The adverse effects and cost of classically used steroids- or NSAID- based drugs as anti-inflammatory drugs have demanded an alternative to existing drugs. The application of herbal medicines as an anti-inflammatory agent would promise safety, better efficacy, and cost-effectiveness. Moreover, the generalised population is more biased towards herbal medicines because of various concerns such as 'natural is better, 'reluctance to allopathy,' religion, or cultural influences. In the current study, efforts have been made to evaluate the anti-inflammatory activity of T. sulcatum.
2.1.Extraction & Fractionation of the Total Crude Extract:
The pharmacologically active components are assessed with extract isolated from the medicinal plant parts, and further fractionation is carried out in different solvents. We first extracted the T. sulcatum leaves in ethanol to yield TSETOH extract. TSETOH extract was further fractionated to yield TSHEX, TSTOL, and TSEA. Soxhlet extraction of T. sulcatum leaves in the three experiments yielded total crude TSETOH extract (56.9 g, 7.59%) as a viscous and dark-coloured semisolid extract. A part of it (46 g) on fractionation yielded hexane soluble fraction (TSHEX, 18.3g, 39.8%), toluene soluble fraction (TSTOL, 14.7 g, 32%), ethyl acetate soluble fraction (TSEA, 4.3 g, 9.3%) leaving the insoluble fraction (TSRES, 8.7 g, 18.9 %).
2.2.Isolation and characterization of the pure compound and its derivatives
Developing novel anti-inflammatory agents with less or no side effects has remained a significant thrust area considering finding alternatives to NSAIDs. Natural products, especially medicinal plants, have remained a very successful avenue for discovering new therapeutic agents. Plants represent an enormous reservoir of new biological active molecules. The chemical structures of secondary metabolites of plants can be used as a model for new synthetic compounds. We attempted to isolate and characterize the active molecules from the leaf extract. Column chromatography of the total crude TSETOH extract yielded a pure crystalline compound. It was characterized by studying physical and spectral data. The details of the structure elucidation are as follows:
Compound I was observed to be white, crystalline, and has a sharp melting point. The elemental analysis suggested the molecular formula C30H52O. Further, the FTIR spectrum exhibited a strong band at 3620 cm− 1, indicating presence of hydroxyl bond in the molecule. 1H NMR spectrum showed a broad one proton multiplet at δ 3.81, indicating the hydroxyl group's presence at the secondary carbon atom. The 13C NMR (Supplementary Table 1) showed a signal of CH at δ 72.8 due to the carbon holding the hydroxyl group. These observations could easily establish the identity of compound I as 'Friedelan-3β-ol'. The structural assignment of compound I as Friedelan-3β-ol was further confirmed by comparing the physical and spectral data with literature (Chama et al., 2020; Gonçalves Pereira et al., 2020). We have identified a triterpene compound Friedelan-3β-ol as the important secondary metabolites in the crude ethanolic extract of T. sulcatum leaves. Thus, first-time we report the occurrence of bioactive Friedelan-3β-ol in T. sulcatum leaf extract. These findings will enrich the natural chemical library of the genus Tetrastigma.
Compound CII and CIII
Further, the Friedelinol acetate (C II) and friedelinol methyl ether (C III) derivatives of Friedelan-3β-ol (CI) were synthesized by known methods, and their structures were elucidated, similarly based on their physical and spectral data. Friedelan-3β-ol, on treatment with acetic anhydride in pyridine, yielded the acetate. Similarly, the reaction with trimethylorthoformate generated methyl ether. Structures of the derivatives were confirmed by recording their spectral data. The FTIR spectrum of acetate compound (C II) showed the diagnostic bands at 1740 and 1380 cm− 1. Further, the 1H NMR spectrum showed three proton singlet at δ 2.0 due to the acetate methyl in addition to the signals of tertiary methyls. The 13C NMR (Supplementary Table 1) and DEPT were in complete agreement with the structure. The spectral data of methyl ether compound (C III) was also in agreement with its structure. FTIR spectrum showed the absence of band due to hydroxyl, while its 1H NMR spectrum showed three proton singlet at δ 3.68 due to the methoxyl group. The 13C NMR (Supplementary Table 1) the spectral data was in complete agreement with those reported in the literature.
