Chemicals and reagents used
Atorvastatin (Medipak pharmaceutical industry Pvt. Ltd Lahore), Lipofundin (B Braun Melsungen AG Germany), Diphenylpicrylhydrazyl (DPPH) (Merck Germany), Ascorbic acid (Sigma-aldrich Germany), D-Fructose, Acetylcholine, Phenylephrine, Potassium chloride, Ketamine chloride and Sodium phenobarbital (Sigma, St. Louis, USA) were used to carry out experimentation.
Selection, collection and authentication of the plant
The plant for this study was selected based upon its traditional repute in various diseases like kidney pain, jaundice particularly in cardiovascular diseases. The aerial parts of Salvia bucharica were collected in the month of April and May 2015 from Harboi, Nichara District Kalat, Baluchistan Pakistan. The plant was authenticated by Dr. Muhammad Amin Ullah Shah, Department of Botany, University of Sargodha. For further references the voucher specimen (SB-16-23) of plant was deposited in College of Pharmacy, University of Sargodha.
Preparation of Aqueous methanol extract
The aerial parts of Salvia bucharica were dried under shade at room temperature and grounded into a coarse powder. Aqueous methanol (30:70) extract of the plant was prepared using cold maceration process. After 3 days, it was filtered through muslin cloth, the filtrate was collected, and the plant material was again soaked for 3 days, twice. At the end, all of three filtrates were combined, filtered through muslin cloth and Whatmann qualitative Grade 1 filter paper. The filtrate of the extract was evaporated under reduced pressure in rotary evaporator at 50 °C. This aqueous methanol (30:70) extract was then air-dried to obtain a solid mass [9, 10].
Experimental animals used
A local breed of rabbits weighing 1—2 kg, adult Sprague dawlley rats (200—250 g) of both sex and mice (25—35g) were procured from local market. Animals were kept under standard environmental conditions (room temperature 22 ± 2ºC, relative humidity 50 ± 5% and 12 h light and dark cycle) in animal resource center, University of Sargodha. Diet and water were provided ad libitum. Experiments were performed in accordance with guidelines for care and use of laboratory animals provided by the National Research council . The experimental protocols were approved from Institutional Animal Ethics Committee, College of Pharmacy, University of Sargodha (Approval No. 43A24 IEC UOS).
Pharmacological studies performed on plant extract
Evaluation of Anti-hyperlipidemic effect of aqueous methanol extract of Salvia bucharica against Lipofundin induced dyslipidemia
Lipofundin cause rise in lipid profile thus this experiment was conducted to demarcate effect of Salvia bucharica on lipofundin lipid abnormalities. For this purpose, Rabbits weighing 1—2 kg were divided into different groups (n = 5). Group I (Normal control) received normal diet and water ad libitum for 23 days, Group II (negative control) received lipofundin (2 mL/kg i.v) for 8 consecutive days then from 9—23th day normal diet and water were given, Group III (positive control), Group IV (SB250) and group V (SB500) initially received lipofundin (2 mL/kg i.v) for 8 consecutive days then from 9—23th day atorvastatin (10 mg/kg p.o), Salvia bucharica aqueous methanol extract 250 mg and 500 mg p.o, administered respectively. Blood samples of animals were drawn on 8th and 23th day through jugular vein of the rabbits. Blood samples were allowed to clot for 20—25 min and centrifuged at 2500 g at 4ºC for 10 min. Serum was collected and aliquots were stored at —70ºC until biochemical analysis .
