alexa Comparative Phytochemical Profiling of Clerodendrum infortunatum L. Using GC-MS Method Coupled with Multivariate Statistical Approaches | Open Access Journals
ISSN: 2153-0769
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Comparative Phytochemical Profiling of Clerodendrum infortunatum L. Using GC-MS Method Coupled with Multivariate Statistical Approaches

Dey P, Dutta S and Chaudhuri TK*

Cellular Immunology Laboratory, Department of Zoology, Life Science Building, University of North Bengal, Raja Rammohunpur, Siliguri - 734013, West Bengal, India

*Corresponding Author:
Chaudhuri TK
Cellular Immunology Laboratory
Department of Zoology, Life Science Building
University of North Bengal, Raja Rammohunpur
Siliguri - 734013, West Bengal, India
Tel: +91-9531563048
Fax: +91-3532699001
E-mail: [email protected]

Received date: July 17, 2015; Accepted date: August 13, 2015; Published date: August 15, 2015

Citation: Dey P, Dutta S, Chaudhuri TK (2015) Comparative Phytochemical Profiling of Clerodendrum infortunatum L. Using GC-MS Method Coupled with Multivariate Statistical Approaches. Metabolomics 5:147. doi: 10.4172/2153-0769.1000147

Copyright: © 2015 Dey P, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Clerodendrum infortunatum L. is extensively used in traditional and folklore medicine. It has also been recognized in Ayurveda and Unani medicinal systems. Different pharmacological properties of C. infortunatum have already been reported. However, the phytochemical constituents remained unknown. Thus, the present study was performed to investigate and compare the phytochemical composition of the major parts of C. infortunatum using gas chromatographymass spectrometry. GC-MS is one of the most reliable biophysical method for its specificity and repeatability, was utilized for the phytochemical profiling C. infortunatum leaf, stem, root, flower and seed. The phytochemical profiles were further compared using multivariate statistical techniques to evaluate the extent of analogy between the phytochemical profiles. Several bioactive chemical species such as limonene, phytol, catechol, hexadecanoic acid, squalene, dodecanoic acid, vitamin E, hydroxymethylfurfural, stigmasterol, etc. were identified in different parts of C. infortunatum. Derivatives of pharmacological supplements such as cinnamic acid, guaiacol, eugenol, vanillic acid, vitamin D etc. were also detected. Moreover, a wide range of phenolics and phenolic acid derivatives were identified in C. infortunatum, which are known to be primarily responsible for bioactivities of herbal medicines. Principal component analysis coupled with hierarchical clustering revealed the extent of correlation and divergence among the major parts of C. infortunatum in terms of phytochemical fingerprints. Flower, leaf and root demonstrated equivalent phytochemical fingerprints.The results provided insights into the presence of several pharmacologically active constituents. The pattern of phytochemical correlation between leaf, stem, root, flower and seed was established as well.

Keywords

Antioxidant; Clerodendrum; Extract; GC-MS; Herbal medicine; Metabolomic profiling; Multivariate statistics; Phytochemical

Introduction

Evidence based pharmacognostic studies on complementary and herbal medicine are booming. Synergistic activities of known phytochemicals or novel bioactive leads are constantly being identified by biophysical screening of medicinal plants. Traditionally known therapeutic uses of such plants are also being established through in vivo trails. However, in spite of recognized bioactivities, most medicinal plants still lack complete phytochemical standardization which would not only correlate with its potent bioactivities, but also serve as its individual phytochemical fingerprint. Besides, in most cases, phytochemical profiling is primarily focused on certain part of a plant and thus, the parallel phytochemical information of other parts of the same plant remain completely obscure. Thus, a comprehensive phytochemical profiling of the major parts of a plant becomes quintessential. This would not only provide a logical candidate for ancillary evaluation of bioactivities but also may be useful for in silico prediction of the bioactivities of ethnopharmacological plants

Clerodendrum infortunatum L. (syn. Clerodendrum viscossum Vent., Clerodendrum calycinum Turcz., Ovieda infortunata (L.) Baill.) is an ethnopharmacological plant, used for the treatment of diverse ailments such as wounds, inflammatory diseases, tumor, malaria, hyperglycaemia, fever, snake bite etc. Details of its therapeutic uses in Ayurveda, Unani, homeopathic and folklore medicine have previously been reported [1-5]. However, in spite of its tremendous medicinal uses, a systematic phytochemical profiling of the major parts of the plant is completely lacking. Therefore, the present study was initiated to investigate the phytochemical profiles of leaf, stem, root, flower and seed of C. infortunatum and to compare the individual phytochemical niche with other parts of the plant. To our knowledge, perhaps, this is the first ever study of complete phytochemical metabolome of any medicinal plant.