2.3.Cytotoxicity assay
For the biomedical application of any compound, the safety of the compound needs to be assessed. The cytotoxic effect of a) TSETOH extract and its fractions and b) Friedelan-3β-ol and its derivatives in RAW 264.7 cells using MTT assay was evaluated. It was observed that TSETOH and its fractions (0-100 µg/mL) were not associated with any toxic effects (Fig. 1A). Similarly, no changes in cell viability were observed by exposing RAW 264.7 cells to different concentrations (0–2.5 µg/mL) of the pure compound and its derivatives (Fig. 1B). This indicates that both extracts and pure compounds have no cytotoxic effect at the concentrations tested in our experimental conditions.
2.4.Evaluation of the in vitro anti- inflammatory effect
We further evaluated the anti-inflammatory effects of plant extracts and pure compounds in vitro using LPS induced inflammatory model. LPS model has been commonly used to study inflammation, as it mimics many inflammatory effects (Grylls et al., 2021).
The activity of crude leaf extract and its fractions
To investigate the anti-inflammatory effects of TSETOH and its fractions, we first examined the inhibitory effects of TSETOH extract and its fractions on LPS induced nitrite (NO) production in RAW 264.7 cells. Treatment of cells with TSETOH and its fractions (TSHEX and TSTOL) demonstrated a dose-dependent decrease in NO production. TSETOH and TSTOL at 100 µg/mL demonstrated the highest reduction in NO production. However, TSEA showed a low or negligible effect on NO production (Fig. 2A). NO is known as a pro-inflammatory mediator in different acute and chronic inflammatory diseases. The stress-induced production of NO in RAW 264.7 cells is related to inflammation. Stressed macrophages generate excessive inducible NO synthase (iNOS), which forms NO as a part of the inflammatory response and causes death by inducing apoptosis (Du et al., 2020).
Inflammation may lead to various cytokines, which act as essential mediators in inducing inflammatory effects. Among these inflammatory cytokines, IL-1β, IL-6, and TNF-α are considered the most crucial in mediating immunity and activating macrophages (Shen et al., 2018; Du et al., 2020). As the highest reduction in NO production was observed at 100 ug /mL, we further evaluated the effect of TSETOH and its fractions (100 µg/mL) on the LPS-stimulated expression of pro-inflammatory cytokines. The mRNA expressions of inflammatory markers IL-1β, IL-6, and TNF-α were measured by qPCR. It was observed that treatment of cells with TSETOH extract and its fractions (TSHEX and TSTOL) resulted in a significant decrease in expression levels of IL-1β, IL-6, and TNF-α compared to LPS stimulated cells which were not treated by extracts (Fig. 2B).
The activity of pure compound Friedelan-3β-ol and its derivatives
Similar to assessing extracts and fractions, the pure compound Friedelan-3β-ol and its derivatives were evaluated for anti-inflammatory effect on LPS induced NO production and expression of pro-inflammatory cytokines levels (IL 1-β, IL-6, and TNF-α) in RAW cells. The result demonstrated that Friedelan-3β-ol and its derivatives significantly inhibited NO production in a dose-dependent manner after treatment. (Fig. 3A). Similarly, expression levels of pro-inflammatory cytokines in LPS stimulated cells significantly reduced compared to non-treated cells. The Friedelan-3β-ol and its derivative exhibited higher inhibitory effects on the expression of TNF-α as compared to the expression of IL 1-β and IL-6 (Fig. 3B).
