Therapeutic Potential of Salvia Bucharica for Dyslipidemia and Vascular Dysfunction Induced through Two Different Pathways

Background: The present study was endeavored to validate the antihyperlipidemic potential of Salvia bucharica (Lamiaceae) in lipofundin induced hyperlipidemia in rabbits and fructose treated hyperlipidemic rats. Method: Extract of Salvia bucharica and atorvastation (10 mg/kg) were given to the animals through oral route. At the end of study period, blood samples were taken for determination of lipid prole. Furthermore, thoracic aorta and liver were studied for vasorexalation and histopathological changes respectively. Results: Salvia bucharica has shown dose dependent antihyperlipidemic activity with a signicant (p < 0.05) decrease in the levels of total cholesterol, low density lipoprotein and triglycerides in both models. In addition, Studies performed on the isolated thoracic aorta and histolopathological studies on liver demonstrated that Salvia bucharica has endothelial dependent vasoprotective and hepatoprotective activity respectively as compared to fructose fed rats. The toxicity studies of Salvia bucharica revealed that plant extract is safer for oral use. Conclusion Hence, it was suggested that Salvia bucharica has the therapeutic potential against dyslipidemia and vascular dysfunction.

(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 [11]. 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 pro le 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 8 th and 23 th 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 [6].  [12][13][14] Effect of Salvia bucharica aqueous methanol extract on Fructose induced hyperlipidemia in rats The purpose of this experiment was to investigate the bene cial effect of Salvia bucharica on dyslipidemia induced by chronic administration of fructose. Wister albino rats of both sex weighing 100-150g were divided into ve 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 15-28 th 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 15-28 th day plant extract (250 and 500 mg p.o) was administered along with fructose (25% w/v). On 29 th day animals were sacri ced to collect the blood samples and were kept 30 min for coagulation, centrifuged at 3000rpm to get serum and the lipid pro le was determined [15]. Aortas of animals were removed to determine the vascular reactivity and liver was dissected for histopathological study [16].
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 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 lled with 10 mL of Krebs-Henseleit solution at 37ºC and gassed with 95% O 2 and 5% CO 2 . 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][17][18][19].
Histopathological examination of liver in fructose treated rats On 28 th day of treatment in fructose induced hyperlipidemia, rats were sacri ced, 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 para n and sections stained with Hematoxylin and Eosin (H&E). For estimation of histopathological changes in liver tissues was observed under light microscope [20,21].

Toxicity pro le
Acute toxicity studies performed in present work This study was performed as per Organization for Economic Cooperation and Development (OECD) guidelines 425 [22].
Study was performed in three phases as following Phase 1 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.

Phase 2
After 24 hours based on the ndings 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.

Phase 3
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 [23].

LD 50 determination
Based on the ndings 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 LD 50 of the Salvia bucharica aqueous methanol extract [23].

Sub-acute Toxicity Test
Sub-acute toxicity study was performed as per the OECD guidelines 407 (OECD, 2008) with slight modi cations. On the bases of ndings of acute toxicity test,1/5 th 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 [24] . Heart, liver and kidneys of each mouse were individually weighed [25].

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 modi ed by Miliauskas [26] 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: Where AB absorbance of the blank sample, and AE absorbance of the plant extract [27].

Phytochemical analysis of Salvia bucharica
Bioactive constituents in crude extract were analyzed through using RP UHPLC-MS by adopting the method of [28] . In brief, UHPLC of Agilent 1290 In nity 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. was used to identify the compounds.

Statistical analysis
Data obtained from all experiments in current study was expressed as means ± S.E.M. For estimating the statistical signi cance, 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 signi cant.

