|Year : 2020 | Volume
| Issue : 3 | Page : 285-298
Comparative botanical and phytochemical studies of ambiguous medicinal plant species of Wedelia and Eclipta (Fam. Asteraceae) used in ASU systems of medicine with special reference to in-silico screening of hepatoprotective potential of marker wedelolactone with acetaminophen targets
C Arunachalam1, R Arunadevi2, S Murugammal3, N Monika2, R Susila4, S N Sunil Kumar5
1 Department of Botany, Captain Srinivasa Murthy Regional Ayurveda Drug Development Institute, (CCRAS, Ministry of AYUSH, Government of India, New Delhi), Anna Hospital Campus, Chennai, Tamil Nadu, India
2 Department of Pharmacology, Captain Srinivasa Murthy Regional Ayurveda Drug Development Institute, (CCRAS, Ministry of AYUSH, Government of India, New Delhi), Anna Hospital Campus, Chennai, Tamil Nadu, India
3 Department of Chemistry, Siddha Central Research Institute, (CCRS, Ministry of AYUSH, Government of India, Chennai), Anna Hospital Campus, Chennai, Tamil Nadu, India
4 Department of Clinical Research, Siddha Central Research Institute, (CCRS, Ministry of AYUSH, Government of India, Chennai), Anna Hospital Campus, Chennai, Tamil Nadu, India
5 Department of Botany, Siddha Central Research Institute, (CCRS, Ministry of AYUSH, Government of India, Chennai), Anna Hospital Campus, Chennai, Tamil Nadu, India
|Date of Submission||15-Feb-2020|
|Date of Acceptance||29-Apr-2020|
|Date of Web Publication||14-Aug-2020|
Dr. R Arunadevi
Captain Srinivasa Murthy Regional Ayurveda Drug Development Institute, (CCRAS, Ministry of AYUSH, Government of India, New Delhi), Anna Hospital Campus, Arumbakkam, Chennai, Tamil Nadu
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Context: In traditional medicine, Kesaraja (Ayurveda) or Manjal karisali (Siddha) is effective for jaundice. Aim: Three species of Asteraceae need to be studied for their therapeutic superiority of their intended claim. They are Wedelia chinensis (Osbeck) Merr. Philipp J., Wedelia trilobata (L.) Hitchc. and Eclipta prostrata (L.) L. (Asteraceae). The present study aimed to screen and characterize the potential species for therapeutic purpose. Materials and Methods: The whole plants, W. chinensis (Osbeck) Merr. Philipp J., W. trilobata (L.) Hitchc. and Eclipta prostrata (L.) (Asteraceae) were collected and botanically identified. Preliminary phytochemical analysis and high-performance thin-layer chromatography finger printing with marker wedelolactone were done for the ethanolic extracts of these plants. Botanical and pharmacognostical diagnostic characters of the plants based on macro-morphological, micro-morphological and powder microscopical characterization were worked out. Comparative in-vitro antioxidant potential of ethanolic extracts of these plant species was carried out. Using ADMET SAR software, the pharmacokinetics of wedelolactone were predicted. Using Autodock 4.2 software, the binding energy of wedelolactone on targets of acetaminophen-induced hepatotoxicity namely PPAR-α, AMPK, JNK-1, EGFR, Nrf2, ALT, ALP, GGT, CAR, Frizzled receptor, FXR, ERK1, LXR, mitochondrial glutamate dehydrogenase, p53, mTOR C1, CYP1A2, CYP2E1, 5-lipoxygenase, thrombin, UCP1, GSK1, RXR and PXR was predicted. Results: All the three plant species were pharmacognostically and chemically different. W. chinensis was found to possess more antioxidant potential than the other two plants. The marker compound wedelolactone was not detected in W. trilobata. Wedelolactone passed the Lipinski's rule of five, and the docking analysis of wedelolactone confirmed high binding affinity toward PPAR-α, AMPK, Nrf2, CYP2E1, EGFR, JNK1, UCP-2, thrombin, 5-lipoxygenase, mTORC1, RXR, FXR, LXR, Frizzled receptor, GDH and Erk-1. Conclusion: Based on the above observations, we conclude that the presence of marker compound wedelolactone might have attributed the potency of W. chinensis and E. prostrata in counteracting acetaminophen toxicity when compared with W. trilobata.