The in vitro study demonstrated that the ethanolic extract, fractions, pure compound Friedelan-3β-ol, and their derivatives significantly inhibited LPS-induced NO production and mRNA expression of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in RAW 264.7 macrophages. Reports suggest that flavonoid presence suppresses the TLR-4/NF-kB p65 signalling pathway, resulting in decreased gene expression of IL-1β, IL-6, and TNF-α and iNOS proteins during anti-inflammatory effect (Andrade et al., 2020). Macrophage activation induced by LPS causes mitogen-activated protein (MAP) kinases and NF-κB intracellular pathway (Hwang et al., 2017; Sanjeewa et al., 2019). This results in pro-inflammatory mediators such as cytokines or regulates iNOS expression by LPS (Hwang et al., 2017; Sanjeewa et al., 2019). The study exhibited a similar pattern of reduced NO production and pro-inflammatory cytokines reported in previous studies, such as P. Subulate aqueous extract (Genc et al., 2019), O. Gratissimum extract (Dzoyem et al., 2021), L. Spinosa leaf extract (Nguyen et al., 2020), showing anti-inflammation activity.
2.5.Acute oral toxicity study
An acute toxicity study of leaf extract and its fractions was carried out. It was found that the animals treated with the T. sulcatum leaf extract showed no mortality or any untoward signs or symptoms or abnormal behavioural changes during the 14 days observation period following dosing. The animals were found normal during the experimental period. The treated mice exhibited a regular pattern of body weight gain during 14 days period. There were no gross pathological changes observed in all animals' treatment groups than control (Table 3, Fig. 4).
Table 3
Acute toxicity of TSETOH extract at 2000 mg/kg as a single oral dose in mice
Parameters
|
7th day
|
14th day
|
Control
|
Experimental
|
Control
|
Experimental
|
Body Weight
|
30.17 ± 0.19
|
30.57 ± 0.73
|
32.30 ± 0.15
|
32.40 ± 0.37
|
SGPT (IU/L)
|
14.63 ± 1.2
|
13.56 ± 0.80
|
14.40 ± 1.06
|
13.60 ± 0.47
|
Creatinine (mg/dL)
|
0.35 ± 0.04
|
0.31 ± 0.03
|
0.39 ± 0.05
|
0.44 ± 0.04
|
Haemoglobin (g/dL)
|
14.76 ± 0.57
|
14.46 ± 0.31
|
15.76 ± 0.21
|
14.73 ± 0.31
|
RBC (million/µl)
|
7.32 ± 0.19
|
7.31 ± 0.15
|
7.65 ± 0.20
|
7.46 ± 0.15
|
2.6.Assessment of anti-inflammatory activity,
in vivo mice model
Encouraged with the in vitro results, we further evaluated the anti-inflammatory effect of selective extracts and pure compounds in vivo. We used the carrageenan-induced mouse paw oedema model for in vivo studies. Carrageenan-induced mice paw oedema is a commonly used model to test systemic anti-inflammatory activity (Banik et al., 2021; Kumar et al., 2020). In vivo studies demonstrated relative inflammatory activity represented as 'the ratio of treated to control measured paw volume.
The activity of crude leaf extract and its fractions
The TSETOH extract and its fraction (TSTOL, TSHEX), which showed significant anti-inflammatory activity in in vitro study, were further evaluated for in vivo anti-inflammatory activity. TSETOH extract and its fraction at the different doses (200, 400 and 600 mg/kg) showed a significant decrease in the Carrageenan induced mice to paw oedema volume in a dose-dependent manner at 1, 3 and 5 h compared with the control group. It was observed that at a higher dose, 600 mg/kg plant extracts showed a significant decrease in oedema volume comparable with standard anti-inflammatory drug such as Dexamethasone at five hours (Fig. 5A-C).
The activity of pure compound Friedelan-3β-ol and its derivatives
Like extract and fractions, Friedelan-3β-ol and its derivatives exhibited a significant decrease in carrageenan-induced paw oedema volume compared to carrageenan control in a dose-dependent manner. Friedelan-3β-ol and its methyl ether derivative at a dose rate of 30 mg/kg showed decreased carrageenan-induced paw oedema volume comparable with standard anti-inflammatory drug dexamethasone at five hours (Fig. 5D-E).
Gene expression study
Analogous to in vitro gene expression studies, reduced mRNA expression of IL-1β, IL-6, and TNF-α was observed in tissue homogenate of inflamed paw compared to the control group (Fig. 6).
