Collection and identification of plant material

Indigofera heterantha is a deciduous shrub growing to 2–3 m (7–10 ft) tall and broad, young branches are angular, with slightly spreading white medifixed symmetrically 2-branched trichomes; stems are greyish brown, terete, with rounded lenticels, and covered in appressed medifixed trichomes. Dry, sunny slopes between 1,500 and 3,000 m in elevation, frequently creating dense brush, and in forests [27, 28]. I. heteroantha is a rather resistant plant; while temperatures between − 5 and − 10 °C might stunt top development, the rootstock is more resilient and can withstand temperatures as low as -15 °C most of the time [29]. Plants grow best in hot seasons with abundant sunshine, not flowering so freely in cool climates [30].

The I. heterantha employed in the study was obtained in Kashmir, India’s Gulmarg region of the Baramulla district during the month of June-July. Dr. Akhter H. Malik, a taxonomist with the Department of Botany at the University of Kashmir, recognized the plant at the Centre of Biodiversity and Taxonomy. A specimen of the plant was kept at the herbarium of the University of Kashmir’s Department of Botany in Srinagar under accession number 2325-KASH Herbarium. Roots, leaves, bark, and flowers were separated from the whole plant. For a period of two weeks, the plant material was dried in the shade. Dried roots, leaves, and bark were crushed into coarse powder by an electric grinder.

Experimental

All chemicals used for phytochemical screening were obtained from CDH chemicals Ltd. Mumbai and were of analytical reagents (AR) grade and reagents were obtained from Medsource Ozone Biomedicals Pvt. Ltd. Delhi. Antibacterial activity was performed under laminar flow cabinet. The reagents used for qualitative and quantitative phytochemical estimation of the plant were obtained from Himedia company. Antioxidant activity was evaluated via UV visible spectrophotometer.

Chemicals and Instruments

All chemicals were of highest purity (≥ 99.0%) and analytical grade. The chemicals used during the study includes Pet. ether, Methanol, Ethyl acetate, Acetic acid, AgNO3, Ammonia, AlCl3, CuSO4, Copper acetate, Diethyl ether, Ethanol, FeCl3, FeSO4, H2SO4, HCl, HNO3, Lead acetate, Magnesium metal strips, Mercury, Methanol, Na2CO3, NaCl, NaOH, n-butanol, Ninhydrin, Paraffin, Phloroglucinol, Picric acid, Hagers reagent, Wagners reagent, Dragendroffs reagent, Potassium dichromate, Potassium thiocyanate, Pyridine, Resorcinol, Sodium citrate, Sodium nitroprusside, TCA, Griess reagent, Potassium acetate, Glucose, Phenol, Gallic acid, AlCl3, Folin Ciocalteu reagent, Mueller Hinton agar (MHA), SDA, Fluconazole discs, Streptomycin discs, Xylene, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Ascorbic acid.

The instruments used during the study include Incubator cum shaker, Hot air oven, Autoclave, Centrifuge, Desiccator, Digital weighing balance, Grinding mill, Oven, Rotary evaporator, UV-spectrometer, Laminar flow cabinet, Inoculating wire, Cork borer, Water bath, and general glass ware.

Extraction yield

The cold extraction method was used to remove the plant material. Methanol and distilled water were the solvents employed in the cold extraction process. A 5000 ml macerating flask containing an accurately weighed 600 g of dried powdered leaves was filled with a 1:4 ratio of extracting solvent, and the mixture was allowed to stand at room temperature for 48 h while being constantly stirred. Once the soluble matter had dissolved, the mixture was strained, the marc was pressed, and the combing liquids were clarified by filtering. With the use of a rotary evaporator operating at low pressure, the extract was concentrated, and the solid extract was kept in a refrigerator for later use. A similar procedure was used to extract leaves aqueously. However, 300 g of powder were taken in a 2000 ml macerating flask. After adding a 1:4 ratio of extracting solvent to a precisely weighed 600 g of dried Root powder, the contents of the 5000 ml macerating flask were left to stand at room temperature for 48 h while being constantly stirred. Once the soluble matter had dissolved, the mixture was strained, the marc was pressed, and the combing liquids were clarified by filtering. With the aid of a rotary evaporator operating at lower pressure, the extract was concentrated, and the solid extract was then kept in a refrigerator for later use. A similar procedure was used to extract Root aqueously. nonetheless, 300 g of powder were consumed in a 2000 ml macerating flask. A precise weight of 300 g of dry Bark powder was weighed and placed in a 2000 ml macerating flask. After adding an extracting solvent in a 1:4 ratio and agitating the contents of the flask frequently for 48 h at room temperature, the soluble matter was dissolved. The combination was then strained, the marc was pressed, and the combing liquids were clarified by filtering. With the use of a rotary evaporator operating at low pressure, the extract was concentrated, and the solid extract was kept in a refrigerator for later use. A similar procedure was used to extract Bark aqueously. However, 200 g of powder was taken in a 2000 ml macerating flask. 200 g of dried flower powder, precisely weighed, was placed in a 2000 ml macerating flask. After adding an extracting solvent in a 1:4 ratio and agitating the contents of the flask frequently for 48 h at room temperature, the soluble matter was dissolved. The combination was then strained, the marc was pressed, and the combing liquids were clarified by filtering. With the use of a rotary evaporator operating at low pressure, the extract was concentrated, and the solid extract was kept in a refrigerator for later use. A similar procedure was used to extract flowers using water. However, 50 gms of the powder was taken in a 1000 ml macerating flask. Using a cold extraction technique using an aqueous and methanolic solvent system, crude extracts of several I. heterantha components were made [31].