Materials and Methods

Chemicals

All chemicals and reagents were procured form HiMedia Pvt. Ltd. (Mumbai, India) unless otherwise indicated. HPLC grade solvents were obtained from Sigma Aldrich (USA). Milli-Q ultrapure water from the departmental facility was used in the experiments.

Plant material

Fresh and disease free plant materials were collected from the campus of University of North Bengal, India (26°42'N, 88°21'E). The plant was identified and authenticated by plant taxonomist Prof. Abhaya Prasad Das of Department of Botany, University of North Bengal. A voucher specimen was stored at the Botany Department Herbarium, University of North Bengal with an accession number of 09618.

Preparation of extract

The extract was prepared according to a standard method [6]. Plant material was washed twice with double distilled water to remove dirt and foreign materials. The whole plant was divided into five major parts, i.e., leaf, stem, root, flower and seed. The parts were chopped into pieces (0.5-1.0 cm) and shade dried at laboratory temperature for 14 days. The dried parts were then grinded to powder using a blender-mixer (Hummer, Lords). The resultant fine powder (100 g) was mixed with 1000 ml 70% methanol (methanol : water 7:3, v/v) and kept in a shaking incubator (160 rpm) at 37°C. The mixtures were centrifuged for 15 min at 5000 rpm (Heraeus) after 12 h. The supernatant liquid was separately stored. The pellet was again mixed with 70% methanol and treated as previously described. After second phase of centrifugation, liquid supernatants of both phases were mixed together and concentrated in a rotary evaporator under reduced pressure (Rotavapor, Buchi). The resultant concentrated extract was lyophilized (SJIA-10N) and the anhydrous extract was stored at -20°C until further use.

GC-MS analysis

C. infortunatum leaf, stem, root, flower and seed extracts were passed through anhydrous Na2SO4 and activated charcoal (2:1; w/w) to remove any trace of moisture and colour. The samples were analysed using Thermo Scientific Trace 1300 gas chromatography instrument attached with Thermo Scientific ISQ QD single quadrupole mass spectrophotometer. The GC was equipped with TG-5MS column (30 m × 0.25 mm × 0.25 μm). The inlet temperature was maintained at 250°C. The initial temperature was set at 60°C (solvent delay 5 min) with a hold of 2 min, followed by a ramp of 5°C to 290°C with a hold of 6 min (54 min programme). Samples were (1 μl) injected in a splitless mode (split flow 50 ml/min) with splitless time of 0.80 min, using a Thermo Scientific AI-1310 auto-sampler. The carrier gas was helium, with a constant flow of 1 ml/min. MS transfer line temperature was set at 290°C with an Ion source temperature of 230°C (electron ionization). The individual samples were analysed at electron energy 70 eV (vacuum pressure- 2.21e-0.5 Torr). The mass analyser range was set to 50-650 amu.

GC-MS data analysis

All samples were analysed thrice for confirmation. MS data analysis was performed by Automated Mass Spectral Deconvolution and Identification System (AMDIS) version 2.70. The major and essential compounds were identified by mass fragmentation patterns using the database of National Institute Standard and Technology (NIST) with a MS library version 2011.

Multivariate analysis

Principal Component Analysis (PCA) based on correlation matrix was performed in order to elucidate the correlated phytochemical profile in leaf, stem, root, flower and seed of C. infortunatum. The results were further analysed by multivariate statistical approach, employing a hierarchical cluster analysis (HCA) associated with proximity score matrix represented as proximity heat-map. PCA and HCA were performed using the IBM SPSS statistics version 20.0 software package for Windows.

Results

The results of the GC-MS analysis revealed the presence of several bioactive constituents in different parts of C. infortunatum. The GC spectra of C. infortunatum leaf, stem, root, flower and seed are presented Figure 1-5 and the identified compounds are enlisted in Table 1-5.

metabolomics-Clerodendrum-infortunatum-leaf

Figure 1: Complete GC chromatogram of Clerodendrum infortunatum leaf (corresponding to table 1).

metabolomics-Complete-GC-chromatogram

Figure 2: Complete GC chromatogram of Clerodendrum infortunatum stem (corresponding to table 2).

metabolomics-Clerodendrum-infortunatum

Figure 3: Complete GC chromatogram of Clerodendrum infortunatum root (corresponding to table 3).

metabolomics-Clerodendrum-infortunatum-flower

Figure 4: Complete GC chromatogram of Clerodendrum infortunatum flower (corresponding to table 4).

metabolomics-Clerodendrum-infortunatum-seed

Figure 5: Complete GC chromatogram of Clerodendrum infortunatum seed (corresponding to table 5).