Our previous study reported that the topical application of TSETOH and its fractions (TSHEX, TSTOL, TSEA) significantly reduced inflammation in the TPA-induced ear oedema model (Waghole et al., 2015). In the view of inflammation, the macrophages are antigen-presenting cells (APCs), which signals the immune system to produce various inflammatory mediators such as cytokines, nitric oxide (Kany et al., 2019). The current study reports increased pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and NO production in LPS stimulated inflammation. Carrageenan-induced inflammation in the paw mouse model revealed a similar pattern of NO production and expression of pro-inflammatory cytokines, including interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumour necrosis factor-α (TNF-α) (Ahn et al., 2020). Various traditionally used herbal extracts were found to be a source of triterpenoids, carrying anti-inflammatory activity (Ou et al., 2019; Wu et al., 2021). Similar to the previous reports, the current study demonstrated significantly reduced IL-6, IL-1β, and TNF-α mRNA levels in tissue homogenates of inflamed paw tissues with anti-inflammatory leaf extract activity.
The pure compound Friedelan-3β-ol (CI) and its derivatives Friedelinol acetate (C II), friedelinol methyl ether (C III) also demonstrated the anti-inflammatory activity. The order of activity observed is Friedelinol > Friedelinol acetate > friedelin methyl ether. These findings suggest the presence of free hydroxyl groups is needed for optimum activity. It was revealed that the triterpenoids exhibited better anti-inflammatory activity, which was found to agree with previous reports (Ou et al., 2019; Wu et al., 2021).
Further, the study report demonstrated the anti-inflammatory effect of leaf extract and its fractions comparative with Dexamethanasone (positive control). Dexamethasone (synthetic pregnane corticosteroid; a cortisol derivative) is a known anti-inflammatory and autoimmune condition drug (Black and Grodzinsky, 2019; Giles et al., 2018; Li et al., 2020). Therefore, the leaf extract of T. sulcatum can be taken as an anti-inflammatory drug candidate for future studies.
2.7.Molecular docking study
A strong correlation is observed in cytokine mediators and inflammation in inflammation-associated diseases such as traumas, autoimmune disease, bone pathologies (Kany et al., 2019). To get a better understanding of the structural-activity relationship, molecular docking was performed. The interaction study of compounds and Dexamethasone (a standard steroidal anti-inflammatory drug) was performed with TNF-α (PDB: 2AZ5), IL-6 (PDB: 1ALU), and IL-1β (PDB: 9ILB). The interaction study of IL-6 with compounds demonstrated that Friedelan-3β-methyl ether shared the most common residues (Thr-89, Glu-93, Thr-137, Asp-140, Asn-144, Leu-147, Asn-63, Tyr-97, Thr-143, Val-96, Pro-139) to the Dexamethasone, with higher docking energy of -7.1 kcal/mol− 1. Moreover, TNF-α docked interaction with ligands showed all the compounds (Friedelinol-3ß-ol, Friedelan-3ß-acetate, and Friedelan-3ß-methyl ether) share the common residues with Dexamethasone. Otherwise, IL-1β interaction revealed the common residues (Asn-7, Ser-5, Lys-63, Leu-62, Glu-64, Lys-65, Tyr-68, Tyr-90, Pro-91, Ser-43) with Friedelan-3ß-methyl ether alone, with docking energy of -7.3 kcal/mol-1 (Table 2, Fig. 7).
Table 2
Revealing the binding affinity (kcal mol− 1) and residues interaction of control compound (Dexamethasone) and other compounds (Friedelan-3-ol, Friedelan-3ß-acetate, and Friedelan-3ß-methyl ether) against anti-inflammatory targets TNF- (PDB: 2AZ5), IL-6 (PDB: 1ALU), and IL-1 (PDB: 9ILB). The residues in bold share common with Dexamethasone at respective targets.