Results
Anti-hyperlipidemic effect of Salvia bucharica against lipofundin induced dyslipidemia Effect on lipid pro le Aqueous methanol extract of S. bucharica showed dose dependent antihyperlipedimc effect. Lipofundin administered to all group except normal control caused signi cant rise in lipid parameters. LIPO group at 8 th day produced signi cant increase in TC (86.6 ± 4.40 mg/dL) and TG (118 ± 7.26 mg/dL) as compared to normal control (65 ± 2.8, 72.66 ± 4.33 mg/dL) with level of signi cance P<0.001. LIPO group at 23 rd day also showed signi cant rise in serum total cholesterol (84.33 ± 4.25 mg/dL) and serum triglyceride (112 ± 6.24 mg/dL) in comparison of normal control with level of signi cance p<0.001 and p<0.01 respectively. Atorvastatin (10 mg/kg) and S. bucharica at dose of 500 mg/kg caused remarkable (p<0.001) reduction in TC and TG level and showed comparable results. Atorvastatin and S. bucharica reduced TC to 61 ± 4.94 mg/dL and 62.66 ± 3.71 mg/dL respectively than LIPO 23 rd day. Atorvastatin and S. bucharica reduced serum TG to 72.66 ± 4.33 mg/dL and 82.66 ± 5.81 mg/dL respectively than LIPO 23 rd day. Both of these groups also showed signi cant reduction of LDL i.e 11.33 ± 2.02 mg/dL and 10.46 ± 1.84 mg/dL respectively than the LIPO 23 rd day (32 ± 2 mg/dL) with level of signi cance p<0.01. SB also caused reduction in HDL and VLDL levels but the effect of extract on these parameters was not signi cant (Fig.1).
Effect on the atherogenic index (AI) and coronary risk index (CRI) Aqueous methanol extract of S. bucharica showed dose dependent effect on AI and CRI. Atorvastatin (10 mg/kg) and aqueous methanol extract of S. bucharica at dose of 500 mg/kg showed signi cant reduction in AI to 0.73 ± 0.06 and 0.75 ± 0.05 respectively in comparison of LIPO 23 rd day (1.63 ± 0.15) with level of signi cance p< 0.01. In case of CRI, Atorvastatin (10 mg/kg) and aqueous methanol extract of S. bucharica at dose of 500 mg/kg showed signi cant reduction in CRI to (1.73 ± 0.06 and 1.75 ± 0.05) than the LIPO 23 rd day (2.30 ±0.46) with level of signi cance p< 0.05 (Fig.2).

Effect of Salvia bucharica as antihyperlipidemic agent against Fructose treated rats
Effect on lipid pro le Aqueous methanol extract of Salvia bucharica showed signi cant dose dependent antihyperlipedimc effect in fructose induce hyperlipidemia. Fructose group produced signi cant increase in total cholesterol (106.66 ± 6.76 mg/dL) and triglyceride (196.66 ± 7.26 mg/dL) as compared to normal group (71 ± 2.30, 91.66 ± 6.00 mg/dL) with level of signi cance P<0.001. The Fructose + SB 250 and Fructose + SB 500 showed signi cant reduction in total cholesterol to 91 ± 4.61 mg/dL and 82 ± 3.75 mg/dL respectively than Fructose group with level of signi cance p<0.05 and P<0.001 respectively. The Fructose + SB 250 and Fructose + SB 500 also showed signi cant reduction in triglycerides to 131.66 ± 5.36 mg/dL and 81 ± 6.24 mg/dL respectively than Fructose group with level of signi cance p<0.05 and P<0.001 respectively. Fructose + ATOR and Fructose + SB500 also caused signi cant reduction of VLDL level to 14.53 ± 0.86 mg/dL and 17 ± 0.57 mg/dL respectively in comparison of Fructose group (39.33 ± 1.45) with level of signi cance p< 0.001 and p< 0.01 respectively (Fig.3).
Effect on atherogenic index (AI) and coronary risk index (CRI) Aqueous methanol extract of Salvia bucharica showed dose dependent effect on atherogenic index and coronary risk index in fructose induced hyperlipidemic rats. Atorvastatin (10 mg/kg) and aqueous methanol extract of Salvia bucharica at dose of 500 mg/kg showed signi cant reduction in AI to 0.93 ± 0.14 and 0.62 ± 0.08 respectively than Fructose treated group (1.43 ± 0.12) with level of signi cance p< 0.01 and p<0.001 respectively. In case of CRI, Atorvastatin (10 mg/kg) and aqueous methanol extract of Salvia bucharica at dose of 500 mg/kg showed signi cant reduction in CRI to 1.93 ± 0.14 and 1.62 ± 0.08 in comparison to Fructose group (2.43 ± 0.12) with level of signi cance p<0.01 and p<0.001 respectively (Fig.4).
Bene cial potential of Salvia bucharica against fructose induced vascular dysfunction Salvia bucharica aqueous methanol extract showed effective improvement in endothelial dysfunction induced by fructose in dose dependent manner. In fructose treated rats, fructose caused vascular endothelium damage as shown by reduction in endothelium dependent vasorelaxation of aorta rings by acetylcholine after phenylephrine induced contraction. While Atorvastatin group and Salvia bucharica 500 mg/kg group protected the vascular endothelium damage and signi cant vasorelaxation was observed by acetylcholine as compared to fructose control group in dose dependent manner with level of signi cance p<0.001. Salvia bucharica 250 mg/kg group do not show remarkable relaxation at lower doses but at dose of 10 -5 mol/L of acetylcholine, it showed signi cance relaxation with p value of 0.01 (Fig.5).

