Table 2. Physical Properties of Isolated Compounds

Table 3. ¹³C and ¹H NMR data (Chloroform-d₁ 700 MHz for 1H and 176 MHz ¹³C) for Compound Ea-7-38

Compound Ea-7-38 was obtained as white solid which appeared green under UV light, Reddish brown when sprayed with MPA and has a melting point of 368^oC and Rf value of 0.42 in a hexane/DCM 9:1 solvent ratio. Compound Ea-9-10 was also obtained as white solid which appeared green under UV light, Reddish brown when sprayed with MPA and has a melting point of 694^oC and Rf value of 0.51 in a hexane/DCM 8:2 solvent ratio. Compound Ea-12-85 was obtained as white solid which appeared green under UV light, brown when sprayed with MPA and has a melting point of 521^oC and Rf value of 0.50 in a hexane/DCM 7:3 solvent ratio (Table 2). Compound Ea-7-38 was obtained as white solid with molecular formula C₃₂H₅₂O₂ from HR-ESIMS m/z 491.2273 [M+Na]⁺  (calculated for C₃₂H₅₂O₂Na 491.2273.

Table 4. ¹³C and ¹H NMR data (Chloroform-d₁ 700 MHz for 1H and 176 MHz ¹³C) for Compound Ea-9-10

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Table 5. ¹³C and ¹H NMR data (Chloroform-d₁ 700 MHz for ¹H and 176 MHz for ¹³C) for Compound Ea-12-85

The proton (¹H) NMR, ¹³C NMR (Table 3) and IR suggested that Ea-7-38 might be a triterpene with the cluster of methylene and methyl protons at δ 2.0 – 0.1 values range. A terminal C=CH₂ protons were conspicuous at δ value 4.69 and 4.68, a δ value of 4.57 corresponds to   O-CH protons. A δ value of 2.3 corresponds to C=C-CH₃ protons, a singlet signal of high intensity at δ value of 2.0 reveal the OCH₃ protons. The carbon-13 NMR revealed a 32-carbon compound with a carbonyl carbon at 171ppm, two peaks at δ150ppm and δ109ppm corresponding to H₂C=CR₂, a moderate peak at δ81ppm revealed an O-CH ring carbon. The peak at δ77ppm with high intensity is the solvent (deuterated chloroform). The 1D carbon-13 NMR, 2D DEPT 90, 45 and 135 (Supplementary Fig S1-S10) revealed 11 CH₂, 8 CH₃, and 6 CH groups, IR spectra revealed a carbonyl (C=O) absorption band at 1732.629cm^-1 C-O stretch at 1245cm^-1 indicating an ester, C-H stretch (broad) at 2942.273cm^-1, C=C-H stretch at 3074.489cm^-1 typical of esters. From the relevant literature, compound Ea-7-38 was identified as a pentacyclic triterpene 3a,5,5b,8,8,11a-hexamethyl-1-(prop-1-en-2-yl) icosahydro-1H-cyclopenta[a]chrysene -9-yl acetate (Lupeol acetate).

Compound Ea-9-10 was obtained as white solid with molecular formula C₃₂H₅₂O₂ from HR-ESIMS m/z 390.2273 [M+Na]⁺ (calculated for C₃₂H₅₂O₂Na 390.2273). The proton (¹H) NMR, ¹³C NMR (Table 4), and IR suggested that Ea-7-38 is a triterpene with the cluster of methylene and methyl protons at δ 2.0 – 0.5 values range. A ring (internal alkene) HC=CH protons were conspicuous at δ value 4.69 and 4.68, a δ value of 4.57 corresponds to   O-CH protons. A δ value of 2.3 corresponds to >C=C-CH₃ protons, a singlet signal of high intensity at δ value of 2.0 reveal the OCH₃ protons. The carbon-13 NMR revealed a 32-carbon atoms compound with a carbonyl carbon at δ171ppm, two peaks at δ144ppm and δ139ppm corresponding to C=C isolated ring alkene, a moderate peak at δ81ppm revealed a O-CH ring carbon. The peak at δ77ppm with high intensity is the solvent (deuterated chloroform). The 1D carbon-13 NMR, 2D DEPT 90, 45 and 135 revealed 11 CH₂, 8CH₃, and 6CH groups. The IR spectrum revealed a carbonyl (C=O) absorption band at 1732.629cm^-1, C-O stretch at 1245cm^-1, C-H stretch (broad) at 2942.273cm^-1, >C=C-H stretch at 3074.489cm^-1 typical of an ester. Table 4 provided the data (carbons position, ¹H multiplicity, coupling constant, ¹H-¹H COSY) for Ea-9-10 while Table 4. 24 avails the comparative data from standard (MestReNova), Literature and isolated compound Ea-9-10. From these data and the library data (Table 6) obtained from both ChemBioDraw Ultra 3.0 and MestReNova application software, compound Ea-9-10 was identified as a triterpene 4,4,6a,6b,8a,10,11,14b, octamethyl 1,1,2,3,4,4a,5,6,6a,6b, 7,8,8a,9,10,11,12,12a, 14,14a,14b icosahydropicen- 3-yl acetate (derivative of β-Amyrin).