1. Dihydroxyacetone C3H6O3 5.08
2. 6-Oxa-bicyclo[3.1.0]hexan-3-one C5H6O2 5.87
3. [1,1'-Bicyclopropyl]-2-octanoic acid, 2'-hexyl-, methyl ester C21H38O2 6.63
4. d-Gala-l-ido-octonic amide C8H17NO8 6.97
5. 4-Amino-1,5-pentandioic acid C7H13NO4 7.16
6. 9-Octadecenamide C18H35NO 7.71
7. 3-[4-(2-Methoxy-ethoxymethoxy)-phenyl]-acrylic acid C13H16O5 8.38
8. Methyl N-(N-benzyloxycarbonyl-beta-l-aspartyl)-beta-d-glucosaminide C19H26N2O10 8.63
9. Acetic acid, 6-morpholin-4-yl-9-oxobicyclo[3.3.1]non-3-yl ester C15H23NO4 9.78
10. R-Limonene C10H16O3 10.03
11. 1,8-Di(4-nitrophenylmethyl)-3,6-diazahomoadamantan-9-one C23H24N4O5 10.29
12. 9,10-Secocholesta-5,7,10(19)-triene-3,24,25-triol, (3β,5Z,7E)- C27H44O3 10.84
13. 2-Myristynoyl pantetheine C25H44N2O5S 11.41
14. 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- C6H8O4 11.67
15. Hydrocinnamic acid, o-[(1,2,3,4-tetrahydro-2-naphthyl)methyl]- C20H22O2 11.76
16. Octadecanoic acid, 4-hydroxy-, methyl ester C19H38O3 12.87
17. Catechol C6H6O2 13.19
18. 6-Methylenebicyclo[3.2.0]hept-3-en-2-one C8H8O 13.74
19. Z-(13,14-Epoxy)tetradec-11-en-1-ol acetate C16H28O3 13.99
20. 2,5-Octadecadiynoic acid, methyl ester C19H30O2 15.85
21. 2-Methoxy-4-vinylphenol C9H10O2 16.39
22. Furan-2-carbohydrazide, N2-(3-indolylmethylene)- C14H11N3O2 18.56
23. 5,8,11-Eicosatriynoic acid, methyl ester C21H30O2 18.86
24. L-Gala-l-ido-octose C8H16O8 19.21
25. Adenosine, 4'-dehydroxymethyl-4'-[N-ethylaminoformyl]-N-[4-nitrobenzyl] C19H21N7O6 19.61
26. Ergosta-5,22-dien-3-ol, acetate, (3β,22E)- C30H48O2 21.42
27. Desulphosinigrin C10H17NO6S 22.76
28. 2,7-Diphenyl-1,6-dioxopyridazino[4,5:2',3']pyrrolo[4',5'-d]pyridazine C20H13N5O2 25.09
29. Pentaerythritol, bis-O-(9-borabicyclo[3.3.1]non-9-yl)-di-O-methyl- C23H42B2O4 25.30
30. 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol C10H12O3 26.54
31. 10-Heptadecen-8-ynoic acid, methyl ester, (E)- C18H30O2 27.22
32. 12-Methyl-E,E-2,13-octadecadien-1-ol C19H36O 28.67
33. 9-Octadecenoic acid, (2-phenyl-1,3-dioxolan-4-yl)methyl ester, cis- C28H44O4 29.54
34. Hexadecanoic acid, methyl ester C17H34O2 30.40
35. n-Hexadecanoic acid C16H32O2 31.05
36. 9,12-Octadecadienoic acid, methyl ester, (E,E)- C19H34O2 33.61
37. Methyl 8,11,14-heptadecatrienoate C18H30O2 33.73
38. Phytol C20H40O 33.94
39. 9,12,15-Octadecatrienoic acid, 2,3-dihydroxypropyl ester, (Z,Z,Z)- C21H36O4 34.37
40. Betulin C30H50O2 47.54
41. Spirost-8-en-11-one, 3-hydroxy-, (3β,5α,14β,20β,22β,25R)- C27H40O4 49.73

Table 1: Phytochemicals identified in Clerodendrum infortunatum leaf (corresponding to figure 1).