Keywords: Antioxidant, Ayurveda, Siddha, botany, docking, Wedelia chinensis, Wedelia trilobata, Eclipta prostrata, wedelolactone, high-performance thin-layer chromatography, macro-morphological, micro-morphological, phytochemical, powder microscopy
|How to cite this article:|
Arunachalam C, Arunadevi R, Murugammal S, Monika N, Susila R, Kumar S N. Comparative botanical and phytochemical studies of ambiguous medicinal plant species of Wedelia and Eclipta (Fam. Asteraceae) used in ASU systems of medicine with special reference to in-silico screening of hepatoprotective potential of marker wedelolactone with acetaminophen targets. Phcog Res 2020;12:285-98
|How to cite this URL:|
Arunachalam C, Arunadevi R, Murugammal S, Monika N, Susila R, Kumar S N. Comparative botanical and phytochemical studies of ambiguous medicinal plant species of Wedelia and Eclipta (Fam. Asteraceae) used in ASU systems of medicine with special reference to in-silico screening of hepatoprotective potential of marker wedelolactone with acetaminophen targets. Phcog Res [serial online] 2020 [cited 2020 Oct 30];12:285-98. Available from: http://www.phcogres.com/text.asp?2020/12/3/285/292043
- In traditional medicine, Kesaraja (Ayurveda) or Manjal karisali (Siddha) is effective for jaundice. Three species of Asteraceae need to be studies for their therapeutic superiority of their intended claim. They are Wedelia chinensis (Osbeck) Merr. Philipp J., Wedelia trilobata (L.) Hitchc. and Eclipta prostrata (L.) L. (Asteraceae). The present study aimed to screen and characterize the potential species for therapeutic purpose with special reference to their antioxidant activity and in silico screening of marker wedelolactone with acetaminophen targets. Based on this study, it is concluded that all the three plants are different. W. trilobata did not have the marker compound wedelolactone. In silico prediction supports the drug likeliness of wedalolactone and its interaction with target proteins. The chemical profiling of W. chinensis is different from that of Eclipta prostrata, but the marker compound is common for both. Because W. chinensis and E. prostrata were interchangeably used for common ailments, the marker compound wedelolactone might have been responsible for their shared efficacy. W. chinensis was observed to be more potent antioxidant than the other two species. Hence, Wedelia chinensis may be a potential species for counteracting acetaminophen toxicity either as a drug or as a supportive therapy.
Abbreviations Used: AAP: Acetaminophen; HPTLC: High-performance thin-layer chromatography; ROS: Reactive oxygen species; NAPQI: N-acetyl-p-benzoquinone imine; ER: Endoplasmic reticulum; PPAR α: Peroxisome proliferator activated receptor α; AMPK: AMP-activated kinase; JNK-1: c-Jun N-terminal Kinase-1; EGFR: Epidermal growth factor receptor; Nrf2: Nuclear factor erythroid 2-related factor 2; ALT: Alanine transaminase; ALP: Alkaline Phosphatase; GGT: Gamma glutamyl transpeptidase; CAR: Cartesenoid activated receptor; FXR: Farnesoid X Receptor; ERK1: Extracellular signal regulated kinase; LXR: Liver X Receptor-alpha; GDH: Mitochondrial Glutamate dehydrogenase; mTORC1: Mammalian target of Rapamycin Complex; CYP1A2: Cytochrome P450 1A2; CYP2E1: Cytochrome P450 2E1; 5-LO: 5-lipoxygenase; UCP-2: Uncoupling protein 2; GSK-1: Glycogen synthase kinase; RXR: Retinoid X receptor; NF-κB: Nuclear factor-kappa B; PXR: Pregnane X receptor; UV: Ultraviolet; W. chinensis: Wedelia chinensis; W. trilobata: Wedelia trilobata; E. prostrata: Eclipta prostrata; DPPH: Diphenyl picrazyl hydrazide; IC50: 50% inhibitory concentration; Ro5: Rule of five; ADMET: Absorption, Distribution, Metabolism, Excretion, Toxicity; HIA: Human intestinal absorption; CYP: Cytochrome P; EDTA: Ethylenediaminetetraacetic acid; Log P: Logarithmic value of partition coefficient; Log S: Logarithmic value of Solubility; H bond: Hydrogen bond; PSA: Polar surface area; BBB: Blood–brain barrier; Ames test: Mutagen identified via toxicity test; FHMT: Fish toxicty; TPT: Tetrahymnema pyriformis toxicity; HBT: Honey bee toxicity; GST: Glutathione transferase; GSH: Glutathione; SULT2: Sulfo transferase; GDH: Glutamate dehydrogenase.
| Introduction|| |
The perennial herbs Wedelia chinensis (Osbeck) Merr. Philipp J., Wedelia trilobata (L.) Hitchc. and Eclipta prostrata (L.) L. belong to the family Asteraceae (formerly Compositae). A decade ago, W. chinensis was often used as a substitute for E. prostrata due to the confusion in their common name. W. chinensis and W. trilobata were confused nowadays due to the similarity in their morphology. Being edible in the form of greens, there is a problem in right identification of the species which were misunderstood with one another. Generally, these plants are distributed all over India, mostly in rainy season and all the year around in wet and watery places. There are three different varieties of Bhringraja in the Nighantus, namely, white, black, and yellow. The white is Eclipta alba or E. prostrata, also named Karicalai in Siddha Formulary of India, and the yellow varieties are W. chinensis and W. trilobata, whereas the black types of Bhringraja seems to be a variety of Eclipta with dark-colored stems. Because the right medicine for the right ailment is a fundamental requirement in herbal medicine, the current investigation was aimed at comparing these three species pharmacognostically, phytochemically, and in silico pharmacologically.
