908
40	Octacosane	31.312	0.834	C28H58	394.7601
41	beta.-Sitosterol	31.464	2.247	C29H50O	414.7067
42	1H-1,2,3-Triazole-4-carboxylic acid, 5-amino-1-(phenylmethyl)-, hydrazide	31.516	1.001	C11H11N3O2	217.22
43	Octacosane	31.960	1.864	C28H58	394.7601
44	2H-Cyclopropa[a]naphthalen-2-one,1,1a,4,5,6, 7,7a,7b-octa hydro-1,1,7,7a-tetramethyl-, (1a.alpha.,7. alpha.,7a.alpha.,7b.alpha.)-	32.354	0.601	C15H24	204.3511
45	Urs-12-en-3-ol, acetate, (3.beta.)	32.632	2.351	C32H52O2

Table 3: Bioactive components of dichloromethane extract of sclerotia of P. tuberregium

S/N	Compound	Retention Time (min)	Percentage of the total	Molecular formula	Molecular weight	Structure
1	Tricyclo[2.2.1.0(2,6)]heptan-3-one, oxime	14.025	2.331	C7H10	94.1543
2	Undec-10-ynoic acid, but-3-yn-2-yl ester	14.095	5.056	C11H18O2	182.2594
3	2,5-Cyclohexa diene-1,4-dione, 2,5-dihydroxy-3-methyl-6-(1-methylethyl)-	15.610	6.192	C10H12O2	164.2000
4	3-Cyclopropen oic acid,1butyl, methyl ester	15.727	0.993	C17H24O4	292.3701
5	4-Pyridinol	16.096	3.539	C5H5NO	95.1000
6	Phthalic acid, 3-chlorobenzyl butyl ester	16.303	25.490
7	Hexadecanoic acid, methyl ester	17.798	2.602	C17H34O2	270.4507
8	2-Methyl-3-(phenylsulfonyl)methyl-2-cyclopenten-1-one	17.892	8.177	C6H8sO	96.1290
9	1,4-Naphtha lenedione, 5,8-dihydroxy-2,7-dimethoxy-	18.060	23.513	C10H6O4	190.15
10	2-Cyclopenten-1-one, 3-methyl-	18.663	19.395	CH3C5H5O	96.1300
11	n-Hexadecanoic acid	28.131	2.713	C16H32O2	256.4241

Table 4: Bioactive components of soxhlet extract of sclerotia of P. tuberregium.

Discussion

The disparity in both compound quantity and concentration observed in the methanol, hexane, dichloromethane and soxhlet extracts of the sclerotia of P. tuberregium shows varying capabilities of different solvents to dissolve and liberate different compounds from a substrate. The ability of methanol to extract only three uncommon polycyclic compounds (2H-Cyclopropa[a]naphthalen-2-one, (3aR,4R,7R)-1,4,9,9-Tetramethyl-3,4,5,6,7,8-hexahydro-2H-3a,7- methane and Urs-12-en-28-oic acid, 3-hydroxy-, methyl ester, (3.beta.)-) from the sclerotia of this mushroom. Table 1 indicates that the use of methanol as an extraction solvent may favour the extraction of polycyclic compounds especially in a lignin based substrate like mushroom. As a very polar compound, the yield and concentration of extracts in methanol extraction may be lower than in a non-polar solvent. This indicates that methanol can dissolve a larger proportion of polar compounds. However, its solubility may reduce when used as solvent for the extraction nonpolar compounds. The result of this study is an indication that methanol may not be a solvent of choice for extraction where both polar and non-polar compounds are needed from a substrate.

Although different solvents has been used for extraction, hexane has been considered ideal for extraction processes because of its non-polar properties, low boiling point (67°C), and its high solubility of lipid compounds. Moreover, Clough and Mulholland [10] also reported that the low reactivity exhibited by hexane also makes it a suitable solvent for the extraction of reactive compounds. The hexane extract of the sclerotia of this mushroom shows the presence of 49 different bioactive components made up of linear, branched, monocyclic and polycyclic compounds (Table 2). The large number of compounds observed in the hexane extract shows the ability of hexane to penetrate, solubilize and release these compounds from the substrate.

The 45 components observed from the dichloromethane extract (Table 4) shows that the volatility and ability of dichloromethane to dissolve a wide range of organic compounds makes it a useful solvent for many chemical processes [11]. The presence of both linear and cyclic compounds in the extract is also an indication that dichloromethane has the potentials to penetrate and extract most constituent compounds from a lignin based sample. Though dichloromethane is the least toxic amongst the simple chlorohydrocarbons it low boiling point also makes it a choice solvent for extraction [11]. As an extraction process, soxhlet extraction is mainly used in the extraction of compounds that are either insoluble or sparingly soluble in water. Though soxhlet extraction is a discontinuous extraction process, the repeated application of hot solvent (ethanol in this case) to the mushroom sample allows for a continuous penetration of the solvent into the sample. However, the yield of the extraction process is dependent on the extraction solvent. This may be responsible for the low yield (11 compounds) observed in the soxhlet extract of the sclerotia of this mushroom.

Conclusion

Hexane and dichloromethane extracts yielded more bioactive components with better nutriceutical and medicinal properties than methanol and soxhlet extracts. Sequel to these findings, hexane and dichloromethane are better solvents for extraction from a lignin based substrate such as mushrooms and other fungi.

References

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