|Ahead of print publication
In vitro cytotoxicity and anti-inflammatory cytokinine activity study of three isolated novel compounds of Prismatomeris glabra
Khaled A A Alkadi1, Kamran Ashraf2, Aishah Adam1, Syed Adnan Ali Shah2, Muhammad Taha3, Mizaton Hazizul Hasan1, Cinimathew John1, Razali Mohamed Salleh4, Wasim Ahmad5
1 Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia
2 Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia; Atta-ur-Rahman Institute for Natural Product Discovery, UniversitiTeknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia
3 Department of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam 31441, Saudi Arabia
4 Faculty of Applied Science UiTM, 40450 Shah Alam, Selangor, Malaysia
5 Department of Pharmacy, Mohammed Al-Mana College for Medical Sciences, Dammam, Saudi Arabia
|Date of Submission||07-Nov-2019|
|Date of Decision||09-Mar-2020|
|Date of Acceptance||24-Aug-2020|
|Date of Web Publication||21-Dec-2020|
Khaled A A Alkadi,
Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam, 42300, Selongor.
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objectives: The aim of the present study was to isolate and evaluate cytotoxicity and anti-inflammatory activities of new novel compounds isolated from Prismatomeris glabra. Materials and Methods: Dried root of P. glabra was extracted under reflux with methyl alcohol, fractionated through the vacuum liquid chromatography technique, and evaporated and then purified the compounds using column chromatography and preparative thin-layer chromatography. THP-1 cells were treated with amentoflavone, 5,7,4′-hydroxyflavonoid, and stigmasterol with various concentrations (0–30 µg/mL) and then incubated with MTS reagent for 2h. Treatment was done for 24, 48, and 72h. Then, effects of these compounds were also tested on PGE2, TNF-α, and IL-6 expression in human THP-1-derived macrophage cells for 24h. Results: Three new compounds such as amentoflavone, 5,7,4′-hydroxyflavonoid, and stigmasterol were isolated. After 24h of incubation, a significant decrease in cell viability was reported with IC50 values of amentoflavone, 5,7,4′- hydroxyflavonoid, and stigmasterol (21 µg/mL ≡ 38 M), (18 µg/mL ≡ 66 M) and (20 µg/mL ≡ 48.5 M), respectively. Whereas for 48 and 72h treatment showed a less decreased cell viability compared with 24h treatment. These compounds also showed a significant reduction in the production of TNF-α, IL-6, and PGE2 in a dose-dependent manner. Conclusions: The isolated new compounds showed significant cytotoxicity and anti-inflammatory effects.
Keywords: Amentoflavone, anti-inflammatory, cytotoxicity, Prismatomeris glabra
|How to cite this URL:|
Alkadi KA, Ashraf K, Adam A, Shah SA, Taha M, Hasan MH, John C, Salleh RM, Ahmad W. In vitro cytotoxicity and anti-inflammatory cytokinine activity study of three isolated novel compounds of Prismatomeris glabra. J Pharm Bioall Sci [Epub ahead of print] [cited 2021 Jan 20]. Available from: https://www.jpbsonline.org/preprintarticle.asp?id=304167
| Introduction|| |
The medicinal plant has been used for many years in the treatment of various diseases and still endures to provide fresh medications to our community. In recent days, one plant can be used to treat different types of diseases because there is always discovery of new characteristics in the medicinal plant. Consequently, screening of plants for biological activity for more than one pharmacological activity is significant. Hence, testing and screening of raw extract of the plants are required for the development of new plant-based entities with high effectiveness and lower side effect in the treatment of various diseases and cancer in particular.,
Prismatomeris glabra (Rubiaceae) or locally known as “Haji Samat,” is usually found in the mixed dipterocarp and keranga forests of up to 700 m altitude in Peninsular Malaysia, Sumatra, and Borneo.,,Prismatomeris glabra has simple and oppositely arranged leaves with white-purplish flowers and green-whitish fruits in the form of berries. In Malaysia, the aqueous extracts of the roots of P. glabra have been used traditionally by the farmers and certain rural Malays for wellness, enhancing stamina, and for its ergogenic effects. In addition, as stated by a local taxonomist, this plant has been used as an aphrodisiac for generations in Kelantan and Terengganu.,,,
| Materials and Methods|| |
General experimental procedures
Melting points were determined on a Yanaco MP-S3 apparatus. UV spectra were measured on a Shimadzu UV 240 spectrophotometer. A JASCO DIP–360 Digital polarimeter was used to measure the optical rotations in chloroform by using a 10-cm cell tube. The 1H-NMR and 2D NMR spectra were recorded on a Bruker Avance III 600 Ascend spectrometer using the BBO probe, while 13C-NMR spectra were recorded on a Bruker Avance III 600 Ascend spectrometer operating at 125 MHz using CDCl3 as a solvent. Chemical shifts were reported in δ (ppm), relative to SiMe4 as internal standard, and coupling constants (J) were measured in Hz. The EI-MS and HREIMS were measured on the Jeol HX 110 mass spectrometer. Thin-layer chromatography (TLC) was performed on silica gel pre-coated plates (PF254 20 × 20, 0.25mm, Merck, Germany). Ceric sulfate in 10% H2SO4 spraying reagent was used for the staining of compounds on TLC. All reagents used were of analytical grades.