Qualitative phytochemical tests

Following the standard procedures described in the literature [32,33,34], the methanolic extracts of I. heterantha leaves, bark, roots, and flowers were subjected to qualitative analysis for secondary metabolites such as alkaloids, tannins, flavonoids, cardiac steroidal glycosides, proteins and amino acids, and carbohydrates.

Infrared analysis

Likewise, infrared (IR) spectra were captured using a Perkin Elmer Spectrum 2 MIR Spectrometer (L1600235). The IR spectra (KBr pellets) between 400 and 4000 cm-1 were recorded using a total of 10 images [35]. We recorded the FTIR spectra to search for potential functional groups [36,37,38].

Antibacterial activity

The bacterial strains used to evaluate the in vitro antibiotic activity of I. heterantha crude extracts were purchased from the Microbial Type Culture Collection (MTCC) at Chandigarh, India’s Institute of Microbial Technology (IMTECH). Escherichia coli (MTCC 739), Proteus vulgaris (MTCC 426), Staphylococcus aureus (MTCC 96), Pseudomonas aeruginosa (MTCC1688), Bacillus subtilis (MTCC441), and Klebsiella pneumoniae (MTCC432) are some of the species that are included. Both aqueous and methanolic extracts had their antibacterial activity evaluated using the Agar Well Diffusion Method. The Mueller Hinton Agar Well Diffusion Method was modified somewhat for use in the susceptibility tests. The bacterial strains were adjusted to a turbidity of 0.5 Mac Farland norm (108 CFU/ml) and suspended in sterile water after growing on nutritional agar for 18 h at 37 °C. The turbidity was measured using a UV spectrophotometer at 625 nm. Mueller Hinton Agar (MHA) medium was prepared using tap water and sterilized in an autoclave at 121 °C and 16 psi for 14 min in the Agar Well Diffusion Process. After sterilization, the medium was added to clean Petri plates in the laminar hood, where the plates were left to harden. Using a sterile cork borer with a 5 mm diameter, wells were created in each Petri plate that were uniform and equally spaced. On the plates, a standardized inoculum was used to inoculate each test bacteria (0.1 ml) of (0.5 McFarland) and spread with a spreader. Plant extracts were loaded into various peripheral wells at different concentrations (10 mg/ml, 30 mg/ml, 50 mg/ml, 80 mg/ml, and 100 mg/ml). Each Petri plate had a positive control of gentamycin (10 mcg/disc), whereas a different Petri plate contained a negative control of 10% dimethyl sulfoxide (DMSO). For 18 to 24 h, the Petri plates were incubated at 37 °C in an incubator. Zones of inhibition were then looked for on the plates. The inhibitory zone diameters, which are measured in millimeters (mm), were used to determine the antibacterial potential.

Antifungal activity

To assess the in vitro antifungal activity of the fungal strains from the Microbial Type Culture Collection (MTCC), Chandigarh, India, crude methanolic and aqueous extracts of I. heterantha were utilized. These strains include Saccharomyces cerevisiae (MTCC 170), Candida albicans (MTCC 227), Penicillium chrysogenum (MTCC 947), and Aspergillus fumigatus (MTCC 426). The antifungal potency of I. heterantha aqueous and methanolic extracts was evaluated using the Agar Well Diffusion Assay. Each test fungus was injected with 0.1 ml of standardized inoculum (0.5 McFarland), homogenized, and then placed into a sterile Petri plate at a constant depth of 4 mm on sterile molten Sabouraud dextrose agar media. The Petri plates were able to harden in the laminar hood. Using a sterile cork borer with a diameter of 6 mm, five wells were created on the periphery and one well in the center of each Petri plate. Plant extract in five different peripheral wells at concentrations of 10 mg/ml, 30 mg/ml, 50 mg/ml, 80 mg/ml, and 100 mg/ml totaled 40 l. A separate Petri plate had one well with nystatin (10 mg/ml) and fluconazole (25 mg/ml) inserted as a disc. In a different well, 10% dimethyl sulfoxide was used as a negative control. The plates were stored at 32 °C for 24 to 36 h. After incubation, the plates were examined for areas of inhibition. The antifungal potential was measured by measuring the widths of the inhibitory zones in millimeters (mm) using a standard measurement scale.

Anthelmintic activityExperimental worms

Because the adult Indian earthworms (Pheretima posthuma) share structural similarities with human intestinal roundworm parasites, these investigations were conducted on them exclusively. After being removed from the damp earth, they had a water wash to get rid of all the excrement.

Administration of albendazole

Using 0.5% w/v of CMC as a suspending agent, albendazole (20 mg/ml) was produced and administered in accordance with the extract technique.

Administration of extract

Using 0.5% w/v of CMC as a suspending agent, the suspension of methanolic and aqueous extracts of I. hethrantha was created in different concentrations (50, 100 mg/ml). The final volume was increased to 10 ml for each concentration. The standard treatment was albendazole. Two earthworms per group, in groups of roughly equal size, were introduced into each 10 millilitre of the required medication and extract concentration in the petridish.

Experimental design

The antihelmintic activity was carried out in compliance with Partap et al., 2012 [39]’s methodology on an adult Pheretima posthuma Indian earth worm, which shares physiological and anatomical similarities with human intestinal roundworm parasites. Pheretima posthuma were put in a petridish with two distinct quantities of ethanolic and aqueous extract from various I. heterantha Sects. (50 & 100 mg/ml). Two worms were added to each petridish, and the worms’ paralysis or death was monitored. After it was determined that worms did not move in response to external stimuli or when they were shook violently, the mean time for paralysis was recorded, which was the point at which no movement of any kind could be seen. The time of worm death (min) was also recorded. The test findings were contrasted with samples treated with the reference chemical albendazole (20 mg/ml).