Chemical Profiling, Chromatographic Fingerprinting and Anthelmintic Screening of a Rare Species of Holly, Ilex khasiana Purkay.from Mizoram, Northeast India

Background: I. khasiana Purkay. is an evergreen tree of the family Aquifoliaceae and is classified critically endangered as only few of them are identified in Northeast India. In Khasi traditional medicine, it is used for the remedy of tuberculosis and viral infections, while it is therapeutically used for all sorts of infections, blood diseases and cancer in the Mizo traditional medicine. As a rare species, there is an overwhelming paucity of knowledge on the chemical and biological properties of this plant. Objectives: The study was designed to garner pharmacognostic information on the secondary metabolites in the leaves of I. khasiana that are likely the factors behind the medicinal uses and test for an anthelmintic activity using a parasitic helminth model. Materials and Methods: I. khasiana leaves were collected from Aizawl, Mizoram, India. Five extracts were prepared, namely Petroleum ether (IKP), Chloroform (IKC), Methanol-Hexane (IKM-H) extracts, Methanol-Chloroform (IKM-C) and Methanol-Butanol (IKM-B) extracts. Chemical profiling was done in a single quadrupole Gas Chromatography-Mass Spectrometry (GC-MS). Bioactive compounds were screened using High-Performance Thin-Layer Chromatography (HPTLC) based on nine established bioactive compounds. Anthelmintic assay was performed against a parasitic nematode, Ascaridia galli. Results: GC-MS data showed that the principal compound was a flavonoid, 7,9-di-tert-butyl-1- oxaspiro[4,5]deca-6,9-diene-2,8-dione from the various extracts. Other major compounds were a terpene lactone, 6-hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydrobenzofuran-2(4H)-one and a phenol, 2,4-di-tert-butylphenol. From HPTLC fingerprinting, two compounds were identified in two extracts, quercetin in IKM-C and β-sitosterol in IKM-H. HPTLC quantification showed that quercetin in IKM-C at an Rf value of 0.49 indicated a total amount of 372.98 ng; while β-sitosterol in IKM-H an Rf value of 0.43 was 257.04 ng. IKM-H with the most diverse compound and containing β-sitosterol, a compound tested for antiparasitic activity, was found to have anthelmintic activity against the helminth model. Scanning electron microscopy revealed anthelmintic effects on the nematode including collapse of the mouth region, contraction throughout the body and localised erosions of the cuticle. Conclusion: I. khasiana leaf extracts contain important bioactive compounds. The principal flavonoid, lactone and phenol are ostensibly the molecules to be attributed to the medicinal properties. The detections of quercetin and β-sitosterol add to the possible diverse pharmacological properties. The findings directly advocate the necessity for actions into the conservation of this rare and medicinally valuable plant.