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Solvent Based Variations in Yield of Bioactive Extracts from the Sclerotium of Pleurotus tuber-regium

Reginald C Ohiri*, Mercy O Ifeanachor, Keku Preye

Department of Biochemistry, Faculty of Science, University of Port Harcourt, Nigeria

Corresponding Author:
Reginald C Ohiri
Department of Biochemistry
Faculty of Science
University of Port Harcourt, Nigeria
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Abstract

Background: The therapeutic effectiveness of herbs and fungi used for medicinal purposes is not only a factor of their bioactive constituents but also a factor of both the extraction solvent and extraction method.

Objective: The objective of this study is to extract and analyze the bioactive components in the sclerotum of Pleurotus tuber-regium using different solvents, as to ascertain the solvent that gives a better yield.

Method: A quantity of 10.0 kg of fresh sclerotia of P. tuber-regium 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 dried at room temperature for fourteen days. After grinding, the bioactive components were extracted by weighing 10 g of sample into three well stopper bottles and each was extracted in 20 mL of specific extraction solvent (methanol, hexane and dichloromethane), while that of soxleth extraction was done in a soxleth apparatus, using ethanol as the solvent. The process was repeated twice and the combined aliquot obtained were concentrated to 5.0 mL and purified. Two milliliters of the extracts were used for gas chromatographic and mass spectroscopy analysis.

Result: The highest peak on the chromatogram for the methanol extract was observed at 32.644 min., while hexane, dichloromethane and soxhlet extracts had their highest peaks at 31.459 min., 14.254 min. and 18.060 min. respectively. The highest bioactive component in methanol extract was (3aR,4R,7R)-1,4,9,9-Tetramethyl-3, 4,5,6,7,8-hexahydro- 2H-3a,7-methane with a value of 62.856 %, while hexane, dichloromethane and soxhlet extracts had Hexasiloxane, tetradecamethyl- , Bis(2-ethylhexyl) phthalate and Phthalic acid, 3-chlorobenzyl butyl ester with values of 29.170 %, 5.092 % and 25.490 % respectively.

Conclusion: Hexane and dichloromethane extracts yielded more bioactive components with better nutriceutical and medicinal properties and may be regarded as better solvents for mushroom and fungi extractions.

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.

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

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

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

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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 image
 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 image
3 Urs-12-en-28-oic acid, 3-hydroxy-,
methyl ester, (3.beta.)-
33.498 23.151 C30H48O3 456.700 image

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

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

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 image
 2 Undec-10-ynoic acid, but-3-yn-2-yl ester 14.095 5.056 C11H18O2 182.2594 image
3 2,5-Cyclohexa diene-1,4-dione, 2,5-dihydroxy-3-methyl-6-(1-methylethyl)- 15.610 6.192 C10H12O2 164.2000 image
4 3-Cyclopropen oic acid,1butyl, methyl ester 15.727 0.993 C17H24O4 292.3701 image
5 4-Pyridinol 16.096 3.539 C5H5NO 95.1000 image
6  Phthalic acid, 3-chlorobenzyl butyl ester 16.303 25.490     image
7 Hexadecanoic acid, methyl ester 17.798 2.602 C17H34O2 270.4507 image
8 2-Methyl-3-(phenylsulfonyl)methyl-2-cyclopenten-1-one 17.892 8.177 C6H8sO 96.1290 image
9 1,4-Naphtha lenedione, 5,8-dihydroxy-2,7-dimethoxy- 18.060 23.513 C10H6O4 190.15 image
10 2-Cyclopenten-1-one, 3-methyl- 18.663 19.395 CH3C5H5O 96.1300 image
11  n-Hexadecanoic acid 28.131 2.713 C16H32O2 256.4241 image

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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