Sl. # Compound name Formula RT
1. Mannosamine C6H13NO5 5.10
2. 6-Oxa-bicyclo[3.1.0]hexan-3-one C5H6O2 5.89
3. L-Gala-l-ido-octose C8H16O8 7.44
4. Acetic acid, 6-morpholin-4-yl-9-oxobicyclo[3.3.1]non-3-yl ester C15H23NO4 9.79
5. Ethyl iso-allocholate C26H44O5 10.30
6. 10,13-Octadecadiynoic acid, methyl ester C19H30O2 10.87
7. 2-Myristynoyl pantetheine C25H44N2O5S 11.41
8. Hydrocinnamic acid, o-[(1,2,3,4-tetrahydro-2-naphthyl)methyl]- C20H22O2 11.77
9. 3,6-Dimethoxy-2,5-dinitrobenzaldehyde oxime C9H9N3O7 13.20
10. 2,5-Octadecadiynoic acid, methyl ester C19H30O2 14.29
11. Methyl 8,10-octadecadiynoate C19H30O2 15.86
12. 2-Methoxy-4-vinylphenol C9H10O2 16.39
13. Octahydrochromen-2-one C9H14O2 18.49
14. 9-Octadecenoic acid, (2-phenyl-1,3-dioxolan-4-yl)methyl ester, cis- C28H44O4 19.19
15. Dodecanoic acid, 3-hydroxy- C12H24O3 21.42
16. Hexadecane, 1,1-bis(dodecyloxy)- C40H82O2 21.99
17. Desulphosinigrin C10H17NO6S 22.72
18. Propanoic acid, 2-(3-acetoxy-4,4,14-trimethylandrost-8-en-17-yl)- C27H42O4 25.09
19. 2,7-Diphenyl-1,6-dioxopyridazino[4,5:2',3']pyrrolo[4',5'-d]pyridazine C20H13N5O2 26.26
20. 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol C10H12O3 26.54
21. 10-Heptadecen-8-ynoic acid, methyl ester, (E)- C18H30O2 27.23
22. Ergosta-5,22-dien-3-ol, acetate, (3β,22E)- C30H48O2 28.41
23. Hexadecanoic acid, methyl ester C17H34O2 68.40
24. Estra-1,3,5(10)-trien-17β-ol C18H24O 31.05
25. Cyclopropanedodecanoic acid, 2-octyl-, methyl ester C24H46O2 32.34
26. Methyl 9-cis,11-trans-octadecadienoate C19H34O2 33.61
27. 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)- C19H32O2 33.73
28. Phytol C20H40O 33.94
29. Heptadecanoic acid, 16-methyl-, methyl ester C19H38O2 34.18

Table 2: Phytochemicals identified in Clerodendrum infortunatum stem (corresponding to figure 2).

Sl. # Compound name Formula RT
1. D-glucosamine C6H13NO5 5.09
2. 6-Oxa-bicyclo[3.1.0]hexan-3-one C5H6O2 5.88
3. 4-Amino-1,5-pentandioic acid C7H13NO4 7.18
4. 3-[4-(2-Methoxy-ethoxymethoxy)-phenyl]-acrylic acid C13H16O5 8.40
5. 4-Methylpiperidine-1-carboxylic acid, phenyl ester C13H17NO2 9.80
6. Acetamide, N-methyl-N-[4-(3-hydroxypyrrolidinyl)-2-butynyl]- C11H18N2O2 10.28
7. 10,13-Octadecadiynoic acid, methyl ester C19H30O2 10.86
8. Desulphosinigrin C10H17NO6S 11.29
9. 2-Myristynoyl pantetheine C25H44N2O5S 11.40
10. 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- C6H8O4 11.67
11. 2-Cyclohexylpiperidine C11H21N 12.59
12. 2-Oxaspiro[5.5]undecane-1,5-dione, 4,4-dimethyl-3-(4-nitrophenyl)- C18H21NO5 13.20
13. 5-Hydroxymethylfurfural C6H6O3 13.98
14. Methyl 6-oxoheptanoate C8H14O3 15.29
15. 2,5-Octadecadiynoic acid, methyl ester C19H30O2 15.85
16. 2-Methoxy-4-vinylphenol C9H10O2 16.39
17. 2,4-Dimethoxyphenol C8H10O3 17.37
18. 9-Hexadecenoic acid C16H30O2 17.55
19. Cyclobuta[1,2:3,4]dicyclooctene, hexadecahydro- C16H28 18.49
20. 9-[2-Deoxy-β-d-ribohexopyranosyl]purin-6(1H)-one C11H14N4O5 19.59
21. Dodecanoic acid, 3-hydroxy- C12H24O3 21.44
22. Hexadecane, 1,1-bis(dodecyloxy)- C40H82O2 21.99
23. 2-Oxabicyclo[3.3.0]oct-7-en-3-one, 7-(1-hydroxypentyl)- C12H18O3 22.52
24. 3-Oxabicyclo[4.1.0]heptane-7-carboxamide, 6-methyl-N-(1-naphthyl)- C18H19NO2 22.65
25. α-D-Galactopyranoside, methyl C7H14O6 22.94
26. 2,7-Diphenyl-1,6-dioxopyridazino[4,5:2',3']pyrrolo[4',5'-d]pyridazine C20H13N5O2 25.08
27. 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol C10H12O3 26.55
28. Hexadecanoic acid, methyl ester C17H34O2 30.40
29. n-Hexadecanoic acid C16H32O2 31.06
30. Gibberellic acid C19H22O6 31.69
31. Hexadecanoic acid, 14-methyl-, methyl ester C18H36O2 32.34
32. 9,12-Octadecadienoic acid (Z,Z)-, methyl ester C19H34O2 33.61
33. 9-Octadecenoic acid (Z)-, methyl ester C19H36O2 33.72
34. 10-Octadecenoic acid, methyl ester C19H36O2 33.82
35. Heptadecanoic acid, 16-methyl-, methyl ester C19H38O2 34.19
36. 8,11,14-Eicosatrienoic acid, (Z,Z,Z)- C20H34O2 34.26

