A bioactive chalcone from the aerial parts of Indigofera conferta Gillet

Abstract Ethnobotanical information indicates that Indigofera conferta is used in northern Nigeria for the management of poisonous snakebites and the methanol extract was previously reported to have antivenin activity. In this study, we report the isolation of an oxyprenylated bioactive secondary metabolite; 2',4'- dihydroxy-4-prenyloxychalcone from the chloroform fraction of the aerial parts of the plant’s methanol extract and the in vivo evaluation of the compound against Naja nigricollis venom. The compound has demonstrated significant (p < 0.05) and dose-dependent antivenin activity against LD99 Naja nigricollis venom. At a dose of 10 mg/kg, the compound protected 60% of the animals tested from death. The observed activity lends credence to the traditional use of the plant in the management of snake bites in northern Nigeria. The compound, 2',4'- dihydroxy-4-prenyloxychalcone, could also serve as a lead in the development of novel antisnake venom agents. Graphical Abstract


Introduction
Indigofera conferta Gillet (Fabaceae) is a grey branching herb that is distributed throughout West Africa, including Northern Nigeria (Hutchinson and Dalziel 1958).Ethnomedicinal information indicates that the Hausa people in northern Nigeria use a decoction of the plant to manage snake bite envenomation, inflammation, treat sore feet, induce labour, and pain (Musa et al. 2008a;Abdulkadir et al. 2011).Other Indigofera species, like I. pulchra, are also used in traditional medicine as prophylactic against snake bites (Sule et al. 2003), to counteract various poisons (Burkill 1995), and to treat infected wounds (Herper 1976;Burkill 1995).The antimicrobial activity of various extracts of I. conferta has been reported (Musa et al. 2008a).The plant methanol extract has shown in vivo antivenin effect (Abdulkadir et al. 2011) and also demonstrated relaxation effect on rabbit jejunum, but without an effect on pregnant rat uterus in vitro (Abdulkadir et al. 2007).In a preliminary communication, we reported the in silico inhibitory potential of an oxyprenylated chalcone and a stilbene, identified in the extracts of I. conferta, against snake venom phospholipase A 2 (PLA 2 ) and metalloprotease using molecular docking and molecular dynamics simulation studies (Isah et al. 2021).As part of our search for bioactive constituents with antivenin properties from Nigerian plants, we report the isolation and characterization of the oxyprenylated chalcone from the aerial parts of I. conferta and evaluation of its antivenin activity in an animal model.