Biochemical parameters measured
Total cholesterol (TC), Triglyceride (TG), high density lipoprotein cholesterol (HDL-C) levels were measured through laboratory testing from the diagnostic laboratory of University Medical and Diagnostic Center of University of Sargodha. VLDL level, Low density lipoprotein (LDL-C), Atherogenic index (AI) and Coronary risk index (CRI) level were assessed by following formulas
VLDL = TG/5
LDL = TC — (HDL + VLDL)
AI = TC — HDL/HDL
CRI = TC/HDL [12-14]
Effect of Salvia bucharica aqueous methanol extract on Fructose induced hyperlipidemia in rats
The purpose of this experiment was to investigate the beneficial effect of Salvia bucharica on dyslipidemia induced by chronic administration of fructose. Wister albino rats of both sex weighing 100–150g were divided into five following groups: Group I (normal control) received normal diet and water ad libitum for 28 days, Group II (negative control) received fructose (25% w/v) in drinking water and normal diet for 28 days, Group III (positive control) received fructose (25% w/v) for consecutive 14 days then from 15—28th day atorvastatin (10 mg/kg p.o) was administered along with fructose (25% w/v). Group IV and V (treatment groups) received fructose (25% w/v) for consecutive 14 days then from 15—28th day plant extract (250 and 500 mg p.o) was administered along with fructose (25% w/v). On 29th day animals were sacrificed to collect the blood samples and were kept 30 min for coagulation, centrifuged at 3000rpm to get serum and the lipid profile was determined . Aortas of animals were removed to determine the vascular reactivity and liver was dissected for histopathological study .
Total cholesterol (TC), Triglyceride (TG), high density lipoprotein cholesterol (HDL-C) levels were measured through laboratory testing. VLDL level, Low density lipoprotein (LDL-C), Atherogenic index (AI) and Coronary risk index (CRI) level were assessed by above mentioned formulas [12-14].
Evaluation of protective effect of Salvia bucharica on fructose induced vascular dysfunction in rats
As chronic administration of fructose lead to progression of endothelial dysfunction therefore, this study was designed to assess protective effect of Salvia bucharica on vascular reactivity. Vascular reactivity was performed to observe the functional integrity of endothelium. Rats were killed by cervical dislocation and dissected .The thoracic aorta was separated and placed in the cold Kreb’s solution of the following composition (mM): NaCl 118, KCl 4.73, KH2PO41.2, MgSO4.7H2O 1.2, CaCl2 2.5, NaHCO3 25, glucose 11 and EDTA 0.026 for 30 seconds. Blood present in the aorta was removed by gently pressing aorta and transferred the aorta to the kreb’s solution having temperature 37ºC. Aorta was separated from the surrounding connective tissue and cut into rings of 3 mm long. Special care was taken to avoid any damage to endothelium. Rings were then mounted in organ-baths by means of two stainless steel wires attached with force-displacement transducer. A resting tension of 2 g was applied to facilitate the measurement of isometric force. The organ chamber was filled with 10 mL of Krebs–Henseleit solution at 37ºC and gassed with 95% O2 and 5% CO2. Preparations were allowed to equilibrate for approximately 1 h with an exchange of Kreb’s solution every 15 min. Following 1h equilibration the active muscle tone of ring segments contraction was then observed by potassium chloride (70—80 mM) and washout was given by changing the Kreb’s solution to return back to its initial tension and allowed to stabilize it for 30 min. Phenylephrine (10−6 M) was added in the chamber and contraction was noticed and washout period was given by changing the Kreb’s solution and allowed to stabilize it at its baseline tension. Again phenylephrine (10−6 M) was added in the chamber and contraction was observed. After a stable contraction plateau was reached, relaxation of the aortic rings was measured in response to cumulative additions of acetylcholine (Ach; 10–9 to 10–4 M) [16-19].
Histopathological examination of liver in fructose treated rats
On 28th day of treatment in fructose induced hyperlipidemia, rats were sacrificed, liver was dissected and washed with normal saline solution in order to clear it from blood and preserved in 10% formalin solution. Fixed liver was sectioned (5 micron thickness), embedded in paraffin and sections stained with Hematoxylin and Eosin (H&E). For estimation of histopathological changes in liver tissues was observed under light microscope [20, 21].
Acute toxicity studies performed in present work
This study was performed as per Organization for Economic Cooperation and Development (OECD) guidelines 425 .
Study was performed in three phases as following
Mice were randomly divided into four groups of two mice per group. Graded doses (500, 1000, 1500, 2000 mg/kg) of the Salvia bucharica was administered to the mice orally. After treatment, Mice were continuously observed during next 24 h (0.25, 0.5, 1, 2, 4, 12 h) for mortality, behavioral changes (restlessness, dullness, agitation, sedation) and signs of toxicity.
After 24 hours based on the findings of phase 1, three groups of mice (n = 2) were given the next doses (2500, 3000, 3500 mg/kg) of Salvia bucharica orally. All the animals were continuously observed for general behavioral changes, symptoms of toxicity and mortality during 24 initially after every 15 min, then every 30 min, then after 1, 2, 4 and 12 h.