E. prostrata is used as liver tonic and hair growth promoter traditionally in the Indian Systems of Medicine. It is also used in the treatment of spleen enlargements, uterine hemorrhages, skin diseases, and respiratory disorders. The plant W. chinensis is called as Kesaraja in Ayurveda and Potrilaikaiyan and Potrilaikaiyanthagarai in Siddha System of Medicine and also has medicinal value in traditional Chinese medicine and Unani. As per the Ayurvedic Pharmacopoeia of India, the whole plant has been used for various ailments such as piles, diarrhea, alopecia, helminthiasis, jaundice, cough, headache, gynecological disorder and ulcer, whereas in Siddha Pharmacopoeia of India, the same plant has been used for night blindness and ulcer.
It is generally considered that W. chinensis is native to India, China, and South-East Asia, whereas W. trilobata is a different species that originates from Caribbean, Central America, and north of South America and was introduced to Asia as an ornamental plant; being highly probable, it escaped cultivation and got naturalized. W. trilobata, as it's name indicates, has distinct three lobes on its leaves, whereas W. chinensis has leaves with more smooth margins, rarely serrated or lobed. Most often, W. trilobata was used in the place of W. chinensis because of its ubiquitous availability.
Most of the edible plant's phytoconstituents possess free radical scavenging ability. Normally, the reactive oxygen species (ROS) generated during physiological process will be cleared by the presence of antioxidants in the body. However, owing to inadequate antioxidant defense, this balance will be disturbed, favoring the ROS increase that end up in oxidative stress, which leads to several disorders. Abundant use of acetaminophen (AAP) for pyrexia especially fevers of viral entity often ended up with toxicity, The mechanisms of AAP-induced liver injury are highly complex, and many intracellular and extracellular events are involved in this pathophysiological process, including metabolism of AAP to its metabolite N-acetyl-p-benzoquinone imine (NAPQI), mitochondrial oxidative stress, ER stress, autophagy, sterile inflammation, and microcirculatory dysfunction. Hence, the key proteins involved in the individual process were selected for the docking study with wedelolactone, a marker compound for E. prostrata, and W. chinensis, and both plants possess hepatoprotective potential., The selected targets (PDB ID) are peroxisome proliferator activated receptor-α (PPAR-α: 3KDU), AMP-activated kinase (AMPK: 6C9F), c-Jun N-terminal kinase-1 (JNK-1: 3ELJ), epidermal growth factor receptor (EGFR: 2RGP), nuclear factor erythroid 2-related factor-2 (Nrf2: 2FLU), alanine transaminase (ALT: 3IHJ), alkaline phosphatase (ALP: 1ZEB), gamma glutamyl transpeptidase (GGT: 4GDX), cartesenoid-activated receptor (CAR: 1XNX), frizzled receptor (4F0A), farnesoid X receptor (FXR: 5Q0I), extracellular signal regulated kinase 1 (ERK1: 2ZOQ), liver X receptor-alpha (LXR: 1UHL), mitochondrial glutamate dehydrogenase (GDH: 1L1FA), p53 (1C26), mammalian target of rapamycin complex 1 (mTORC1: 5H64), cytochrome P450 1A2 (CYP1A2: 2HI4), cytochrome P450 2E1 (CYP2E1: 3E6I), 5-lipoxygenase (5-LO: 308Y), thrombin (2A2X), uncoupling protein 2 (UCP-2: 2LCK), glycogen synthase kinase (GSK -1: 1GNG), retinoid X receptor (RXR: 4K4J), nuclear factor-kappa B (NF-κB: 1SVC), and pregnane X receptor (PXR: 2QNV). Hence, the present study aimed to characterize the plants, test their antioxidant and free radical scavenging ability, and explore in silico hepatoprotective potential against acetaminophen-induced liver damage.
| Materials and Methods|| |
Three plant species in Asteraceae family, namely Wedelia trilobata (Osbeck) Merr. Philipp J. (syn. of Solidago chinensis Osbeck; Wedelia calendulacea Less), W. trilobata (L.) Hitchc. (syn. of Sphagneticola trilobata L.), and Eclipta prostrata (L.) L. (syn. Eclipta alba L.) were collected from Chennai and nearby areas and taxonomically identified at CSMRADDI (CCRAS), Arumbakkam, Chennai, and its voucher specimens (W. chinensis (00473), W. trilobata (00626), and E. prostrata (00530)) were preserved in the Department of Botany, CSMRADDI, Chennai. The plants were subjected to botanical testing by macro-morphological, micro-morphological, and powder microscopical characters. Plant powders were treated with benzene, chloroform, ethyl acetate, methanol, acetone, ethanol, and water for fluorescence characters under ultraviolet (UV) light of 366 nm.
All the three plants were collected, shade dried, powdered, and extracted with ethanol (80°C) for 48 h under reflux by Soxhlet extraction. It was filtered and concentrated under reduced pressure, made free of solvent to obtain a semisolid mass. The preliminary phytochemical screening of W. chinensis, W. trilobata, and E. prostrata was determined.