Table 3: Phytochemicals identified in Clerodendrum infortunatum root (corresponding to figure 3).

Sl. # Compound name Formula RT
1. 6-Oxa-bicyclo[3.1.0]hexan-3-one C5H6O2 5.94
2. 2-Propyl-tetrahydropyran-3-ol C8H16O2 7.19
3. Oxirane, [(2-propenyloxy)methyl]- C6H10O2 7.52
4. Dithiocarbamate, S-methyl-,N-(2-methyl-3-oxobutyl)- C7H13NOS2 9.35
5. Thymine C5H6N2O2 9.92
6. 1-Hexanethiol, 2-ethyl- C8H18S 10.34
7. n-Nonaldehyde C9H18O 10.57
8. 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- C6H8O4 11.71
9. 5-Methoxypyrrolidin-2-one C5H9NO2 12.61
10. Desulphosinigrin C10H17NO6S 13.36
11. Benzofuran, 2,3-dihydro- C8H8O 13.75
12. 2-Methoxy-4-vinylphenol C9H10O2 16.38
13. Carbamic acid, N-methyl-N-[6-iodo-9-oxabicyclo[3.3.1]nonan-2-yl]-, ethyl ester C12H20INO3 18.57
14. 2-Hydroxy-5-methylbenzaldehyde C8H8O2 19.64
15. Dodecanoic acid, 3-hydroxy- C12H24O3 20.45
16. 2-Myristynoyl pantetheine C25H44N2O5S 22.05
17. Undecanoic acid C11H22O2 22.55
18. 3-tert-Butyl-4-hydroxyanisole C11H16O2 22.69
19. 2-Methyl-9-β-d-ribofuranosylhypoxanthine C11H14N4O5 24.62
20. 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol C10H12O3 26.57
21. Tetradecanoic acid C14H28O2 26.99
22. Hexadecanoic acid, 1-(hydroxymethyl)-1,2-ethanediyl ester C35H68O5 27.22
23. Ethyl iso-allocholate C26H44O5 28.67
24. 2-Bromotetradecanoic acid C14H27BrO2 29.06
25. Phen-1,4-diol, 2,3-dimethyl-5-trifluoromethyl- C9H9F3O2 29.29
26. Hexadecane, 1,1-bis(dodecyloxy)- C40H82O2 29.47
27. Hexadecanoic acid, methyl ester C17H34O2 30.39
28. n-Hexadecanoic acid C16H32O2 31.09
29. Estra-1,3,5(10)-trien-17β-ol C18H24O 31.81
30. 2-Hexadecanol C16H34O 33.37
31. 7,10-Octadecadienoic acid, methyl ester C19H34O2 33.60
32. 9,12-Octadecadienoyl chloride, (Z,Z)- C18H31ClO 33.71
33. Phytol C20H40O 33.93
34. cis-Vaccenic acid C18H34O2 34.40
35. Octadecane, 3-ethyl-5-(2-ethylbutyl)- C26H54 37.15
36. Hexanoic acid, 2-ethyl-, hexadecyl ester C24H48O2 40.00
37. Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester C19H38O4 40.51
38. 9,12-Octadecadienoic acid (Z,Z)-, 2,3-dihydroxypropyl ester C21H38O4 43.19
39. Lup-20(29)-en-3-ol, acetate, (3β)- C32H52O2 43.60
40. Squalene C30H50 45.20
41. Heptacosane C27H56 46.11
42. 2-[4-methyl-6-(2,6,6-trimethylcyclohex-1-enyl)hexa-1,3,5-trienyl]cyclohex-1-en-1-carboxaldehyde C23H32O 47.55
43. Vitamin E C29H50O2 49.35
44. Stigmasterol C29H48O 51.42

Table 4: Phytochemicals identified in Clerodendrum infortunatum flower (corresponding to figure 4).