Result and discussion
The spectra and tables showing that the compound was identified correctly were reported in Supplemental Material.
Compound R2 was obtained as a yellow amorphous solid which reacted positive for shinoda and ferric chloride test implying the presence of a flavonoid nucleus (Silva et al. 1998).The UV spectrum (MeOH) showed absorption bands k maxima at 365 nm and two other weak bands at 285 and 283 nm characteristic of phenyl nucleus (Andersen and Markham 2006).The IR spectrum showed strong absorption bands at 3340 cm À1 (OH), 2985 cm À1 and 2885 cm À1 (C-H), 1736 cm À1 (C ¼ O), 1628 cm À1 (C ¼ C aromatic functions),1235 and 1042 cm À1 (C-O-C stretching band of ether) a good correlation to those of flavonoid derivative (Roberta et al. 2003;Lee et al. 2007).
The HSQC spectra of R2 were used to assign protons on their respective carbons.The HMBC spectra were used to establish the connectivity between different fragments of the molecule.In the HMBC spectrum, a common 3 J correlations between d H 6.27 (H-3 0 ) and that at d H 6.40 (H-5 0 ) to a quaternary carbon d 112.8 confirmed its assignment as C-1 0 , it also showed a 3 J correlations between d H 7.02 (H-3 & 5) and d H 7.77 (H-2 0 '') to a quaternary carbon d 127.1 confirmed its assignment as C-1 and also suggest that the chain of olefinic carbons is attached to ring B at C-1.A 3 J correlation observed between hydrogen at d H 8.17 (H-6 0 ) and quatenary carbonyl carbon confirmed that C ¼ O is linked to C-1 0 of ring A. In the prenyloxy side chain the methyl hydrogens showed 3 J correlation to the methyl carbons of their respective partners and to a methine carbon d 119.5, they also show 2 J correlation to the quaternary carbon (d 137.5, C-3 0 '), on which they are attached.The oxymethylene hydrogens d 4.62 of the prenyloxy side chain showed 3 J correlation to oxygen bearing quaternary carbon (d 160.6, C-4), this indicated that the prenyloxy side chain is joined to the molecule through C4 carbon (Figure 1).Based on the spectral data and by comparison with literature (Musa et al. 2011;Dauda et al. 2019), compound R2 was confirmed to be 2 0 4 0 -dihydroxy-4-prenyloxychalcone (Figure 1).In addition, a minor metabolite, identified as 3,5-dimethoxy-4 0 -O-(2 0 ',3 0 '-dihydroxy-3 0 '-methylbutyl)-dihydrostilbene (R1) (Musa et al. 2008b) was also discovered in the plant.
Indigofera conferta has been employed ethnomedicinally in the treatment of snake bite, inflammation, pain and induction of labour.The plant methanol extract has shown in vivo antivenin effect, validating its traditional use in the treatment of snake bite (Abdulkadir et al. 2011).The isolated chalcone showed superior binding interactions, than the stilbene, with the active site residues of snake venom PLA 2 and metalloprotease enzymes in computer models (Isah et al., 2021).Thus, to further investigate this promising antivenom potential, compound R2 was evaluated for in vivo antivenin activity.No mortality was observed in the acute toxicity studies, in groups of mice treated with 4, 6, 8, and 10 mg/kg of the isolated compound, indicating that R2 had no apparent toxicity at these concentrations.The minimum lethal dose (LD 99 ) of the N. nigricollis venom was found to be 5.75 mg/kg.The prenylated chalcone demonstrated significant and dose dependent in vivo antivenom activity with 20% survival rate at the lowest and the medium doses (4 mg/kg and 6 mg/kg, respectively) while the compound offered 60% protection to mice against toxic effects of the N. nigricollis venom at the highest (10 mg/kg) dose as shown on Table 1.

General procedures
NMR spectra were obtained on Bruker AVANCE III spectrometers (600 MHz for 1 H and 150 MHz for 13 C), using a 5 mm BBOZ probe.The spectra were referenced to residual solvent peaks, d H 2.50, d C 39.53 for DMSO-d6.Mass spectra were recorded on a TOF Waters Micromass LCT Premier mass spectrometer using ESI-ionization in negative or positive modes in MS-grade acetonitrile and methanol solutions.For thin-layer chromatographic analyses, pre-coated TLC silica gel 60 F 254 (Merck) plates were used.Column chromatography was performed on Merck silica gel (230-400) mesh, spots on TLC plates were observed under UV light at 254 and 365 nm, and visualised by spraying with p-anisaldehyde/H 2 SO 4 spray reagent (0.5 mL of p-anisaldehyde, 10 mL of glacial acetic acid, 4 mL of concentrated H 2 SO 4 acid and 85 mL of MeOH), followed by heating at 100 C for 5 min.

Collection, identification and preparation of plant material
The aerial parts of Indigofera conferta were collected at area BZ, Samaru, Zaria, Nigeria in October 2018.The plant was authenticated by Namadi Sanusi, a taxonomist, at the herbarium section in the department of botany, Ahmadu Bello University Zaria.A sample was deposited and voucher specimen number 01084 was assigned.The plant material was air-dried and pulverized using a mortar and pestle.

Experimental animals
Swiss albino mice of either sex weighing 18-24 g, were obtained from the Animal House Facility of the Department of Pharmacology and Therapeutics, Ahmadu Bello University, Zaria, Nigeria.They were fed with laboratory diet and water ad libitum and maintained under standard conditions (12 h light and 12 h dark cycle) in propylene cages at room temperature.All experimental procedures were approved by the Animal Right and Ethics Community of the University.

