The researchers collected latex from wild-growing plants near their institution by snapping stems and gathering the dripping sap drop by drop. They then split the latex into five solvent extracts using acetone, methanol, ethanol, isopropyl alcohol and isoamyl alcohol, each mixed with water in an 8:2 ratio. The choice of solvent matters enormously, because polarity determines which phytochemicals dissolve out of the raw sap. After vortexing, overnight incubation and centrifugation at 10,000 rpm, the team had five distinct fractions, labeled CAE, CME, CEE, CIPE and CIAE, ready for chemical fingerprinting and biological testing.

Fourier-transform infrared spectroscopy and fluorescence spectroscopy provided the first look at what each extract contained. The FT-IR spectra revealed a broad band between 3400 and 3200 cm−1 in every extract, a signature of hydroxyl groups that pointed to the presence of phenolic compounds. All extracts except the acetone fraction showed a distinct band near 2800 to 2900 cm−1 from alkane C–H stretching, and the complex fingerprint region below 1500 cm−1 differed across solvents. Fluorescence spectra showed a distinct emission peak near 500 nm in every extract, consistent with polyphenolic compounds such as anthocyanins, phenolics, alkaloids and aromatic benzenoids, confirming that the latex is loaded with fluorescent secondary metabolites.

Quantitative assays then measured exactly how much of each phytochemical class each solvent had pulled out. The ethanol extract, CEE, was the clear winner, delivering the highest phenolic content at 0.59 mg gallic acid equivalents per milliliter, the highest flavonoid content at 0.18 mg rutin equivalents per milliliter, and the highest tannin content at 3.8 mg tannic acid equivalents per milliliter. The pattern followed solvent polarity: the most polar solvent extracted the most polar phytochemicals most efficiently, while the less polar acetone pulled out fewer. This solvent-dependence echoes earlier findings in plants such as Limonium delicatulum and Azanza garckeana, where alcohol-based extraction consistently outperformed less polar alternatives for polyphenol recovery.

Gas chromatography with a flame ionization detector then catalogued the volatile bioactive compounds in each fraction. The ethanol extract yielded the most peaks, with 29 phytocompounds detected, followed by the isoamyl alcohol extract with 23. In the acetone extract, which contained 14 peaks, the dominant compound was phytol at 43.61 percent abundance, followed by ethion at 18.67 percent and diisobutyl phthalate at 11.68 percent. Benzhydrazide emerged as a prominent compound across fractions, ranging from 5.31 percent in methanol to 67.5 percent in isoamyl alcohol, while phytol reached 64 percent in the isopropyl fraction. Several of these molecules carry documented pharmacological credentials: phytol and its derivatives have been linked to antioxidant, neuroprotective, antibacterial, anticancer and anti-inflammatory effects, while 2-phenylindole derivatives show antiviral, analgesic and anti-parasitic activities.

The antioxidant performance of the extracts was striking. Using three complementary assays, the team measured IC50 values for nitric oxide scavenging, hydroxyl radical scavenging and iron chelating activity. The ethanol extract again led the pack, with IC50 values of 14 µg/ml for nitric oxide scavenging, 15 µg/ml for hydroxyl radical scavenging and 117 µg/ml for iron chelation, all far below the corresponding values for the ascorbic acid standard, which came in at 115.23, 523.22 and 456.21 µg/ml respectively. Correlation analysis reinforced the link between chemistry and function: the R² value between total flavonoid content and nitric oxide scavenging in the ethanol extract was 0.94, while phenolic content and iron chelation correlated at 0.95, and tannin content tracked hydroxyl radical scavenging at 0.94.

Anti-inflammatory activity was assessed through a protein denaturation assay, a widely used in vitro proxy in which bovine serum albumin is heat-denatured and the protective effect of test compounds is measured by tryptophan fluorescence. Every extract showed substantial protection, but the acetone extract stood out. Adding CAE to the BSA solution dropped fluorescence intensity from 3.6 × 10⁴ INT units to 0.1 × 10⁴ INT units, and the extracts outperformed the synthetic anti-inflammatory drugs paracetamol, dexona and combiflame used as positive controls. The authors attribute this protection to complex mixtures of polyphenolics that may act cooperatively to stabilize protein structure against thermal denaturation.