High-performance thin-layer chromatography fingerprinting of Wedelia chinensis, Wedelia trilobata, and Eclipta prostrata extracts
Powders of W. chinensis, W. trilobata, and E. prostrata (4 g) were extracted with 40 ml of ethanol (80°C) under reflux for 8 h. The extracts were concentrated to 10 ml solution. Different volumes of these solutions were applied on a precoated silica gel 60 F254 of 2 mm thickness aluminum plates to a bandwidth of 6 mm using CAMAG HPTLC system equipped with TLC Linomat IV applicator and TLC scanner 3, and win CATS 1.4.4. Software (Camag, Muttenz, Switzerland) was used for HPTLC analysis. The plate was developed in a solvent system of toluene:ethyl acetate:formic acid (7:2.5:0.5) up to 80 mm. The developed plates were visualized and scanned under UV 254 nm (D2 lamp) and 366 nm (Hg lamp) and under white light at 520 nm (tungsten W lamp) after derivatization in vanillin-sulfuric acid spray reagent. Rf values of the spots were recorded.
In vitro antioxidant activity
Determination of diphenyl picrazyl hydrazide radical scavenging activity
To 1 ml of ethanolic extracts of the three plants at various concentrations, 1 ml of 10 μM DPPH was added and incubated at 37°C for 30 min. Change in the absorbance of reaction mixture was read at 517 nm in an UV-visible spectrophotometer. The percentage radical scavenging activity was calculated by the following formula:
Determination of iron chelating activity
A volume of 0.1 ml of 2.0 mM ferrous chloride was added to 1.0 ml of ethanolic extracts of the three plants at different concentrations. After vortexing, the mixture was incubated for about 10 min and the reaction was initiated by the addition of 0.4 ml of 5 mM ferrozine solution. The mixture was vigorously shaken and left to stand for 10 min at room temperature. The absorbance of ferrozine–Fe2+ complex formation was measured at 562 nm in the UV-visible spectrophotometer. The percentage inhibition was calculated as follows:
In silico screening
The marker compound, wedelolactone, was subjected to predict the drug-likeliness properties as per Lipinski's rule of five (Ro5). Lipinski's screening was performed using Molinspiration web server, and the physicochemical properties of ligand were calculated.
Wedelolactone was subjected to docking studies with acetaminophen target proteins using Autodock 4.2 (Molecular Graphics Laboratory (aka Olson Laboratory), U.S.A), and binding energies were calculated. Further, wedelolactone was subjected to ADMET prediction for pharmacokinetics and pharmacodynamics properties.
The three-dimensional crystal structures of the target proteins were retrieved from the Protein Data Bank. The ligand molecule, wedelolactone, was retrieved from Pub Chem server and was refined using ACD Chem Sketch software (Advanced Chemistry Development, Canada), a tool that offers functionalities such as structure refining and optimization.
The target proteins were docked with wedelolactone using Auto Dock 4.2. The free energy of binding between the ligand and proteins was calculated. Auto Dock 4.2. uses charge-based desolvation force fields and well-defined improved models of the unbound state. The docking analysis attempts to bind the ligand to the obtained binding sites of the target protein and produces the best docked conformations with minimal energy, as the output. A semi-flexible docking protocol was applied, wherein the target proteins were kept rigid, while the phytochemical ligand was kept flexible for being docked upon. A 5A° grid was built surrounding the binding pocket. Grid map dimensions were set to yield the receptor model that included atoms within 0.5A° of the grid center. All the other parameters were kept at default, and Lamarckian genetic algorithm was chosen to predict the best conformers. The protein–ligand complexes were viewed using Molegroviewer software (Molegro Molecular Viewer, CLC bio company, Denmark). Each protein consists one or more chains; the active sites were predicted based on their ligands which were previously docked; if not, each chain of the protein was individually docked and the chain which has less binding energy with ligand was reported.
Absorption, Distribution, Metabolism, Elimination, and Toxicity studies
The molecular structure of ligand was submitted to ADMET-SAR web tool to examine their drug likeliness and different pharmacokinetic and pharmacodynamic parameters including human intestinal absorption (HIA); Caco-2 permeability; aqueous solubility; blood–brain barrier penetration; renal organic cation transportation; cytochrome P (CYP) inhibitory promiscuity; cytochrome P450 inhibition; AMES toxicity; fish toxicity; rat acute toxicity; Tetrahymena pyriformis toxicity; human ether-ago-go-related gene inhibition; and mutagenic, tumorigenic, and reproductive risks.
| Results|| |
Pharmacognostic characterization of the herbs
Macro-morphology of the plants
Comparative macro-morphology of the three plants is depicted in [Figure 1].
|Figure 1: Comparative macro-morphology of W. chinensis, W. trilobata, and E. prostrata|
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Stem cylindrical, branched with pubescence at surface, small roots at the lower nodes. Leaf 2.7 to 7.5 cm long and about 1.8 to 2.5 cm wide. Leaves opposite, sub-sessile, and lanceolate to oblong in shape. Margin entire, sparsely mucronulate-serrulate, base tapering. Apex acute, scabrous, covered with short, white trichomes. Flower had a solitary head, slender with tetragonous rayed flower yellow in color, in axillary or at the terminal heads.
