Plant material
In plant material, similar size stems collected as ofT.cordifolia (Willd) Hook. F & Thoms. from medicinal plant garden, SPPU campus, Pune (18.55598° N, 73.82128° E), T.crispa (L.) Hook. F. & Thoms. from Odisha University of Agriculture & Technology, Bhubaneswar, Odisha, (20.2650° N, 85.8117° E) and T.sinensis (Lour.) Merr from College of Agriculture, Dapoli, Maharashtra (17.7501° N, 73.1802 °E) during winter 2021. The botanical authentication of all samples was done at Regional Cum Facilitating Centre (RCFC) for Western Region, Pune, India. All the samples were powdered, dried at room temperature in the shade, and then kept there until further use.
Extraction
Three times using 80% ethanol in water at room temperature, the air-dried and powdered stems of T. cordifolia, T. sinensis, and T. crispa(100g each) were extracted. To get dry brown extract (1.48 g), the mixed percolations were concentrated under reduced pressure below 45°C. The dried brown extract was fractionated with methanoland kept at 4℃ for further HPTLC use.
HPTLS analysis
Chromatographic conditions
A CAMAG HPTLC system (Muttenz, Switzerland) comprising of a CAMAG Linomat 5 semi-automatic sample applicator, CAMAG TLC Scanner IV, CAMAG TLC visualizer, and twin-trough developing chamber (20 X20 cm), UV cabinet with dual-wavelength UV lamp, Hamilton syringe (100 µL), and CAMAG winCATS software was used in the study.
HPTLC was performed on 20 cm×20 cm normal phase TLC aluminum plates pre-coated with 200 mm layer thickness of silica gel 60F254. The bands were applied as 6 mm wide at 10 mm from the bottom and 15 mm from the sides with 11.3 mm space between the two bands. Ascending development of the plate, mobile phase migration distance 130 mm was performed at 27 ± 2°C temperature and 50 ± 5% relative humidity with chloroform/ethyl acetate/methanol (13:8:0.3 v/v) as mobile phases, in a saturated chamber for 40 min. After development, the plate was dried with the help of TLC heater at 100°C. TLC plates were dipped in freshly prepared anisaldehyde sulfuric acid detection reagent and heated at 100°C for 5 min. Here, the plate was scanned at 366 nm before spraying and at 600 nm after spraying with a detection reagent. The densitometry scanning was performedin absorption/reflectance mode with winCATS software, using deuterium, halogen–tungsten, and high-pressure mercury lamps, slit width 6.0×0.45 mm2, scanning speed 20 mm s− 1 and data resolution 100 µm step− 1.
Preparation of standard solutions
A stock solution (1 mg/ml each) of three bioactive isolated compoundscardifolioside, tinosporaside, and berberine was prepared by dissolving accurately weighed amounts in HPLC-grade methanol.
HPTLC finger Printing
The dried extracts were re-dissolved in the mobile phase to prepare a 10 mg/ml concentration of all samples, filtered through 0.45 µm Millipore membrane, and an aliquot was subjected to HPTLC analysis. The peak areas were recorded. Calibration curves of all standards were obtained by plotting peak areas viz. applied concentrations of compounds.The developed plate was derivatized, then scanned and quantified densitometrically at 254 nm, 220 nm, and 366 nm respectively for cardifolioside, tinosporaside, and berberine.
Organoleptic tests in Tinospora species
Stem morphological traits: Stem thickness, color, and outer appearance have been noted from five random samples among collected material immediately after harvest in all three species separately.
Physiochemical evaluation: All the samples were subjected to the physiochemical parameters viz.drying %, ash %, water, acid, and alcohol soluble extractive in three replicates according to WHO, 2011.
Drying percentage: In a measuring vial that has been precisely weighed, add 10 g of the sample and weigh it precisely. After that, dry it at 105°C for 5 to 6 hours, and then cool it in desiccators using silica gel. The percentage of drying loss is calculated once the material has dried to a consistent weight.
Ash percentage: In a crucible that has already been dried and weighed, put 2 to 4 g of the sample. It must be heated gradually and kept at 550°C for about 4 hours to get rid of compounds that are easily carbonizable. After cooling in desiccators, it is weighed and then calculates weight from the percentage of total ash.
Acid insoluble ash: The remaining insoluble material is ignited after the complete ash was thoroughly boiled with diluted hydrochloric acid. The insoluble matter was obtained in a crucible or on a filter paper with no ash, boiled with 25 ml of diluted hydrochloric acid for 5–10 minutes, ignited, and weighed. Now, using the air-dried drugs as a reference, we estimated the percentage yield of acid-insoluble ash as follows.
$$\% Acid insoluble ash value=\frac{ Wt.of acid insoluble ash}{Wt.of ccrude drug taken} X 100$$
Water soluble extractive: In a closed flask, 1 g of air-dried powder was macerated with 100 ml of distilled water for 24 hours while being continuously shaken. The filtered solution was then evaporated in a shallow dish with a flat bottom and tar coating, dried at 100°C, and weighed. With reference to the air-dried drugs, the percentage of water-soluble extractives was estimated.
Alcohol soluble extractive:Macerated 5 g accurately weighed coarse powdered drug with 100 ml of alcohol (90% v/v) in a stoppered flask for 24 h, stirring frequently during the first 6 h. Filtered quickly through filter paper taking safety measures against excessive loss of alcohol.Evaporated 25 ml of alcoholic extract to dryness in a tarred dish and weighed it.
Microscopic observations: The anatomical observation on the transversely cut sections of the stem of samples were observed for different parts viz. pith, xylem, phloem, pericycle, epidermis, cortex, medullary rays under the light microscope (Leica).