βαβ
|
Protein
|
Ligands
|
Binding affinity (kcal mol− 1)
|
Pocket residues
|
IL-6
(PDB: 1ALU)
|
Dexamethasone
|
-6.6
|
Leu-147, Tyr-97, Asn-144, Val-96, Pro-139, Asp-140, Thr-138, Thr-89, Thr-137, Glu-93, Thr-143, Asn-63
|
Friedelan-3β-ol
|
-7.3
|
Asp-134, Leu-133, Lys-70, Thr-89, Glu-93, Lys-86, Asp-71, Val-85, Thr-82, Glu-81
|
Friedelan-3β-acetate
|
-7.1
|
Thr-89, Ile-136, Thr-137, Lys-70, Thr-82, Asp-71, Leu-133, Asp-134, Val-85, Lys-86
|
Friedelan-3β-methyl ether
|
-7.1
|
Glu-93, Asp-140, Asn-144, Leu-147, Lys-150, Leu-62, Asn-61, Asn-63, Tyr-97, Thr-143, Val-96, Pro-139
|
TNF-α
(PDB: 2AZ5)
|
Dexamethasone
|
-8.1
|
Leu-(C)-57, Ile-(C)-58, Tyr-(C)-59, Gly-(C)-122, Leu-(D)-57, Tyr-(D)-59, Gly-(C)-121, Leu-(C)-120, Tyr-(C)-119, Tyr-(D)-151, Gln-(D)-61, Tyr-(D)-119, Ser-(D)-60, Leu-(D)-120, Gly-(D)-121
|
Friedelan-3β-ol
|
-9.7
|
Leu-(D)-120, Leu-(C)-57, Gly-(C)-121, Leu-(D)-57, Tyr-(C)-59, Leu-(C)-120, Tyr-(D)-59, Leu-(B)-55, Gly-(D)-121, Tyr-(D)-119, Tyr-(C)-119
|
Friedelan-3β-acetate
|
-9.5
|
Tyr-(D)-151, Tyr-(C)-59, Gly-(C)-121, Val-(B)-123, Val-(D)-123, Leu-(B)-157, Leu-(D)-57, Leu-(B)-55, Leu-(C)-120, Tyr-(C)-119, Tyr-(D)-59, Ser-(D)-60, Leu-(D)-120, Gly-(D)-121
|
Friedelan-3β-methyl ether
|
-10.4
|
Leu-(B)-55, Tyr-(D)-119, Leu-(C)-120, Ile-(C)-58, Gly-(C)-121, Leu-(C)-57, Gly-(C)-122, Tyr-(C)-59, Leu-(D)-57, Tyr-(D)-59, Tyr-(C)-119, Gly-(D)-121, Leu-(D)-120, Leu-(B)-55,
|
IL-1β
(PDB: 9ILB).
|
Dexamethasone
|
-7.3
|
Leu-67, Leu-62, Tyr-90, Tyr-68, Gly-61, Pro-91, Ser-5, Asn-7, Glu-64, Lys-63, Ser-43, Lys-65
|
Friedelan-3β-ol
|
-7.5
|
Thr-137, Gly-136, Gly-135, Trp-120, Leu-134, Leu-80, Pro-78, Phe-133, Val-132, Pro-131, Thr-79
|
Friedelan-3β-acetate
|
-7.9
|
Lys-77, Leu-134, Pro-78, Leu-80, Ser-125, Asp-142, Met-130, Pro-131, Phe-133, Thr-79, Trp-120
|
Friedelan-3β-methyl ether
|
-7.3
|
Arg-4, Ala-1, Asn-7, Ser-5, Lys-63, Leu-62, Glu-64, Lys-65, Tyr-68, Tyr-90, Pro-91, Ser-43
|
The docking study and experimental results demonstrated that significant inhibition of cytokines by CI and CIII. The in-silico data suggests TNF-α shares most interaction residues of either CI, CII or CIII. The significant reduction of TNF-α mRNA expression levels in vitro is in concurrence with in-silico data. Moreover, the highest binding energy (-10 kcal/mol− 1) was observed to be associated with TNF-α interactions. A similar study reported (Dayakar et al., 2017) the higher selectivity towards TNF-α, suggesting NF-kB pathway activation by anti-inflammatory agents. In the present study, the reduction of pro-inflammatory cytokines has been observed, suggesting the NF-kB pathway's involvement (Andrade et al., 2020).