Stem decumbent, branched and cylindrical in shape, with nodes bearing roots which had lateral or secondary branched small hairs. Leaf three lobed with a serrated margin had small petioles. Flower solitary, yellow in color with slender and long peduncles.
Stem erect and cylindrical; pubescence at the surface and branched with distinct nodes and internodes. Leaf 2–9 cm long and about 1–2.5 cm wide, opposite, sub-sessile to sessile, lanceolate to oblong in shape, dentate margin, acute apex and pubescence surface. Lower surface paler. Flower solitary with tubular disc-rayed flower white in color.
Comparative micro-morphology of the three plants is given in [Figure 2].
|Figure 2: (a) Comparative micro-morphology of stem midrib, lamina, stomata, veins and trichomes of W. chinensis, W. trilobata, and E. prostrata. e/ep: Epidermis; p: Parenchyma; u.e: Upper epidermis; st: Stomata; h: Hypodermis; v.b: Vascular bundles; l.e: Lower epidermis; pa: Palisade cells; c: Cortex; pi: Pith; t: Trichome; v: Vein; v.t: Vein terminal|
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In stem, epidermis had a thin-layered cuticle covered with three-celled unicellular trichomes and glandular trichomes with eight-celled heads. Hypodermis had 3–5 rows of walled polygonal collenchymatous cells yellow in color. Cortex composed of aerenchyma with certain large intracellular space. Endodermis and pericycle layer were distinct. Leaf dorsiventral, covered with a prominent cuticle had thin-walled epidermis in the upper and lower surfaces. Palisade cells were present in a single layer on the upper surface. It consisted of well-developed spongy parenchyma and amphicribal vascular bundles situated at the center surrounded by a thick-walled bundle sheath. Palisade ratio was about 3–4 and the stomatal index was 12–14 on the upper surface and 22–25 on the lower surface.
In stem, epidermis covered with a thin-layered cuticle, glandular trichomes with eight-celled heads and unicellular trichomes with three-celled wide heads. The hypodermis consisted of thin-walled polygonal collenchymatous cells. Cortex consisted of aerenchyma cells with large intercellular spaces. Endodermis and pericycle layer were distinct. Leaf was dorsiventral. Epidermis showed a prominent cuticle. It had lower and upper epidermis and a single layer of palisade cells on upper epidermis. It also consisted of well-developed spongy parenchyma and amphicribal vascular bundles were present at the center surrounded by thick-walled bundle sheath. The palisade ratio was about 3–5 and the stomatal index was 3–5 for the upper surface and 18–22 for the lower surface.
Upper epidermis had thin-walled rectangular cells covered with a thick cuticle. Hypodermis had eight rows of collenchymatous cells. Cortex had thin-walled parenchymatous cells embedded with air cavities followed by a single layer of endodermis. Endodermis had central stellar region with collateral wedge discontinuous ring of vascular bundles capped with lignified pericyclic fibers combined with narrow intra fascicular parenchyma cells. Vascular bundles composed of usual elements. Pith had thin-walled parenchymatous cells with a few prismatic calcium oxalate crystals. In leaf, epidermis composed of a layer covered with warty, tubercle, pointed stiff uniserate trichome followed by a single layer of palisade layer. Hypodermis had thin-walled polygonal collenchymatous layer of about 2–5 cells. Central regionhad five bicollateral vascular bundles of usual elements. The lamina was dorsiventral. Mesophyll differentiated into palisade and spongy parenchyma cells. Epidermis composed of a single layer of palisade cells with warty, tubercle, pointed stiff uniseriate trichomes. Endodermis consisted of spongy parenchyma cells of about seven layers transversed with fibrovascular strands with a few oil globules and a few prismatic calcium oxalate crystals. Palisade ratio 3.2–4.5 and the stomatal index 18–20.5 for the upper surface and 24–27.3 for the lower surface.
Comparative powder microscopy of the three plants is given in [Figure 3].
|Figure 3: Comparative powder microscopical study of W. chinensis, W. trilobata and E. prostrata. tri: Trichomes; pv: Pitted vessel; sv: Spiral vessels; f: Fibres; pa: Parenchyma; cic: Cicatrix; tr: Tracheids; st: Stomata; pv: Pitted vessel; ep: Epidermis; og: Oil globules; cr: Crystals of calcium oxalate; av: Annular vessels; p: Parenchyma; v: Vessel|
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In powder microscopy, W. chinensis appeared greenish brown. Trichomes were long, straight, or bent multiseriate had basal cells. Stomata anisocytic type at the upper and lower epidermis on surface view. Mesophyll had cluster and prismatic calcium oxalate crystals embedded in the parenchyma cells. Lamina had fragments of laminar layer with a single layer of epidermal cells covered with thin sinuous cuticle and a single palisade layer of spongy parenchyma cells. Vessel elements were annular type associated with spiral and laticiferous cells. Fibers were long with narrow lumen. Cicatrix type of cells were present at the lower base of trichomes.