Plant material and extraction
The root of P. glabra species was collected from Pasoh-Negeri Sembilan. Voucher specimens were prepared, identified, and deposited at the Herbarium of the Forest Research Institute Malaysia (FRIM), Kepang.
The dried root of P. glabra was extracted under reflux with MeOH for 6–8h. The obtained MeOH extract was fractionated by the vacuum liquid chromatography (VLC) technique using different polarities. Evaporation of the respective solvents and purified the compounds through column chromatography and preparative TLC.,,
THP-1 cells were grown in T75 cell culture flasks in a total volume of 20mL of complete cell culture medium. Cells were kept in an incubator at 37°C and 5% CO2 in humidified air.
Cell seeding into 96-well plate
Confluent (80–90%) cells were taken out from the incubator for plating. Cells were viewed under an inverted microscope to check the percentage of confluence and to confirm the absence of bacterial and fungal contaminations. The cells were centrifuged for 5min at 4000rpm, the pellet was formed, the supernatant was removed, and 1mL of complete medium was added into the centrifuge tube. The cells were re-suspended and 10 μL was taken and added into an Eppendorf tube containing 100 μL of trypan blue for the determination of cell viability. Then, 30 μL of the mixture was pipetted onto both sides of a hemocytometer and the number of cells was counted. Cells were seeded at a density of 3 × 105 per well in a 96-well plate, and incubated with the desired drug concentration in triplicates, to a total of 200 μL, at 37°C in a humidified atmosphere of 5% CO2 for 24h. We used two negative controls for the tests: (1) THP-1 cells in drug-free culture media. (2) THP-1 cells treated for 24, 48, and 72h with the maximum concentration of the drug’s solvent used in the experiments (4% DMSO).,,,
The following equation was used to calculate the volume of cells and the volume of media needed to get 2 × 104 cells/mL in each well plate.
where d.f. is the dilution factor.
The volume of media per well to get 2 × 104 cells/mL = 50 µL − volume of the cells per well.
The levels of cytokines TNF-α, IL-6, and PGs were determined by specific enzyme-linked immunosorbent assay (ELISA) techniques according to the manufacturer’s instructions (R&D Systems Europe, France). The minimum detectable doses were less than 5 pg/mL for TNF-a and IL-1b, and less than 20 pg/mL for IL-6. The serum samples were incubated without dilution. In comparison with a standard cytokine curve, the concentrations of cytokines in serum or tissue extracts were determined spectrophotometrically (Bio-Tek EL 808, Bio-Tek Instruments Inc., Colmar, France) by reading the absorbance at 420 nm.,,
All data were expressed as mean ± SD. The statistical analysis was performed using Sigma Stat software (SPSS Science, NC, USA). The global course of each parameter was evaluated by an ANOVA ranks test (Kruskal–Wallis test) and Dunnett’s test, which is used for multiple comparison procedures, to compare each of many treatments with a single control.
| Results and Discussion|| |
The dried stem root of P. glabra weighing 800 g was turned into the powdered form on a dry grinder successively. Extraction with a maceration was done using 500 g of the powder in 1L of methanol and kept the mixture on a sealed tank for 3 days. The MeOH extracted solution was evaporated under pressure to yield a gummy extract (45 g). Twenty grams from this extract was subjected to vacuum liquid chromatography on a Sephadex LH-20 eluted with n-hexane–EtOAc (50:50→0:100) system and MeOH to give six fractions: fr.1, 1.5 g; fr. II, 2.8 g; fr. III, 4.5 g; fr. IV, 5.2 g; fr. V, 4.1 g; fr. VI, 1.7 g. Fractions III (4.5 g), fr. IV (5.2 g) and fr. V (4.1 g) were further separately subjected to silica gel and a Sephadex LH-20 column eluted with n-hexane–EtOAc and MeOH system. Evaporation of the respective solvents and purification through preparative TLC and Sephadex LH-20 provided β-stigmasterol (1) (7 mg), apigenin (2) (4 mg), and amentoflavone (3) (6 mg) [Figure 1].