The computational phase of the study focused on the three most abundant compounds in the acetone extract: phytol, ethion and diisobutyl phthalate. Using the CB-Dock2 platform, the team docked these ligands against the crystal structures of inducible nitric oxide synthase (PDB 3E7G, 2.20 Å resolution) and cyclooxygenase-2 (PDB 1CX2, 3.00 Å resolution), the two enzymes most closely associated with inflammatory signaling. Redocking a co-crystallized inhibitor into HSP90 produced an RMSD of 0.102, validating the docking protocol. The results were remarkable: diisobutyl phthalate scored −8.3 kcal/mol against iNOS, matching the −8.3 score of the standard drug combiflame, and scored −7.5 against COX-2, close to combiflame’s −7.8. Phytol followed with −7.1 against iNOS and −6.7 against COX-2, while ethion scored −5.1 and −5.6 respectively.

The interaction analysis revealed why these plant molecules bind so well. The most stable diisobutyl phthalate–iNOS complex was held together by two hydrogen bonds, three alkyl interactions, one pi-alkyl interaction and seven van der Waals contacts involving residues including Pro350, Ala351, Asn370, Leu209, Ile244, Tyr489 and Met355. Against COX-2, the same compound formed a pi-sigma interaction with Ala527, alkyl contacts with Val523 and Leu352, and five pi-alkyl interactions with residues including His90, Phe518, Trp387 and Val349. Crucially, the phytocompound occupied the same binding pose as the synthetic drug and targeted conserved residues in both active sites, with ligands docking into the oxygenase domain of iNOS and the C-terminal catalytic domain of COX-2. This dual-target engagement suggests a plausible molecular mechanism for the anti-inflammatory effects observed in the wet-lab assays.

Drug-likeness and ADMET predictions added a final layer of cautious optimism. All three compounds satisfied Lipinski’s Rule of Five, with consensus log P values between 3.56 and 6.25 indicating good oral absorption potential and predicted bioavailability scores around 0.55. Toxicity screening with ProToxII placed phytol and diisobutyl phthalate in toxicity classes 5 and 6, indicating low toxicity, and neither compound showed organ-level toxicity, though diisobutyl phthalate was flagged as active against carcinogenicity, a caveat the authors acknowledge. PASS analysis predicted anti-inflammatory activity for all three ligands with probability-of-activity scores above 0.5. The team is careful to note that these findings are preliminary: the docking scores and in vitro assays must now be validated through cell-based experiments and in vivo studies before any of these milkweed molecules can be considered genuine drug leads. Still, the convergence of chemical profiling, enzyme docking and pharmacokinetic screening paints the humble giant milkweed as a promising starting point for developing safer, plant-derived alternatives to the NSAIDs that currently dominate anti-inflammatory therapy, at the cost of gastrointestinal and cardiovascular side effects.

Subject of Research: Phytochemical profiling and molecular docking of Calotropis gigantea latex compounds against inflammatory enzymes iNOS and COX-2

Article Title: Chemical profiling and in silico analysis of phytocompounds extracted from milky-latex sap from giant milkweed Calotropis gigantea (Linn.) targeting nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins

Article References: Sharma, A. D., Kaur, I., & Chauhan, A. (2026). Chemical profiling and in silico analysis of phytocompounds extracted from milky-latex sap from giant milkweed Calotropis gigantea (Linn.) targeting nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins. Discover Chemistry, 3(1), Article 497. https://doi.org/10.1007/s44371-026-00951-8

Image Credits: AI Generated

DOI: 10.1007/s44371-026-00951-8

Keywords: Calotropis gigantea, latex, phytochemicals, anti-inflammatory, iNOS, COX-2, molecular docking, GC-FID, antioxidant, phytol, diisobutyl phthalate, ADMET