W. trilobata was greenish brown. The trichomes had two-celled cicatrix pointed trichomes. The tracheids were thick walled. The lamina had a single layer of palisade cells at the epidermis.
Stomata was anisocytic/anamocytic stomata embedded at the surface of the epidermal layers. Vessel elements were spiral, spongy parenchyma cells and mesophyll cells embedded with a few prismatic calcium oxalate crystals and oil globules.
The results of fluorescence characters are presented in [Figure 4]. W. chinensis appeared red, red, red, pink, creamy pink, creamy pink, and no fluorescence with different solvents namely benzene, chloroform, ethyl acetate, acetone, methanol, Ethanol and water, respectively. With the same solvent, W. trilobata assume red, red, red, red, red, red, and no fluorescence, respectively. In case of E. prostrata, with the same solvent, the powder assumed no fluorescent, light blue, bluish pink, pink, bluish white, bluish white and green, respectively.
|Figure 4: Fluorescence characters observed under ultraviolet light of W. chinensis, W. trilobata, and E. prostrata|
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Preliminary phytochemical screening
The alcoholic extracts of three species were subjected to qualitative phytochemical analysis. In preliminary phytochemical screening, W. chinensis revealed the presence of phenol, steroids, tannin, flavonoids, and coumarins. E. prostrata revealed the presence of phenol, cardiac glycosides, coumarin, tannin, saponin, and flavonoids. W. trilobata revealed the presence of phenol, steroids, glycosides, tannin, saponin, and flavonoids.
High-performance thin-layer chromatography fingerprinting profile
The result of the HPTLC analysis is given in [Figure 5].
|Figure 5: (a) High-performance thin-layer chromatography fingerprinting profile of W. chinensis (track 1: 5 μl, track 2: 10 μl; track 3:15 μl) and W. trilobata (track 5: 5 μl, track 6: 10 μl; track 7: 15 μl) with marker wedelolactone (Track 4: 5 μl) under (a) 254 nm, 366 nm and 520 nm|
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HPTLC analysis with specific solvent system of Toluene:ethyl acetate:formic acid (7:2.5:0.5) revealed 12 and 12 spots at various Rf when viewed under UV 254 nm and 366 nm, respectively. After derivatization, 13 spots were observed. Whereas W. trilobata revealed 10 and 7 spots at various Rf when viewed under UV 254 nm and 366 nm, respectively. Thirteen spots were observed after derivatization. The marker compound wedelolactone was visible in 254 nm and 366 nm but not visible after derivatization of the plate with vanillin–sulfuric acid reagent. The compound wedelolactone was present in W. chinensis and not in W. trilobata. Because wedelolactone is a marker for E. prostrata, all the three plants were subjected to HPTLC fingerprinting and their chemical profiles were compared, which is represented in [Figure 6].
|Figure 6: Comparative high-performance thin-layer chromatography fingerprinting profile of W. chinensis (track 1: 5μl, track 2:10 μl), E. prostrata (track 3: 5 μl, track 4: 10 μl) and W. trilobata (track 5: 5 μl, track 6: 10 μl) under 366 nm|
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Evaluation of antioxidant potential
The liver is the most frequent target organ in terms of drug toxicity. In response to oxidative stress, the production of radical species (ROS and reactive nitrogen species) increased, which induces oxidative stress including increase of cellular oxidants, lipid peroxidation and depletion of antioxidants in the liver with consequent release of marker enzymes of hepatotoxicity. Hence, thein vitro assay was carried out to evaluate its antioxidant potential through DPPH and iron chelating assay. In DPPH assay, IC50 value of E. prostrata was 190 μg/ml and that of W. chinensis was 1.24 μg/ml, whereas that of W. trilobata was 1411.7 μg/ml. For the reference drug quercetin, IC50 was 11.87 μg/ml. In iron chelating activity assay, the IC50 value of E. prostrata was 340 μg/ml and that of W. chinensis was 755.555 μg/ml, whereas that of W. trilobata was 2,892 μg/ml. For the reference drug, sodium ethylene diamine tetra acetic acid, IC50 was 10 μg/ml.
In silico studies
Studies are available stating that both W. chinensis and E. prostrata possess hepatoprotective activity and contain a common marker wedelolacatone. It was proved that the wedelolactone has hepatoprotective activity in CCl4 and concanavallin A-induced liver injury. The biological mechanism of these plants on hepatoprotection remains elusive. Hence, the in silico studies were carried out to predict the role of wedelolactone in ac etaminophen-induced hepatotoxicity. The drug likeliness and pharmacokinetics of wedelolactone were predicted, which are represented in [Table 1] and [Table 2].
|Table 2: Absorption, Distribution, Metabolism, Excretion, Toxicity predicted properties of wedelolactone|
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The key protein targets involved in acetaminophen toxicity were selected and docked with the marker compound, wedelolactone [Figure 7] whose binding energy (docking score), interacting residues, and hydrogen bond interactions are tabulated in [Table 3].