Sl. # Compound name Formula RT
1. Cyclopentanone, 2-methyl- C6H10O 5.97
2. 2-Furanmethanol, 5-methyl- C6H8O2 6.64
3. 2-Propyl-tetrahydropyran-3-ol C8H16O2 7.22
4. β-Allyloxypropionic acid C6H10O3 7.65
5. 2-Hexene, 1-(1-ethoxyethoxy)-, (E)- C10H20O2 7.84
6. Desulphosinigrin C10H17NO6S 8.33
7. β-Hydroxybutyric acid C4H8O3 8.73
8. 2,5-Dimethyl-4-hydroxy-3(2H)-furanone C6H8O3 9.49
9. 13,16-Octadecadiynoic acid, methyl ester C19H30O2 10.86
10. 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- C6H8O4 11.81
11. 5-Methoxypyrrolidin-2-one C5H9NO2 12.80
12. Cyclohexanone, 4-ethoxy- C8H14O2 12.93
13. Catechol C6H6O2 13.35
14. β-Hydroxydodecanoic acid C12H24O3 13.55
15. 5-Hydroxymethylfurfural C6H6O3 14.07
16. D-Tyrosine, 3-hydroxy- C9H11NO4 14.98
17. 2-Methoxy-4-vinylphenol C9H10O2 16.39
18. 2,4-Dimethoxyphenol C8H10O3 17.37
19. 2-Cyclohexen-1-one, 2-methyl- C7H10O 18.69
20. Octan-2-one, 3,6-dimethyl- C10H20O 19.11
21. Benzeneethanol, 4-hydroxy- C8H10O2 19.37
22. Octanoic acid, 7-oxo- C8H14O3 20.45
23. 2-Dodecenoic acid C12H22O2 21.69
24. Cyclobutanecarboxylic acid, decyl ester C15H28O2 22.16
25. Oleic Acid 22.58 22.58
26. Phenol, 2,6-dimethoxy-4-(2-propenyl)- C11H14O3 25.79
27. Benzeneacetic acid, 4-hydroxy-3-methoxy-, methyl ester C10H12O4 26.35
28. Tetradecanoic acid C14H28O2 27.06
29. 13-Heptadecyn-1-ol C17H32O 28.67
30. Pentadecanoic acid C15H30O2 29.09
31. 1-Hexadecanol, 2-methyl- C17H36O 29.47
32. Hexadecanoic acid, methyl ester C17H34O2 30.40
33. n-Hexadecanoic acid C16H32O2 31.66
34. 9-Octadecenoic acid (Z)-, methyl ester C19H36O2 33.73
35. cis-Vaccenic acid C18H34O2 35.58
36. trans-13-Octadecenoic acid C18H34O2 39.80
37. Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester C19H38O4 40.61
38. 9-Octadecenoic acid (Z)-, 2,3-dihydroxypropyl ester C21H40O4 43.39
39. 7,8-Epoxylanostan-11-ol, 3-acetoxy- C32H54O4 44.22
40. 9-Octadecenoic acid (Z)-, 2-hydroxy-1-(hydroxymethyl)ethyl ester C21H40O4 44.71
41. Squalene C30H50 45.21
42. Ethyl iso-allocholate C26H44O5 46.80
43. Pregna-3,5-dien-9-ol-20-one C21H30O2 47.31
44. Stigmasterol C29H48O 51.46

Table 5: Phytochemicals identified in Clerodendrum infortunatum seed (corresponding to figure 5).

Discussion

Phytochemical profiles of plants vary heavily depending on variation in season, soil constituents as well as cultivar. Complexity and variation in metabolites regarding polarity, molecular weight, abundance and different physiochemical properties makes it impossible to extract the whole metabolome using a single solvent extraction method. However, in the present study, phytochemical extraction was performed using a combination of water and methanol, because hydro-methanolic extraction method is one of the most reliable and highly preferred methods for extraction of phenolic metabolites which are known to be responsible for bioactivities of herbal medicines. Moreover, traditional plant based medicines are mostly prepared using polar solvents such as tinctures in Ayurveda.