The study, published open access in Discover Chemistry, set out to close a conspicuous knowledge gap. While close relatives such as Isodon rubescens have been extensively characterized—yielding celebrated diterpenoids like oridonin that have attracted attention for anti-inflammatory and cytoprotective effects—I. coesta has remained comparatively obscure despite its wide distribution across Bangladesh, Nepal, India, China, Myanmar, the Philippines, Indonesia, Sri Lanka, and Thailand. The researchers harvested whole plants, excluding roots, in September 2023, air-dried them for two to four days, ground them into a fine powder, and extracted the material separately with methanol and hexane over ten to fifteen days of soaking. The polar solvent proved far more generous: the methanolic extract yielded 4.02 percent by weight, while the nonpolar hexane extract yielded only 1.10 percent, a difference that foreshadowed the chemistry to come.

Gas chromatography–mass spectrometry, performed at the Advanced Instrumentation Research Facility at Jawaharlal Nehru University in New Delhi on a Shimadzu GCMS-QP2010 Plus system with a DB-5 capillary column, identified 24 compounds in each extract. In the methanolic extract, these accounted for 81.93 percent of total composition, with the acyclic diterpene alcohol phytol dominating at 12.22 percent, followed by the steroid precursor pregnenolone acetate at 7.54 percent, squalene at 6.83 percent, and methyl linolenate at 6.51 percent. The hexane extract told a different story: squalene, a triterpene hydrocarbon famous for its antioxidant and skin-protective properties, led at 11.79 percent, with the long-chain hydrocarbon 2-methylhexacosane close behind at 9.68 percent, and hexatriacontane, propylene glycol monooleate, and palmitic acid rounding out the major peaks. Seven compounds appeared in both extracts, including phytol, squalene, neophytadiene, and vitamin E, suggesting overlapping biochemical pathways that persist regardless of solvent polarity.

The identity of the dominant molecules matters because several of them carry well-documented biological credentials. Phytol serves as a biosynthetic precursor to vitamins E and K1 and has reported antioxidant, anti-inflammatory, and anticancer activities. Pregnenolone acetate sits upstream in the biosynthesis of steroid hormones and has been studied for neuroprotective and anti-inflammatory effects, making it a molecule of interest in neuropharmacology and endocrinology. Squalene, meanwhile, is an intermediate in sterol biosynthesis and a widely used emollient in cosmetics and pharmaceuticals. The presence of gamma-sitosterol, tocopherol, and linoleic acid derivatives in the methanolic extract further enriches the profile of a plant whose chemical inventory had previously been little more than a footnote in the Isodon literature.

Quantitative colorimetric assays confirmed that polarity drives the phytochemical harvest. The methanolic extract registered a total phenolic content of 31.37 milligrams of gallic acid equivalents per gram, a total flavonoid content of 313.28 milligrams of quercetin equivalents per gram, and a total antioxidant capacity of 8.61 milligrams of ascorbic acid equivalents per gram. The hexane extract trailed on every measure, with 17.19 milligrams GAE per gram of phenolics, 117.57 milligrams QE per gram of flavonoids, and 2.07 milligrams AAE per gram of antioxidant capacity. The pattern is chemically intuitive: methanol, being polar, efficiently pulls phenols and flavonoids out of plant tissue, whereas hexane preferentially dissolves lipids, waxes, and terpenoid hydrocarbons. Flavonoids, in particular, are major contributors to plant antioxidant activity, so their concentration in the methanolic fraction is consistent with the rest of the data.

The radical-scavenging results, however, temper any temptation to crown the plant a super-antioxidant. In the DPPH assay, which tracks the reduction of a stable violet free radical by measuring absorbance at 517 nanometers, the methanolic extract posted an IC50 of 748.38 micrograms per milliliter and the hexane extract 794.13 micrograms per milliliter. Those figures indicate moderate to weak activity compared with ascorbic acid, the reference standard, which achieved an IC50 of just 74.72 micrograms per milliliter. Two-way ANOVA showed that concentration strongly influenced scavenging activity, with a highly significant effect, while the difference between the two extract types alone did not reach statistical significance, though a significant interaction between extract type and concentration revealed concentration-dependent divergence in their responses. The authors caution that direct comparison with the standard should be interpreted carefully because different concentration ranges were used for the plant extracts and the reference compound.