|Figure 7: Structure of wedelolactone: molecular formula: C16H10O7 IUPAC name: 1, 8, 9-trihydroxy-3-methoxy-6H-benzofuro[3,2-c] chromen-6-one|
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|Table 3: Binding energy, interacting residues and hydrogen bond interactions of Wedelolactone with selected target proteins|
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It is considered that, the lesser the G score (binding energy) value, greater is the binding of the ligand with the protein. Those target proteins, which have binding energy of −6 and below with wedelolactone were considered as probable targets for wedelolactone. From the G score values, it is observed that wedelolactone showed lesser G score value, i.e., high affinity toward target proteins in the following order: PPAR-α, AMPK, Nrf2, CYP2E1, EGFR, JNK1, UCP-2, thrombin, 5-lipoxygenase, mTORC1, RXR, FXR, LXR, frizzled receptor, GDH and Erk-1. Hydrogen bond interactions of wedelolactone at the active site of target proteins were observed, which influence high binding affinity toward the protein. The molecular representation of docking proteins with wedelolactone are depicted in [Figure 8]a-c.
| Discussion|| |
Pharmacognostical studies revealed that the three plants were entirely different by macro-morphology, micro-morphology, and by powder microscopy. Fluorescence characters with the different solvents were also different from each other except the marker compound wedelolactone which is present both in W. chinensis and E. prostrata, not in W. trilobata. This was supported by HPTLC fingerprinting profile of the plants. In comparative fluorescence study, red color was common for both W. chinensis and W. trilobata, but not in E. prostrata. Bluish white was observed in E. prostrata, but in W. chinensis, pink and bluish white were mixed together forms a creamy pink color. The same was observed with HPTLC fingerprinting profile, where the marker compound wedelolactone appeared bluish white fluorescent band in E. prostrata. The same wedelolactone was observed in W. chinensis; some pink color bands were very nearer to wedelolactone making it creamy pink. However, low polar blue color-emitting other compounds were also observed in HPTLC fingerprinting of E. prostrata. The preliminary phytochemical screening also revealed the presence of various major groups of phytoconstituents. Reports stated that the following compounds were present in W. chinensis: α-pinene (21.7%), spathulenol (20.3%) limonene (14.3%); luteolin, apigenin, and indole-3-carboxyaldehyde; wedelolactone;, norwedelolactone; bisdesmodic-osidicoleanolic acid; bisdesmosidicoleanolic acid and β -D-glucopyranosyl-3-o-[o-β-D-xylopyranosyl-(1 → 2)- β -D-Glucu r o n o P y r a n o s y l] o l e a n o l a te (IV); and β -D-glucopyranosyl 3β-[(o-β-D-xylopyranosyl-(1→)-(pD-glucuronopyranosyl)]-olean-12-en-28 oate).,, In W. trilobata, the following compounds were reported: grandiflorenic acid; 1α-acetoxy-6α, 9β-dihydroxy-4, 10α-dimethyl- 5αH, 7αH, 8αH-endesm-3-en-8, 12-olide; 1β-acetoxy-4α-hydroxy-6β-isobutyryloxy-9α- isovaleryloxyprostatolide; 16α-hydroxy-ent-kauran-19-oic acid; (3R, 4R, 6R)-3, 4-dihydroxy-1-menthene; trilobolide-6-O-isobutyrate; 1β-acetoxy-4α, 9α-dihydroxy-6β-isobutyroxyprostatolide; 16β, 17-dihydroxy-ent-kauran-19-oic acid; daucosterol; protocatechualdehyde, caffeic acid, and syringaresinol-4-O-β-D-glucopyranoside; pinoresinol-4-sulfate; pinoresinol-4-O-β-D-glucopyranoside; 1H-indole-3-carboxylic acid; 1H-indole-3-carbaldehyde; 2,6-dimethoxy-4-hydroxyphenol-1-O-β-D-glucopyranoside; 3,5-dimethoxy-4-hydroxyphenol-1-O-β-D-glucopyranoside, germacrene D; α-phellandrene; α-pinene; E-caryophyllene; bicyclogermacrene; limonene α-humulene; 3α-Angeloyloxy-16α-hydroxy-ent-kauran-19-oic acid; 3α-Angeloyloxy-16α,17-dihydroxy-ent-kauran-19-oic acid; 3α-Tigloyloxy-16α-hydroxy-ent-kauran-19-oic acid; 3α-Tigloyloxy-16α,17-dihydroxy-ent-kauran-19-oic acid; 3α-Dihydrocinnamoyloxy-ent-kaur-16-en-19-oic acid; 3α-Cinnamoyloxy-ent-kaura-9 (11), 16-dien-19-oic acid and 3α-Cinnamoyloxy-9β, 17-dihydroxy-ent-kaur-15-en-19-oic acid. In E. prostrata, the following compounds were reported: wedelolactone, demethyl wedelolactone, demethyl wedelolactone 7-o – glucoside, apigenin, luteolin, luteolin – O- glucoside, eclalbasaponins I-X, eclalbatin, ursolic acid, oleanolic acid, strychnolactone, stearic acid, lacceroic acid, 3, 4, dihydroxy benzoic acid, stigmasterol, β- sitosterol, α- formylterthineyl, acetoxymethyl eneterthienyl, angeloyloxy methylene terthienyl, senecioyloxymethy leneterthienyl, tigloyloxymethyle neterthienyl, terthienyl, α-terthieny lmethanol, 5'- isovaleryloxymethylene-2-(4-isovaleryloxybut-3-ynyl) dithiopene, 2- acetoxymethylene-5'-(but-3-en-1-ynyl) dithiopene, 