Chemicals and reagents
Chemicals such as gallic acid, quercetin and LC/MS grade solvents were obtained from Sigma Aldrich, Bangalore, India. All other reagents used were of analytical grade of Merck, Bangalore, India.
Collection of plant materials
Bergenia ciliata was obtained from Khari Baoli market, New Delhi. All other materials were collected from different zones of Kerala and were authenticated by the Plant Systematics and Genetic Resources division, CMPR, Arya Vaidya Sala, Kottakkal, Kerala. The voucher specimens were deposited at CMPR Herbarium (Table 1).
Extraction of materials
The shade dried materials (250 g) of B. ciliata, A. lanata, R. aquatica and P. amboinicus were extracted with water using soxhlet extraction method for 72 h. The extracts were evaporated to dryness at 40o C on a rotary evaporator (Heidolph, Germany) and it was stored under refrigerator until the various phytochemical and pharmacological studies.
Estimation of Total polyphenols
Polyphenols such as phenolics and flavonoids were estimated spectrophotometrically. The total phenolic content (TPC) was determined using Folin-Ciocalteu reagent. TPC was expressed as gallic acid equivalents (mg GAE) in mg / g of sample. Total flavonoid content (TFC) was measured by aluminium chloride colorimetric assay and expressed as mg quercetin equivalents (mg EQ) [14,15,16].
High performance thin layer chromatographic (HPTLC) analysis
HPTLC analysis was performed by Camag HPTLC system (Switzerland). Samples were applied using Camag ATS 4 on aluminium backed pre-coated silica gel 60F254 HPTLC plate (Merck India). Mobile phase was standardized as toluene, ethyl acetate, methanol and formic acid in the ratio of 7:3:1:0.2. The chromatogram was developed in a saturated Twin Trough chromatographic chamber (Camag, Switzerland). The developed plate was visualized under UV 366 nm [17].
LC/MS analysis
LC-ESI/MS analysis was conducted on Agilent 6520 accurate mass Q-TOF LC/MS coupled with Agilent LC 1200 equipped with Extend-C18 column of 1.8 μm, 2.1 × 50 mm. Gradient elution was performed with LC/MS grade Acetonitrile (A) and 0.1% acetic acid in methanol (B) at a constant flow rate of 0.8 ml/ min, with an increase in the volume of B%; 5–20%, 12–30%, 19–40%, 26–50%, 30–40%. The MS analysis was performed using ESI in negative mode. The conditions for mass spectrometry were: drying gas (nitrogen) flow 5 L/min; nebulizer pressure 40 psig; drying gas temperature 325 °C; capillary voltage 3000 V; fragmentor volt 125 V; Oct RF Vpp 750 V. The mass fragmentation was performed with varying collision energy 4 V/ 100 DA with an offset of 8 V [17, 18].
Acute Oral toxicity study
The experiment was conducted on Wistar rats (females) weighing 139 – 155 g and aged 8 to 9 weeks obtained from the Animal House, J.S.S. College of Pharmacy, Ooty, Tamil Nadu, India. The rats were distributed into 5 groups with 6 animals in each group. The experimental procedures relating to the animals were authorized by Committee for the Purpose of Control and Supervision of Experiments on Animals (Approval No.: JSSCP/IAEC/OT/Ph.D/Ph.Cology/06/2017–18) before starting the study and were conducted under the internationally accepted principles for laboratory animal use and care.
The extracts were prepared from plant material having a high safety margin and hence it was decided to use 2000 mg/kg (Limit test) for this study. The test item was prepared immediately prior to administration on respective treatment days. A quantity of 2 g of the test item was dissolved in distilled water and the volume made up to 10 ml to get a test item concentration of 200 mg/ml. Homogeneity of the test item in the vehicle was maintained during treatment by constant stirring and mixing. The test substance was administered soon after preparation.
The prepared test item solutions were administered once orally as gavage to the fasted (17–19 h) rats at the dose volume of 10 ml/kg b.wt. to deliver a dose of 2000 mg/kg b.wt. Food was offered about 3–4 h after dosing. Water was not withheld.
The treated rats were observed five times during day 1 (day of administration) i.e., at 30 min and four times at hourly (post-administration) intervals and once daily, and thereafter for a total of 14 days. The clinical signs were recorded on all working days. The body weights of rats were recorded on test day 1 (pre-administration), day 8 (7 days post-administration) and day 15 (14 days post-administration. The rats were euthanized by using diethyl ether anesthesia and necropsied [17].
Antiurolithiatic activity
Adult Male Wistar albino rats were used for the study. The prior approval of the Institutional Animal Ethical Committee (Approval No. JSSCP/OT/IAEC/05/2018–19) was obtained for conducting this study. The animals were housed in polypropylene cages in a controlled environment (Temperature 23 ± 2 °C and 12 h dark and light cycle) with standard laboratory diet and water ad libitum in the animal house of the institution.
The plant extracts, BC, AL, RA and PA were prepared as solution in distilled water at two different concentrations of 10 and 20 mg/ml and administered at a dose volume of 10 ml/kg, body weight as low (100 mg/kg) and high (200 mg/kg) dose, respectively.
Wistar rats were acclimated for 7 days before starting the experiment. Hyperoxaluria and calcium oxalate deposition in the kidneys was induced by ethylene glycol in the drinking water to a final concentration of 0.75%, with 1% ammonium chloride for 3 days, to accelerate lithiasis. The rats were then given only ethylene glycol for 3 weeks. All the rats became nephrolithic by the end of the third week.
After randomization animals were divided into 10 groups of 6 each (Group I consisted of age matched normal animals). Group-1 &2 served as Normal and Disease Control, respectively, and received only vehicle (10 ml/kg, p.o.). Group-3 and 4 received BC extract at a dose of 100 and 200 mg/kg, p.o., respectively. Group-5 and 6 received AL extract at a dose of 100 and 200 mg/kg, p.o., respectively. Group-7 and 8 received RA extract at a dose of 100 and 200 mg/kg, p.o., respectively. Group-9 and 10 received PA extract at a dose of 100 and 200 mg/kg, p.o., respectively. All groups were received assigned treatments for 28 days. During the treatment period all the groups except Group-1, received 0.75% ethylene glycol in drinking water.
During the study period clinical signs, mortality and weekly body weights were measured. On day 27, urine was collected using metabolic cages to analyze urine biochemistry. At the end of the study blood was collected from retro-orbital plexus under light ether anesthesia and used for estimation of serum urea and creatinine levels. Following the blood collection animals were culled by deep ether anesthesia and kidneys were harvested and fixed in formal buffered saline (10% v/v) for histopathological analysis.
Statistical analysis
The data were represented as mean ± SD and analyzed by one-way ANOVA followed by Dunnett’s multiple comparison tests using Prism software (Version 4). P values ≤0.05 were considered significant.