After 24 hours, based on the observations of phase 2, Two groups of mice (n = 2) were given the next higher doses (4000, 5000 mg/kg) of Salvia bucharica orally. Mice were observed for 24 h post-treatment for mortality, behavioral changes (restlessness, dullness, agitation, sedation) and signs of toxicity .
Based on the findings of the acute toxicity study, the lowest dose that killed one mice and the maximum dose that had not killed any mice was recorded. The geographic mean of these doses gave the LD50 of the Salvia bucharica aqueous methanol extract .
Sub-acute Toxicity Test
Sub-acute toxicity study was performed as per the OECD guidelines 407 (OECD, 2008) with slight modifications. On the bases of findings of acute toxicity test,1/5th of the highest dose of acute toxicity study protocol of Salvia bucharica extract was selected and administered orally on daily basis for 14 days to a group of mice (n = 6). Control group of mice (n = 6) was received only vehicle for the same duration. All the experimental animals were observed after treatment daily for any abnormal clinical signs and mortality for 14 days. At the end of 14 day’s observation period, body weight of mice was measured. The mice were anaesthetized, dissected and their blood samples were collected immediately through cardiac puncture with and without anticoagulant (EDTA), for hematological and biochemical studies respectively . Heart, liver and kidneys of each mouse were individually weighed .
Determination of anti—oxidant activity
Free radical scavenging by use of DPPH (1,1—Diphenyl—2—picryl hydrazyl) radical
DPPH radical scavenging capacity of aqueous methanol extract Salvia bucharica was determined according to the method of Brand- Williams modified by Miliauskas  DPPH radicals have an absorption maximum at 515 nm, which disappears with reduction by an antioxidant compound. DPPH solution (6 × 10-5 M) in methanol was prepared, and 3 mL of this solution was mixed with 100 μL of each methanol solutions of salvia bucharica extracts and standard solution of Ascorbic acid. Sample and the standard Ascorbic acid solution were incubated for 20 min at 37 °C in a water bath, and then decrease in absorbance at 515 nm was measured (AE). A blank sample containing100μL of methanol in the DPPH solution was prepared, and its absorbance was also measured (AB). The experiment was carried out in triplicate. Radical scavenging activity was calculated using the following formula:
(% inhibition)= [(AB-AE)/AB] × 100
Where AB absorbance of the blank sample, and AE absorbance of the plant extract .
Phytochemical analysis of Salvia bucharica
Bioactive constituents in crude extract were analyzed through using RP UHPLC-MS by adopting the method of  . In brief, UHPLC of Agilent 1290 Infinity liquid chromatography system coupled to Agilent 6520 Accurate-Mass Q-TOF mass spectrometer with dual ESI source was used. Column used in this experiment was Agilent Zorbax Eclipse XDB-C18, narrow-bore 2.1×150 mm, 3.5 μm (P/N: 930990-902). The Column was kept at 25°C while 4°C was set for auto-sampler. Flow rate was maintained at 0.5ml/min and sample was injected as 1.0 μL. 0.1% formic acid in water (A), 0.1% formic acid in acetonitrile (B) were used as mobile phase. Sample was run for 25 min and an extra of 5 min were given. Full scan mass spectrometry analysis was conducted on m/z 100–1000 employing electro-spray ion source in negative fashion. Whereas, obtained results were processed with Agilent Mass Hunter Qualitative Analysis B.05.00 (Metabolomics-2017- 00004.m). Search Database: METLIN_AM_PCDL-Ne 170502.cdb, with parameters as: Match tolerance: 5 ppm, Positive Ions: +H, +Na, +NH4, Negative Ions: H. was used to identify the compounds.
Data obtained from all experiments in current study was expressed as means ± S.E.M. For estimating the statistical significance, data was analyzed using, one way or two way analysis of variance (ANOVA) followed by an appropriate posttest either Dunnet or Bonfferoni. All statistical analysis were performed and graphs drawn using GraphPad Prism version 5.0 and 6.0 for windows (GraphPad Software, San Diego, CA, USA). The results showing a probability of < 0.05 were considered as statistically significant.