Extraction and isolation
The powdered plant material (1400 g) was extracted by maceration in methanol (5 L) with occasional shaking for 72 hours.The solvent was removed under reduced pressure using a rotary evaporator to afford the crude methanol extract (150 g).The crude extract (130 g) was reconstituted in 10% methanol and successively partitioned with n- hexane, chloroform, and ethyl acetate to give three fractions.The chloroform fraction (7 g) was chromatographed over silica gel and eluted with a gradient solvent system comprising n-hexane 100%, n-hexane-ethyl acetate 45:65, and Methanol 100%.A total of 110 fractions, 100 mL each, were collected and were pooled together on the basis of similarities of their TLC profiles to obtain 14 sub-fractions subFr A-N.SubFr A and B (500 mg) composed of fatty material and were not further purified.Similarly, subFr C, D, E, and F (220 mg) contained waxy material based on proton NMR spectrum and were also not further purified.Purification of subFr G (1.5 g), which showed one major spot and two minor spots on TLC, by preparative thin layer chromatography using 3:1 hexane-ethyl acetate as the solvent system, led to the isolation of a yellow amorphous solid (520 mg) coded R2.The relatively low yield of compound R2 could be due to binding of its hydroxy groups to the stationary phase used, in this case silica.A stilbene (R1, 8.0 mg) was isolated from subFr M (1.3 g) by repeated column chromatography using gradient elution technique; starting with hexane 100% followed by 95:5 hexane: ethylacetate to ethylacetate 100% which afforded a white amorphous material as a minor metabolite.Fraction N (2.0 g) composed of very polar substances, soluble only in water, so its purification was not further pursued too.3.3.Antisnake venom studies

Snake venom
The milking method described by Macfarlane (1967) was used to collect the venom of an adult Naja nigricollis.The collected venom which was pooled, lyophilized was subsequently referred to as the crude venom of N. nigricollis and stored at 4 C until required.

Acute toxicity studies
The acute toxicity studies was carried out using Turner's method (Turner 1965).Eight mice were divided into four groups containing two mice each and group 1, 2, 3 and 4 received 4, 6, 8 and 10 mg/kg of the isolate (R2), respectively.The animals were observed for mortality for 24 h and the LD 50 was calculated by probit analysis

Lethality assay of the venom
The method described by Theakston and Reid (1983) was employed with slight modification to determine the LD 99 of the venom.The venom of the snake used was reconstituted with normal saline and concentrations ranging from 0.1 to 0.6 mg/mL were obtained.Thirty-five (35) mice were divided into seven (7) groups of five (5) mice each and were injected i.p with different doses of the reconstituted venom.The control group received only normal saline (0.2 mL each, i.p).The LD 99 was calculated by probit analysis (Finney 1971) of death occurring within 24 h of venom injection.

Venom detoxification by the isolated compound
The method described by Abubakar et al. (2000) was employed with slight modification.Briefly 20 mice were divided into four groups containing five mice each (n ¼ 5).Group 1 (control) received 0.2 ml of the MLD (LD 99 , 5.75 mg/kg) of N. nigricollis venom only.Groups 2, 3 and 4 (treatment groups) received an equivalent of the MLD of the venom containing 4, 6 and 10 mg/kg of the isolated compound (R2).The mixture of venom and chalcone was incubated at 37 C for 10 min prior to injection.The mixture (0.2 ml) was injected into each mouse in the treatment groups.The route of administration was intraperitoneal and mortality observed for 24 hours.

Statistical analysis
Chi-Square and One-Way ANOVA were used to analyzed the data, followed by a post hoc test using the SPSS software (version 22).Values of p < 0.05 were considered significant.

Conclusion
The first investigation of the chemical constituents of Indigofera conferta has resulted in the isolation of an oxyprenylated chalcone, as 2 0 ,4 0 -dihydroxy-4-prenyloxychalcone (R2) from the chloroform fraction of the aerial parts of the plant.The prenylated chalcone showed an ability to reduce the toxicity of Naja nigricollis snake venom in vivo and reduced mortality in mice pre-treated with the compound before envenomation.This is in agreement with reports on compounds with flavonoidal nucleus to have had antivenin activity (Chippaux et al. 1997).Also, the observed activity of 2 0 ,4 0 -dihydroxy-4-prenyloxychalcone lends credence to the traditional use of the plant in the management of snake bites in northern Nigeria.

Table 1 .
Detoxifying effect of administration (i.p) of incubated mixture of venom of Naja nigricollis and isolate of I. conferta in adult albino mice.