C. infortunatum is an ethno pharmacological plant which is extensively utilized in traditional medicinal system to ameliorate a wide range of diseases. Moreover, several evidence based reports are available which have already demonstrated the therapeutic potentialities of C. infortunatum [4]. Previously, in a preliminary phytochemical analysis, Dey et al. [7] had quantified some major chemical species such as tannin, phenol, ascorbic acid, riboflavin, thiamine, alkaloid, flavonoid, sugar, lipid, protein etc. present in C. infortunatum. Moreover, Ghosh and his group [8] studied the phytochemical profile of the methanolic extract of leaves using chromatographic approaches and identified few compounds.

Limonene, catechol, p-vinylguaiacol (2-methoxy-4-vinylphenol), 5,8,11-eicosatriynoic acid, stigmasterol, desulphosinigrin, guaiacol (2-methoxyphenol), tyrosol (4-hydroxy- benzeneethanol), vaccenic acid, hexadecanoic acid, phytol, betulin, hydroxymethylfurfural were the major bioactive constituents recognized in C. infortunatum. Metabolic intermediates and derivatives of several compounds such as vitamin D (9,10-secocholesta-5,7,10(19)-triene-3,24,25-triol,(3β,5Z,7E)-); eugenol (phenol, 2,6-dimethoxy-4-(2-propenyl)-); cinnamic acid (hydrocinnamic acid, o-[(1,2,3,4-tetrahydro-2-naphthyl)methyl]-); and vanillic acid (benzeneacetic acid, 4-hydroxy-3-methoxy-, methyl ester) were identified as well. Previously, Dey et al, [7] also reported the presence of high amount of thiamine, riboflavin and ascorbic acid in the leaves of C. infortunatum. Moreover, Erukainure [9] also reported the presence of different vitamins including vitamin D in Clerodendrum species. Identification of hydrocinnamic acid has profound importance because cinnamic acid derivatives are integral part of our diet and has been attributed to the prevention of different diseases related to oxidative stress like atherosclerosis, inflammatory injury, cancer, and cardiovascular diseases [10]. The presence of 4H-pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl- and n-hexadecanoic acid was supported by a previous study which reported both the compounds in C. infortunatum leaves [8]. The present study, revealing different phenolic species in C. infortunatum, also corroborated the previous findings that C. infortunatum leaf, stem and root contained high quantity of phenolic and flavonoid compounds [11]. These phytochemicals are considered to be the major determinant of bioactivities of herbal medicines.

Multivariate statistical analysis in the present study, revealed interconnected correlation patterns between the phytochemical profiles of C. infortunatum. Partial convergence of phytochemical profiles resulted due to synonymous primary and secondary metabolic pathways, giving rise to identification of common phytochemicals or their derivatives in multiple parts of the plant. For instance, several compounds were identified in multiple parts of C. infortunatum such as phytol, 6-oxa-bicyclo [3.1.0] hexan-3-one, 2-myristynoyl pantetheine, 2-methoxy-4-vinylphenol, desulphosinigrin, 4-((1E)- 3-Hydroxy-1-propenyl)-2-methoxyphenol, hexadecanoic acid, etc. representing common or overlapping metabolic pathways. Spatial arrangement of the phytochemical profiles of leaf, stem, root, flower and seed of C. infortunatum in the double-positive quadrant signifies presence of interrelated phytochemical species (Figure 6A). The loading plot of principal component 1 and 2 accounted for 97.60% and 1.78% variance, respectively. Moreover, clustering of phytochemical profiles of leaf, flower and root demonstrated similar phytochemical fingerprint with the phytochemical profile of flower highly overlapping with leaf and root. A previous report of antioxidant and free radical scavenging activities of leaf, stem and root of C. infortunatum [11] also demonstrated the comparable bioactivities of leaf and root extracts. Furthermore, the presence of fairly distinct phytochemical species in seed and stem of C. infortunatum are highlighted by their isolated spatial location in the component plot as well as through the correlation matrix (Table 6). It is interesting to note that the clustering analysis using hierarchical method (Figure 6B) also correlated with the PCA. Phytochemical profiles of the major parts of C. infortunatum were grouped into various statistically significant clusters according to their proximities. The proximity heat-map (Figure 6C) corroborates the correlation matrix demonstrating similar phytochemical profile in C. infortunatum leaf, flower and root.

metabolomics-Multivariate-statistical-analysis

Figure 6: Multivariate statistical analysis of the phytochemical profiles of C. infortunatum leaf, stem, root, flower and seed. (A) Principal component loading plot corresponding to Table 1. Clustered spatial arrangement of leaf, flower and root in the double positive quadrant signifies comparable phytochemical profile whereas, unalike profile in case of seed and stem. (B) Dendrogram describing the hierarchical clustering of the phytochemical profiles. Phytochemical profiles of leaf, root and flower remains similar as shown by the clustered branching pattern. However, separation of the phytochemical profile of seed from the very beginning demonstrates highest variation. (C) Proximity heat map generated from proximity scores corresponding to Figure 6B. Shades of red to green colour demonstrates low to high proximity scores. Highest proximity phytochemical proximity resided between root and leaf whereas, lowest proximity between seed and stem.

