Collection and authentication of the test drug
The roots of Saad Kufi (Cyperus scariosus R.Br) were procured from the local market and identified in the Pharmacognosy Section, Department of Ilmul Advia, Ajmal Khan Tibbiya College. They were also authenticated by the National Institute of Science Communication and Information Resources, New Delhi (NISCAIR/RHMD/Consult/2017/3064-13-1). The sample of the test drug was submitted to Mawalid-e-Salasa Museum of the Department of Ilmul Advia after identification, for future reference with the voucher no. SC-0220/17.
Preparation of plant extract
Saad Kufi (Cyperus scariosus) was cleaned from the earthy material, washed with double distilled water, and dried at 45 °C in a hot air oven and powdered by an electrical grinder. Extraction was done in 50% ethanolic solvent by Soxhlet’s apparatus for 6 h. The extract was filtered and dried by evaporation on a water bath. The yield percentage was calculated with reference to a crude drug and was found to be 13.07%. A fresh suspension of the extract was prepared in distilled water (as per calculation w/v) at the time of the experiment.
Ethical statement
All experimental procedures and protocols used in the study were reviewed and approved by the Institutional Animal Ethical Committee (IAEC) on August 26, 2017, Jawaharlal Nehru Medical College, and care of laboratory animals was taken as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). The registration number is 401/GO/Re/S/2001/CPCSEA.
Study design
-
Group I: Normal control (20 ml/kg distilled water)
-
Group II: Negative control (adrenaline 0.05 mg/100 g b. w. i.p.)
-
Group III: Standard group (metoprolol tartrate 0.5 mg/100 g/day, p.o.)
-
Group IV: Test group (low dose): Saad Kufi (C. scariosus R.Br): 15 mg/100gm, p.o.
-
Group V: Test group (high dose): Saad Kufi (C. scariosus R.Br): 25 mg/100gm, p.o.
Experimental procedure
The in vivo anti-hypertensive (non-invasive method) effect of the test drug on systolic blood pressure (SBP) was observed in experimentally induced hypertension in rats by the indirect tail-cuff method [15]. Urethane was used as an anesthetizing agent, and adrenaline (0.1 ml) was injected intraperitoneally (I.P) into rats by 1 ml disposable syringe for 4 consecutive days to induce hypertension. Induction of hypertension was confirmed as the systolic pressure was detected and subsequently recorded with Data Acquisition System (Model: ML125, Serial: NI0502) on a power lab (4/30 Model: ML866, Serial: 430-1612 AD Instruments, Australia).
Measuring of SBP using PowerLab with tail-cuff apparatus
SBP was measured by the tail-cuff method using the NIBP Controller with Data Acquisition System (Model: ML 125, Serial: NI0502 AD Instruments, Australia) and PowerLab (4/30 Model: ML866, Serial: 430-1612 AD Instruments, Australia). To reduce spontaneous variations in blood pressure, animals are adjusted to the experimental cage by bringing them into the restraining cage 3–4 times before the start of the experiment for a period of 30–60 min and the ambient temperature was maintained at 31–32 °C. The blood pressure was measured by a tubular inflatable cuff, placed around the base of the tail, and a piezo-electric pulse detector was positioned distal to the cuff. Normal blood pressures of animals before treatment were recorded as baseline blood pressure, and then, rats were treated with their respective treatments and again blood pressure was recorded. To evaluate anti-hypertensive effect of drugs, adrenaline was injected after an hour of the treatment in standard and test groups and again the blood pressure was recorded. This was made to find the protective effect of Saad Kufi on the SBP induced by the administration of adrenaline. Finally, the mean increase in SBP from the pre-treatment SBP and the percent inhibition of SBP with respect to negative control were calculated.
Experimental animals
Wistar albino rats (n = 6) of either sex weighing 220–300 g were used for the present study. The animals were procured from the animal house, Department of Pharmacology, JNMC, AMU, Aligarh, India, and were allocated randomly to treatment groups and kept in polypropylene cages with paddy husk as bedding. Animals were housed at a relative humidity of 30–70% and temperature of 24 ± 20 °C along with light and dark cycles. A standard balance diet and water ad libitum were provided.
After the experiments, all animals were alive and were gone in the washing out period.
Sample size
Animals were divided into 5 groups, and each group has 5 animals.
Allocating animals to the experimental groups
Animals were randomly allocated to the experimental groups.
Experimental outcome
Animals have an increased systolic blood pressure.
Statistical analysis
One-way ANOVA with Tukey-Kramer multiple pair comparison test was used to determine the degree of significance. The analysis was carried out by using the GraphPad version 8.4.3 InStat Software.