|Figure 1: Compounds isolated from Prismatomeris glabra β-stigmasterol (1), apigenin (2), and amentoflavone (3)|
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The 1H and 13C assignments of compound 1 were verified by 2D Heteronuclear Single Quantum Coherence (HSQC) experiments, while connectivities were verified by Heteronuclear Multi Bond Coherence (HMBC). The hydroxyl group was attached to C-3 due to long-range correlations between this carbon and the methylene protons at H-1 and H-2. One of the double bonds was placed at C-5 because long-range correlations were observed between this carbon and H-4 and H-19. The second double bond was placed at C-22 due to long-range correlations between this carbon and H-20 and H-21. The position of the isopropyl group was deduced from the correlations between C-24 with H-28 and H-29. All long-range correlations observed are consistent with the structure of compound 1.
The relative stereochemistry of compound 1 was deduced from the NOESY spectrum and is as drawn in the structure. NOESY correlations indicate that the protons in the β-position (above the plane) are as follows: H-8/H-18/H-19/H-20/ H-21/H-23, while for the protons in the α position (below the plane) are as follows: Hla/H-3/H-9/H-14/H-17/H-22/H-24. The literature search revealed that compound 1 is β-stigmasterol as evident by its similar 1H and 13C NMR spectral data. Compound 2 was obtained as a yellow powder and its molecular formula was established as C15H10O5 from its molecular spectrum (MS) data, which showed [M + H]+ ion at m/z 271. The molecular formula of 2 was further supported by its 13C NMR spectral data. The UV spectrum of 2 also showed absorption maxima at 268, and 337 nm suggested a flavonoid structure. The 1H NMR spectrum of 2 showed the presence of two meta coupled aromatic doublets at δ 6.68 and 6.88 corresponding to H-6 and H-8 protons, two doublet of doublets at δ 6.95 and 7.81 for H-3′/H-5′ and H-2′/H-6′ protons of ring B, and a singlet at δ 6.62 corresponding to H-3 protons, characteristic for a 5, 7, 4′-trisubstituted flavone. The 13C NMR spectrum showed the presence of 12 aromatic carbons; 7 quaternary carbons, 5 methine carbons, and an unsaturated carbonyl carbon. The 1H and 13C NMR values for all the carbons were assigned based on HSQC and HMBC correlations. A search in the literature suggested that the spectral data of 2 were consistent to 4′, 5, 7-trihydroxyflavone, also known as apigenin. The structure was further supported by the COSY and HMBC correlations as shown in [Figure 2].
|Figure 2: HMBC correlations of compound 1 and COSY and HMBC correlation of compound 2|
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Compound 3 was obtained as a colorless powder and its molecular formula was established as C30H18O10 from its MS data that showed [M + H]+ ion at m/z 539. 1H NMR spectrum of 3 showed two signals at δ H 13.09 and 12.96 due to chelated phenolic hydroxyls, and two doublets at δ 7.56 (J = 9.0 Hz, H-2‴, H-6‴), and 6.70 (J = 9.0 Hz, H-3‴, H-5‴) assigned to an AA′BB′ aromatic system. Signals of a trisubstituted aromatic ring containing one oxygenated carbon were observed at δH 8.00 (d, J = 2.5 Hz, H-2′), 7.14 (d, J = 9.0 Hz, H-5′), and 7.99 (dd, J = 9.0; 2.5 Hz H-6′), along with doublets of a tetrasubstituted aromatic ring containing three oxygenated carbons at δH 6.18 (J = 2.5 Hz, H-6) and 6.45 (J = 2.5 Hz, H-8). Analysis of the HMBC spectra evidenced correlations of the hydroxyl group with hydrogen bonded by a cross peak at δH 13.09 (OH-5) with C-5″ (δ 161.1), C-6″ (δ 98.7), and C-10″ (δ 103.6) and δH 12.96 (OH-5″) with C-5 (δ 162.1), C-6 (δ 98.9) and C-10 (δ 103.7), which are in agreement to those of amentoflavone.