Sl. # Compound name Formula RT
1. Cyclopentanone, 2-methyl- C6H10O 5.97
2. 2-Furanmethanol, 5-methyl- C6H8O2 6.64
3. 2-Propyl-tetrahydropyran-3-ol C8H16O2 7.22
4. β-Allyloxypropionic acid C6H10O3 7.65
5. 2-Hexene, 1-(1-ethoxyethoxy)-, (E)- C10H20O2 7.84
6. Desulphosinigrin C10H17NO6S 8.33
7. β-Hydroxybutyric acid C4H8O3 8.73
8. 2,5-Dimethyl-4-hydroxy-3(2H)-furanone C6H8O3 9.49
9. 13,16-Octadecadiynoic acid, methyl ester C19H30O2 10.86
10. 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- C6H8O4 11.81
11. 5-Methoxypyrrolidin-2-one C5H9NO2 12.80
12. Cyclohexanone, 4-ethoxy- C8H14O2 12.93
13. Catechol C6H6O2 13.35
14. β-Hydroxydodecanoic acid C12H24O3 13.55
15. 5-Hydroxymethylfurfural C6H6O3 14.07
16. D-Tyrosine, 3-hydroxy- C9H11NO4 14.98
17. 2-Methoxy-4-vinylphenol C9H10O2 16.39
18. 2,4-Dimethoxyphenol C8H10O3 17.37
19. 2-Cyclohexen-1-one, 2-methyl- C7H10O 18.69
20. Octan-2-one, 3,6-dimethyl- C10H20O 19.11
21. Benzeneethanol, 4-hydroxy- C8H10O2 19.37
22. Octanoic acid, 7-oxo- C8H14O3 20.45
23. 2-Dodecenoic acid C12H22O2 21.69
24. Cyclobutanecarboxylic acid, decyl ester C15H28O2 22.16
25. Oleic Acid 22.58 22.58
26. Phenol, 2,6-dimethoxy-4-(2-propenyl)- C11H14O3 25.79
27. Benzeneacetic acid, 4-hydroxy-3-methoxy-, methyl ester C10H12O4 26.35
28. Tetradecanoic acid C14H28O2 27.06
29. 13-Heptadecyn-1-ol C17H32O 28.67
30. Pentadecanoic acid C15H30O2 29.09
31. 1-Hexadecanol, 2-methyl- C17H36O 29.47
32. Hexadecanoic acid, methyl ester C17H34O2 30.40
33. n-Hexadecanoic acid C16H32O2 31.66
34. 9-Octadecenoic acid (Z)-, methyl ester C19H36O2 33.73
35. cis-Vaccenic acid C18H34O2 35.58
36. trans-13-Octadecenoic acid C18H34O2 39.80
37. Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester C19H38O4 40.61
38. 9-Octadecenoic acid (Z)-, 2,3-dihydroxypropyl ester C21H40O4 43.39
39. 7,8-Epoxylanostan-11-ol, 3-acetoxy- C32H54O4 44.22
40. 9-Octadecenoic acid (Z)-, 2-hydroxy-1-(hydroxymethyl)ethyl ester C21H40O4 44.71
41. Squalene C30H50 45.21
42. Ethyl iso-allocholate C26H44O5 46.80
43. Pregna-3,5-dien-9-ol-20-one C21H30O2 47.31
44. Stigmasterol C29H48O 51.46

Table 6: Correlation matrix of phytochemical profiles of C. infortunatum leaf, stem, root, flower and seed corresponding to Figure 1. Where, P < 0.001 (1-tailed).

Conclusion

Evaluation of a particular bioactivity of different parts of C. infortunatum has never been performed before. In fact, this remains a common scenario in case of most other ethnomedicinal plants. However, phytochemical analysis coupled multivariate statistical approach provides a simple yet comprehensive method for prediction of correlated bioactivities of different parts of a plant. The present study revealed occurrence of several bioactive phytochemicals in C. infortunatum. Moreover, comparable phytochemical profile of leaf, root and flower also predicts similar bioactivities of those parts as well as dissimilar bioactivities of seed and stem due to their distant spatial arrangement.

Acknowledgements

The authors are thankful to Mr. Bijoy Mahanta for his assistance during experimental works. The research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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