2- (3'-acetoxy-4'- chlorbut-1-ynyl)-5-(pent-1,3-diynyl) thiophene, heptacosan-14-ol, hentriacontan-1-ol, nicotine, ecliptalbine, verazine, 20-epi-3-dehy droxy-3-oxo-5,6 – dihydro-4, 5, -dehydroverazine, (20R)- 4β-hydroxy verazine, 4β-hydroxyverazine, (20R)- 25β-hydroxyverazine and 25β-hyd roxyverazine. The methanolic extracts of the three plants were screened for antioxidant property using DPPH assay and iron chelating assay. All the three plants have antioxidant property, but the IC50 was lower for W. chinensis in DPPH assay, which shows that W. chinensis has more potency than the other two plants and standard quercetin in antioxidant potential. In iron chelating activity, E. prostrata was found to be more potent than other two plants. The mechanism of DPPH scavenging assay is direct scavenging of radical by antioxidant. Whereas the iron chelating activity is related to Fenton's reaction where the chelator binds with the metal and thereby prevents radical formation. The difference in potency observed with W. chinensis and E. prostrata can be exploited for plant preference in specific ailments. In case of acetaminophen toxicity, the metabolite NAPQI is a radical. Hence, the radical scavenging capacity will help in the prevention and progression of injury. In silico screening revealed that the marker compound wedelolactone has more affinity toward the following proteins: PPAR-α, AMPK, Nrf2, CYP2E1, EGFR, JNK1, UCP-2, thrombin, 5-lipoxygenase, mTORC1, RXR, FXR, LXR, Frizzled receptor, GDH, and Erk-1. Activation of liver X receptor prevented acetaminophen-induced liver injury by induction of Phase II conjugation enzymes, especially enzymes such as glutathione transferase (GST) involved in glutathione (GSH) conjugation. It suppresses Phase I CYP2E1 enzyme which is involved in the conversion of acetaminophen into NAPQI, which forms covalent binding with sulfydryl groups in cellular and mitochondrial proteins, results in mitochondrial oxidative stress and dysfunction, ultimately leading to hepatocyte necrosis. The nuclear receptor RXR-α is involved in the upregulation of CYP2E1. Downregulation of RXR-α will be beneficial in alleiviating the APAP toxicity. Suppression of 5-lipoxygenase induces Phase II detoxification enzyme sulfo transferase (SULT2), subsequently causing the reduction of NAPQI formation. JNK-1 amplifies mitochondrial ROS by a self-sustained activation loop, which, in turn, activates signal transduction pathway, which leads to apoptosis. Nrf2 activation induces enzymes involved in the synthesis of GSH. Activation of AMPK pathway leads to energy generation and promotes survival signaling pathway. Fatty acid β-oxidation is inhibited by acetaminophen treatment. PPAR-α encodes peroxisomal and mitochondrial enzymes, which promotes fatty acid catabolism., UCP-2 is a target gene of PPAR-α, which functions as an antioxidant. The inhibition of thrombin at early stage of acetaminophen toxicity may be a protective mechanism. EGFR plays a dual role both in the initiation of hepatotoxicity and subsequent regeneration in acetaminophen-induced hepatotoxicity. Frizzled receptors are involved in liver regeneration process. The FXR provides hepatoprotection by inducing the expression of several genes involved in Phase II metabolism. ERK-1 is involved in detoxification process of liver damage against oxidative stress. GDH is a mitochondrial enzyme that is involved in the metabolism of glutamate to oxoglutarate. GDH is a marker for hepatocyte damage mainly of centrilobular damage. Docking studies revealed the information about the binding of the ligand with the target and not giving the information about the outcome of an interaction. Either the interaction may be induction or inhibition, which needs to be confirmed by wet lab analysis.
| Conclusion|| |
From the present study, it is concluded that all the three plants are different. W. trilobata did not have the marker compound wedelolactone. The chemical profiling of W. chinensis is different from E.prostrata, but the marker compound is common for both. Because W. chinensis and E.prostrata were interchangeably used for common ailments, the marker compound wedelolactone might have been responsible for their shared efficacy. W. chinensis was observed to be more potent antioxidant than the other two. Hence, W. chinensis may be a potential species for counteracting acetaminophen toxicity either as a drug or as a supportive therapy.
The authors are highly thankful to Director General, CCRAS; Director General, CCRS; Assistant Director Institute In-Charge, CSMRADDI; and Assistant Director, Institute In- Charge, SCRI for providing necessary facilities to carry out the study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
[Table 1], [Table 2], [Table 3]