Cytotoxicity of the compounds isolated from P. glabra using THP-1 macrophage cell line
Cytotoxicity of stigmasterol (1), 5, 7, 4′-hydroxyflavonoid (2), and amentoflavone (3), with various concentration (0–30 µg/mL) on THP-1 cells for 24h incubation, x̄ ± ±S.D. (n = 3). IC50 values for stigmasterol was 20 µg/mL (≡48.5 M), 5, 7, 4′-hydroxyflavonoid was 18 µg/mL (≡66 M), and amentoflavone was 21 µg/mL (≡38 M), as shown in [Figure 3].
|Figure 3: Cytotoxicity of the compounds with various concentrations (0–30 μg/mL) on THP-1 cells for 24 h incubation|
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Activity of proinflammatory cytokines (PGE2, IL-6, and TNF-α)
Effect of stigmasterol, 5, 7, 4′-hydroxyflavonoid, and amentoflavone on PGE2 expression in human THP-1-derived macrophages cells were pretreated with different concentrations (0–30 µg/mL) for 24h. At the end of the treatment, the cell culture medium was collected. PGE2 was measured using an ELISA kit. x̄ ± S.E.N (n = 3). Significantly different *P << 0.0001 (ANOVA + Dunnett’s test) [Figure 4].
|Figure 4: Effect of stigmasterol (1), 5,7,4′-hydroxyflavonoid (2), and amentoflavone (3) on PGE2 expression in human THP-1-derived macrophage cells were pretreated with different concentrations (0–30 μg/mL) for 24 h|
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The effect of stigmasterol, 5, 7, 4′-hydroxyflavonoid, and amentoflavone on IL-6 expression in human THP-1-derived macrophages cells was pretreated with different concentrations (0–30 µg/mL) for 24h. At the end of treatment, the cell culture medium was collected. IL-6 was measured using an ELISA kit. x̄ ± S.E.N (n = 3). Significantly different *P << 0.0001). (Anova + Dunnett’s tests) [Figure 5].
|Figure 5: Effect of stigmasterol (1), 5,7,4′-hydroxyflavonoid (2), and amentoflavone (3) on IL-6 expression in human THP-1-derived macrophage cells were pretreated with different concentrations (0–30 µg/mL) for 24 h|
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Tumor necrosis factor (TNF-α)
The effect of stigmasterol, 5, 7, 4′-hydroxyflavonoid, and amentoflavone on TNF-α expression in human THP-1-derived macrophages cells were pretreated with different concentrations (0–30 µg/mL) for 24h. At the end of the treatment, the cell culture medium was collected. TNF-α was measured using an ELISA kit. x̄ ± S.E.N (n = 3). Significantly different *P < <0.0001 (ANOVA + Dunnett’s tests) [Figure 6].
|Figure 6: Effect of stigma sterol (1), 5,7,4′-hydroxyflavonoid (2), and amentoflavone (3) on TNF-α expression in human THP-1-derived macrophage cells were pretreated with different concentrations (0–30 µg/mL) for 24 h|
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The effects of the compounds on PGE2, TNF-α, and IL-6 expression in human THP-1-derived macrophage cells were pretreated with different concentrations showed significantly reduced production of TNF-α, IL-6, and PGE2, in a dose-dependent manner.
Prismatomeris glabra is a well-known Malaysian plant. In this research, we isolated and identified three novel compounds with good cytotoxicity and anti-inflammatory activity. Some literature also showed the presence of saponins, flavonoids, alkaloids, terpenoids, tannins, and glycosides in P. glabra. Burkill also identified the ergogenic effects of P. glabra. As the study showed, pro-inflammatory cytokines such as TNF-a, IL-6, PGs play a key role in the process of inflammatory diseases. Too much production of these pro-inflammatory cytokines may cause a systemic inflammatory response syndrome, shock. The present study could help open a window for the development of a new drug entity.
| Conclusions|| |
The effects of the compounds (5, 7, 4′-hydroxyflavonoid, amentoflavone, and stigmasterol) on PGE2, TNF-α and IL-6 expression in human THP-1-derived macrophage cells showed significantly reduced production of TNF-α, IL-6, and PGE2, in a dose-dependent manner. Thus, the isolated compounds showed significant cytotoxicity and anti-inflammatory effects and it can be used as an entity for drug discovery in the future.
Authors would like to acknowledge the Ministry of Higher Education (MoHE) for financial support under the Fundamental Research Grant Scheme (FRGS) with sponsorship reference numbers FRGS/1/2019/STG05/UITM/02/9. The author would also like to acknowledge Universiti Teknologi MARA for the financial support under the reference number 600-IRMI/FRGS 5/3 (424/2019).
Financial support and sponsorship
Conflict of interests
The authors declare no conflict of interests
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]