To probe how the major constituents might behave at the molecular level, the team turned to computational docking against two enzymes that sit at opposite ends of the oxidative-stress balance. Human peroxiredoxin-5, resolved by X-ray crystallography at 1.50 angstrom resolution, is a thioredoxin-family antioxidant enzyme that detoxifies hydrogen peroxide and peroxynitrite, defending cells against oxidative injury. NADPH oxidase, resolved at 1.80 angstroms, is the enzymatic engine of reactive oxygen species generation, and its overactivation drives inflammatory damage. Using PyRx 0.8 with AutoDock Vina, the researchers docked phytol, pregnenolone acetate, squalene, and 2-methylhexacosane into both binding pockets. Pregnenolone emerged as the clear frontrunner, binding peroxiredoxin-5 at minus 6.1 kilocalories per mole and NADPH oxidase at minus 8.4 kilocalories per mole through a mix of hydrogen bonds, pi-anion, and pi-alkyl contacts. Phytol followed with moderate affinities, while 2-methylhexacosane performed worst, managing only minus 3.0 and minus 5.7 kilocalories per mole through weak van der Waals and alkyl interactions.

The ADMET analysis added a sobering pharmacokinetic dimension. All four compounds passed the molecular-weight criterion of Lipinski’s rule of five, but squalene, phytol, and 2-methylhexacosane showed MLOGP values far above the acceptable ceiling of 4.15, signaling extreme lipophilicity that predicts poor aqueous solubility and low gastrointestinal absorption. Pregnenolone was the only compound predicted to have high GI absorption, the only one expected to cross the blood–brain barrier, and the only one that is not a P-glycoprotein substrate, making it the most drug-like molecule in the set. Toxicity predictions from ProTox 3.0 were reassuring across the board: none of the compounds showed hepatotoxicity, carcinogenicity, or AMES mutagenicity, and squalene and phytol carried LD50 values of 5,000 milligrams per kilogram, indicating generous safety margins. The authors suggest that nanoemulsions, liposomes, solid lipid nanoparticles, or phospholipid-based delivery systems could rescue the bioavailability of the lipophilic constituents.

The researchers are careful to frame the docking results as hypothesis-generating rather than proof of pharmacological activity, and they recommend in vivo validation, molecular dynamics simulations, and quantitative phytochemical confirmation by HPLC or LC–MS as the necessary next steps. Even with those caveats, the study positions Isodon coesta as a flavonoid-rich reservoir of bioactive metabolites from one of the world’s great biodiversity hotspots—the Indian Himalayan region harbors roughly half of India’s flowering plant species with nearly 30 percent endemism, and Uttarakhand alone supports more than 5,000 vascular plant species. As computational screening increasingly complements fieldwork in natural-product drug discovery, this aromatic undershrub of the Himalayan slopes may yet translate its ethnobotanical reputation into molecules worthy of the laboratory bench and, eventually, the clinic.

Subject of Research: Phytochemical profiling and computational antioxidant evaluation of Isodon coesta from the Uttarakhand Himalaya

Article Title: Comprehensive phytochemical profiling, molecular interaction studies, and ADMET analysis of hexane and methanol extracts of Isodon coesta from Uttarakhand’s Himalayan Region

Article References: Mawri, D., Pujari, R., Bahuguna, A., Rawat, D. S., Prakash, O., Kumar, R., & Dubey, S. K. (2026). Comprehensive phytochemical profiling, molecular interaction studies, and ADMET analysis of hexane and methanol extracts of Isodon coesta from Uttarakhand’s Himalayan Region. Discover Chemistry, 3(1), Article 558. https://doi.org/10.1007/s44371-026-01003-x

Image Credits: AI Generated

DOI: 10.1007/s44371-026-01003-x

Keywords: Isodon coesta, phytochemistry, GC-MS, antioxidant, molecular docking, ADMET, Uttarakhand, Himalayan medicinal plants, phytol, pregnenolone, squalene, Lamiaceae