Table 6. BST Assay Results of Compound 1

Table 7: BST Assay Results of Compound 2

Table 8. BST Assay Results of Compound 3

Compound Ea-12-85 was obtained as white solid and molecular formula proposed to be C₃₀H₄₉O from HR-ESIMS m/z 390.2273 [M+Na]⁺(calculated for C₃₀H₄₉ONa, 337). The proton (¹H) NMR, ¹³C NMR (Table 5) and IR suggest that Ea-12-85 might be a triterpene with the cluster of methylene and methyl protons at δ 2.0 – 0.1 values range. A ring (internal alkene) HC=CH protons were conspicuous at δ value 4.69 and 4.68, a δ value of 4.57 corresponds to   O-CH protons. A δ value of 2.3 corresponds to C=C-CH₃ protons, a singlet signal of high intensity at δ value of 2.0 reveal the OCH₃ protons. The carbon-13 NMR revealed a 32-carbon compound with a carbonyl carbon (acid) at δ179ppm, two peaks at δ144ppm and δ139ppm corresponding to C=C isolated ring alkene, a moderate peak at 81ppm revealed a O-CH ring carbon. The peak at δ77ppm with high intensity is typical of the solvent (deuterated chloroform). The 1D carbon-13, 2D DEPT90, 45 and 135 revealed 11 CH₂, 8 CH₃, and 6 CH groups. The IR spectrum revealed a carbonyl (C=O) absorption band at 1732.629cm^-1, C-O stretch at 1245cm^-1 revealing an ester, O-H stretch (broad) at 3242.273cm^-1 revealing an alcohol, C=C-H stretch at 3074.489cm^-1 indicating an alkene. From the relevant literature compound Ea-12-85 was identified as betulinic acid. The earlier work reported by Oliver et al., 2015 on the isolation of triterpene from plants was in agreement and supported this submission with comparative data.

3.2 Brine Shrimp Lethality Test

The result of the in vitro cytotoxicity studies (Tables 6 - 8) revealed that Compounds 1 and 2 (Fig. 1) were non-toxic (LC₅₀(µg/mL) ≥1000) to the nauplii while compound 3 (Fig. 1) was toxic (LC₅₀(µg/mL) = 136.5) to the nauplii. In similar studies terpenes have been reported to exert anti-inflammatory effects by inhibiting various proinflammatory pathways in ear edema, bronchitis, chronic obstructive pulmonary disease, skin inflammation, and osteoarthritis [12-15, 16]. A natural compound linalool found in essential oils of aromatic plants, inhibited cigarette smoke-induced acute lung inflammation [17]. The findings of this study suggest that the plant extracts are reliable natural sources of terpenes terpenoids and their derivatives. A report revealed that structurally related monoterpenes p-Cymene, carvacrol and thymol isolated from essential oil from leaves of Lippia sidoides cham. (Verbenaceae) protected mice against elastase-induced emphysema [18]. Also based on the reported antiplasmodial studies carried out on solvent extracts of this plant [19], the use of this plant by traditional medicine practitioners especially at lower doses to cure jaundice and malaria becomes scientifically valid especially in rural communities where orthodox drugs are unaffordable because of the costs.

The three isolated compounds were found to be nontoxic to the shrimps except one. However, the study revealed that the stem bark extract of this plant contained important secondary metabolites which include but not limited to triterpenes and their derivatives. A report by [12] revealed that pentacyclic lupane-type triterpenes, possess beneficial effects as a therapeutic and preventive agent for a range of disorders which include anti-inflammatory and anti-arthritic activities both in in vitro and in vivo systems. There has been a tremendous effort by researchers worldwide to develop the use of triterpenes toward the treatment of a variety of disorders especially the mechanism of action of lupeol and suggest that it is a multi-target agent with immense anti-inflammatory potential targeting key molecular pathways which involve nuclear factor kappa B, phosphatidylinositol-3-kinase in a variety of cells. It was reported that lupeol at its effective therapeutic doses exhibits no toxicity to normal cells and tissues. The efficacy of triterpenes as antitumor (carcinogenesis) by the use of either natural or synthetic substances individually or in combination therapy was promising. However, there was no report on the use of triterpenes as remedy to malaria disease.

Figure 1. 1D and 3D structures of the Isolated Compounds

4.     Conclusions

Our current study revealed that dichloromethane and methanol extracts of Echinaceae angusifolia DC (Compositae) contained significant amount of some secondary metabolites with the methanol extracts indicating the presence of most of the secondary metabolites. The Secondary metabolites present includes flavonoids, terpenes, saponins, steroids, alkaloids and tannins. The revealed cytotoxicity activity in this study is due to the triterpenes isolated from the plant extract. Consequently, this scientific information can serve as an important baseline data for the development of safe and effective natural medicine.

Supplementary Data

Supplementary Fig S1-S10

DOI Link: https://doi.org/10.58985/jeopc.2023.v01i02.18

Authors’ contributions

AZ: conducted the research work, SJ: assisted in chromatography and spectral analysis, FMO: Supervision of the research work and helped for the structural elucidation, MHS: assisted in the purification and TLC experiment, AS: assisted in VLC and general extractions.

Acknowledgements

The authors wish to acknowledge the Tertiary Education Trust Fund (TETFund – Nigeria) for the research grant. The government of the Sultanate of Oman for granting travel VISA and Sultan Qaboos University for the Bench space to carry out the research. The tireless effort of the staff of Central Analytical and Applied Research Unit (CAARU) Sultan Qaboos University Oman in carrying out the MS and NMR analysis is appreciated.

Funding

The Tertiary Education Trust Fund (TETFUND) Nigeria funded the Three (3) years PhD program and a one-year oversea Bench-Work via FUK/R/SS/SF/184/100 and FUK/R/SS/SF/184/101 respectively.

Conflicts of interest

The Authors wish to declare that there is no conflict of interest.

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