LC-ESI-MS analysis, antioxidant, anti-diabetic and molecular docking studies on Corchorus depressus (L.) C.Chr

Abstract The present study encompasses the ethnomedicinal consumption of Corchorus depressus (L.) C.Chr. (C. depressus) for diabetes. Samples were subjected to LC-ESI-MS analyses. The n-hexane, methanolic and water extracts were screened for α-glucosidase inhibition and in vivo anti-diabetic studies. Further, antioxidant (DPPH) and anti-inflammatory study was performed via luminol-enhanced chemi-luminescence assay. The identified compounds were docked against the target enzymes of diabetes. The n-hexane fraction (CD-J1) showed IC50 of 8.4 ± 0.1 µg/mL against α-glucosidase enzyme. The sub fractions CD-12 and CD-13 of CD-J1 obtained after flash column chromatography displayed further reduced IC50 values of 4.3 ± 0.1 and 6.3 ± 0.1, respectively, as compared with standard drug acarbose (IC50 values of 37.5 ± 0.2 µg/mL). Simultaneously, dereplication of most active sub-fraction CD-12 by LC-ESI-MS led to the identification of strophanthidin and some other active metabolites responsible for anti-diabetic activity. Molecular docking of strophanthidin with α-glucosidase and α-amylase revealed high affinity for these target enzymes. Graphical Abstract


Introduction
Early history of men reveals the use of herbs and plants for remedial purposes.In many cultures the use of plants for therapeutic purposes evolved into well-documented systems of medicines (Ayaz et al. 2017;Mir et al. 2019).The present study focuses on the medicinal plant of Cholistan desert.This barren sandy region covers an area of 26,000 km 2 , and it is 112 km above sea.Recent ethnopharmacological studies reported 29 families, 55 genera and 67 plant species are used by local people for the treatment of different ailments (Malik et al. 2015).One of the most pharmacologically important plant Corchorus depressus (L.) C.Chr.(C.depressus) was selected in this study to unveil its anti-diabetic potential scientifically.
Limited ethnobotanical and ethnopharmacological data of the plants of Cholistan desert is reported in the literature.Ethnopharmacological data of C. depressus Stock (locally called Bhauphali) was collected.This plant is known for aphrodisiac properties, antioxidant, acetylcholinesterase, butyrylcholinesterase, antimalarial, antibacterial, antipyretic, antifungal, a-glucosidase and anti-diabetic activities.Plant mucilage is used for the treatment of gonorrhea and applied as a poultice for wounds healing (Kataria et al. 2013;Malik et al. 2015;Afzal et al. 2017).Decoction of C. depressus seeds and leaves with milk is used as a tonic to cure fever.A special focus was to assemble information on anti-diabetic activity of C. depressus.The information was collected from 105 folk practitioners (29 females and 76 males of age 44-73 years) operating the PRA method (Martin and Ethnobotany 1995).Data collected included parts of the plant used (either leaves, stem or roots), number of diseases cured by this plant, and method of herbal preparation and administration.Its herbal use was quoted by at least 92 folk therapists against diabetes, malaria, bacterial infections, and it also possesses analgesic and antipyretic activities (Kataria et al. 2013).Some species of genus Corchorus also exhibit activities against diabetes, convulsion, estrogenic, cancer, pyretic, oxidant, liver disorders and microbial activities (Do et al. 2021).However, scientific evaluation of the therapeutic effect of C. depressus against diabetes was not reported before.Hence, folklore uses of C. depressus prompted us to scientifically evaluate this plant against diabetes mellitus (DM).

Results and discussion
The hexane extracts (CD-J1) of the C. depressus were subjected to flash column chromatography with increasing polarities of ethyl acetate and hexane (Ahmad et al. 2020).Several fractions were collected from CD-1 to CD-23 .Among all these fractions CD-12 and CD-13 were found active against a-glucosidase with IC 50 values of 4.3 ± 0.1 and 6.3 ± 0.1 mg/mL, respectively.Subsequently, fraction CD-12 was subjected to LCMS to analyse the secondary metabolites present in it.The CD-12 fraction obtained from the flash column chromatography after elution with EtOAc:Hexane (7:3).Typical chromatograms (Figures File S1) with mass spectrometric detection in negative ionisation mode showed mainly six phytochemicals present in it.
Similarly, triterpenes such as corchorusin D2 5 and corosolic acid 6 is reported for its good a-glucosidase inhibition (Zhang et al. 2017) and Phuwapraisirisan et al. also reported the other triterpene, capsugenin 3 as a good inhibitor of a-glucosidase (Phuwapraisirisan et al. 2009).However, strophanthidin 2 is a cardenolidethis class of triterpenes was never tested for a-glucosidae inhibition.Therefore, in silico studies and binding of strophanthidin 2 with a-glucosidase and a-amylase were conducted.Traditional practitioners use C. depressus Linn. to treat DM in Cholistan desert.Therefore, scientific evaluation of its traditional uses was required.Consequently, bioassay-guided in vitro and in vivo screenings of C. depressus were done to prove ethnic claims.Various extracts of plants exhibited significant inhibitory activity against a-glucosidase (Table S2).Initially, respective plant material was collected in three different seasons, to check their in vitro efficacy against a-glucosidase.The plant material was exhaustively extracted with MeOH, n-hexane and water.Among all extracts of C. depressus, the hexane extracts showed a good inhibition against a-glucosidase, as compared with extracts from polar solvents Table S3.
The potent inhibition of a-glucosidase was observed in hexane extracts (CD-J1) of the C. depressus collected in January 2016 that showed IC 50 of 8.4 ± 0.1 mg/mL.This clearly indicates that volatile constituents of C. depressus play a role in enhanced a-glucosidase inhibition, even greater than standard drug acarbose that showed IC 50 value of 37.5 ± 0.2 mg/mL.The aqueous extracts of some of the Nigerian species of Corchorus have been reported to have significant a-glucosidase inhibitory activities (Ademiluyi et al. 2015).However, in our case polar extracts (methanolic and water) of C. depressus were not found as active as the non-polar (n-hexane) extract (Table S4).
The binding mode of strophanthidin 2 was investigated by molecular docking simulation with a-amylase and homology model of a-glucosidase (Ayaz et al. 2022;Mahnashi et al. 2022).Analysis of the docking results revealed that the compound was well fitted in the binding site of both proteins.The binding mode of the compound with a-glucosidase showed a number of noticeable interactions including hydrophilic, hydrophobic and p-stacking interactions.Figure S3 showed that the hydroxyl group on ring A mediates two hydrogen bonds with the side chain NH of His351 and Arg446.The other residues involved in the formation of hydrogen bonds that stabilised the compound are Asp69 and Val216.The p-stacking interactions between Tyr158 and the ligand further stabilised the contacts.
Binding affinity of compound 2 was also examined for a-amylase.The binding mode of the compound displayed prominent hydrophobic and hydrophilic contacts with crucial residues including Trp59, Gln63, Leu165, His201, Ile235, His305 and Gly306 within the active site of a-amylase.Hydrophilic contacts especially Gln63 and Gly306 were found to be accountable to stabilised the compound.The compound further facilitates two important salt bridges and p-stacking interactions with Lys200 and His201 (Figure S4).
Aforementioned in vitro and in silico studies encouraged to evaluate in vivo anti-diabetic potential of methanol extract of C. depressus collected in January 2018.Three doses of C. depressus extracts were injected intraperitoneally to diabetic rats at 50, 75, and 100 mg/kg.At the beginning of the experiment, glucose levels in the normal control group were 102.7 ± 2.8 mg dL À1 , while these were 474.9 ± 3.8 mg dL À1 in the positive control group and 477.9 ± 2.5 mg dL À1 in the standard control group.The glucose levels of extracts at 50, 75, and 100 mg/kg were noted as 476.8 ± 6.3, 483.8 ± 4.2, and 479.6 ± 5.2 mg dL À1 , respectively.After 24 h little change was observed in the normal control group (107.2 ± 1.8 mg dL À1 ), and the positive control group (484.5 ± 2.3 mg dL À1 ).However, the standard control group exhibited significant lowering of glucose level (90.7 ± 6.4 mg dL À1 ).Interestingly, C. depressus extracts treated groups at 50, 75 and 100 mg kg À1 showed anti-diabetic effect in a dose dependent manner as 290.0 ± 20.0, 149.0 ± 4.0, and 115.8 ± 6.4 mg dL À1 , respectively.More pronounced effects were noted on the fourth day of the experiment when glucose level of the extracts treated group at 50, 75, and 100 mg/kg results followed the same sequence as 208.5 ± 3.5, 129.5 ± 7.5 and 106.0 ± 2.2 mg dL À1 , respectively.This dose-dependent anti-diabetic activity of C. depressus was found to be similar to that of the standard drug, glibenclamide.It was also noticed that highest dose (200 mg kg À1 ) of C. depressus caused death to the experimental rats.Present study validated the folklore use of C. depressus for diabetes but it should be used at lower concentration.The polar fraction of the respective plant was found less active against a-glucosidase.This study also leads the pathway to the scientific community regarding the prominence of strophanthidin that provides a rationale to identify and isolate other imperious secondary metabolites from C. depressus.Thus, nature might provide new oral hypoglycemic lead compounds from medicinal plants, which can curb the high cost, and scant availability of medicines in the developing countries.Nevertheless, extensive studies on the efficacy, mode of action, and safety of herbal extracts are required for further development.

Conclusions
C. depressus Linn. is used for the treatment of diabetes mellitus in the Cholistan desert since ancient times.However, scientific evidence to support their therapeutic uses are non-existing.Present study deals with bioassay-guided evaluation of anti-diabetic activities (in vitro) of C. depressus in a more scientific way.Such as, careful extraction of respective plant material in three different seasons led to the finding of an active fraction CD-12, a good inhibitor of a-glucosidase.The fraction CD-12 was de-replicated through LCMS.Plant material collected in January showed considerable potential against diabetes and presence of Strophanthidin in it showed the best binding affinity with a-amylase and a-glucosidase enzymes.In-vitro studies coupled with anti-diabetic studies in animal models revealed considerable results and thus further detailed studies are warranted for potential use of the plant in Diabetes.