Effect of Salvia bucharica on liver histopathology
The histopathology of liver section of normal rats showed normal cell structure while the liver section of fructose fed rats showed the presence of destructive alteration and extensive fatty changes characterized by focal necrosis and damaged sinusoidal spaces and narrowing of central vein. The liver section of rats treated with Salvia bucharica at dose of 250 mg/kg showed less effect on necrosis, central vein damage and fatty changes. While Salvia bucharica at dose of 500 mg/kg and atorvastatin at dose of 10 mg/kg showed hepatoprotective activity in rats and histopathological changes signi cantly attenuated by these groups (Fig.6) Toxicity pro le Acute toxicity study and determination of LD 50 At graded doses, from 250 mg/kg up to 5000 mg/kg body weight of aqueous Methanol extract of Salvia bucharica did not show any sign of adverse reactions and no changes in animals up to 24 h after the administration of the extract to mice. LD 50 value of aqueous Methanol extract of Salvia bucharica was found to be greater than 5000 mg/kg.

Sub-acute Toxicity Test
No signi cant difference in body weight, and weight of liver, heart and kidney was observed between the control and extract-administered groups after 14 days in sub-acute study. In addition, no mortality was recorded throughout the period of observation. There was the decrease in level of serum ALT (U/L) and AST (U/L) but the creatinine (mg/dL) and urea (mg/dL) levels were comparable to normal control ( Phytochemical analysis of aqueous methanol extract of Salvia bucharica using LC-MS LC-MS analysis of aqueous methanol extract of Salvia bucharica revealed the presence of several compounds of which 49 were identi ed. The major compounds found in chemical analysis of Salvia bucharica were belonging to avonoid, phenol, alkaloids and tannins class. As shown in gure Methyl xanthine, glyceric acid, nonic acid, octadecenoic acid, araloside and tri methyluric acid were commonly identi ed in aqueous methanol extract of Salvia bucharica.

Discussion
Lipofundin is a fat emulsion which is used as an energy source in parentral nutrition, but experimental studies evidenced the development of atherosclerotic lesion, lipid peroxidation and oxidative stress in rabbits [29,30]. In the present investigation, it was observed that lipofundin caused signi cant increase in total cholesterol and triglyceride level (86.6 ± 4.40 and 118.33 ± 7.26 mg/dL respectively) in rabbits after intravenous administration for 8 days. In order to assess the possible effect of S. buchrica on lipid pro le, aqueous methanol extract in doses of 250 and 500 mg/kg was administered in lipofundin treated hyperlipidemic rabbits. 500 mg/kg dose of extract produced signi cant (p<0.001) reduction in level of triglycerides, total cholesterol and LDL as compared to lipofundin treated negative control group (Table   3.1, Fig.3.1). It is well known that elevated total cholesterol level plays major role in development of coronary artery diseases [31] and triglycerides play key role in regulation of lipoprotein interaction thus maintaining metabolism of lipids [32]; Hence, attenuation of these factors by extract may contribute to the possible antihyperlipidemic potential of Salvia bucharica which is in agreement with the previous ndings [33]. Formerly, it has been established on the basis of clinical and animal studies that chronic administration of fructose contributes to the development of various abnormalities such as dyslipidemia, hypertension, insulin resistance, oxidative stress and decreased glucose utilization [34]. Present study demonstrated that administration of fructose (25% w/v) in drinking water for 28 days provoked marked increase (p<0.001) in the level of TC, TG and VLDL, which is an indication of development of hyperlipidemia. Further, the results showed that fructose treatment also caused endothelium dysfunction in aorta of treated rats [35].
Subsequently administration of aqueous methanol extract of SB remarkably decreased the levels of lipid pro le in serum of treated animals indicating antihyperlipidemic potential, however, the HDL-C level remains unaffected. This valuable effect might be related to enhanced activity of endothelium bound lipoprotein lipase which is involved in hydrolysis of triglycerides to fatty acids [36] or by inhibition of pancreatic lipase as previously, it has been reported that high fructose diet increases serum pancreatic lipase activity, which is involved in cleavage of dietary triglycerides and thus leads to digestion of lipids [16]. Therefore, inhibition of pancreatic lipase can cause reduction in fat absorption hence, cholesterol level in blood could be decreased. LC-MS analysis of S. bucharica evident the presence of arasolide, derivative of oleanolic acid, Somova and its colleagues [37] had established the antihyperlipidemic effect of olenolic acid therefore, it could be assumed that lipid lowering effect offered by S. bucharica may at least be partially linked to the presence of araloside. Further, Lipoic acid derivative, 3,3-Dimethyl-1,2dithiolane, has been identi ed as an active constituent of S. bucharica and former reports [38] evinced the noticeable cholesterol lowering effect exerted by lipoic acid in rabbits, in addition dithiolane also improves the insulin sensitivity and reduced serum triglyceride level [39]. Therefore, it could be deduced that S. bucharica mediated antihyperlipidemic effect observed in present study may be due to the presence of these active principles. Cholestrol lowering effect exerted by S. bucharica could be attributed to the presence of saponins and methylxanthines as previous studies have demonstrated the hypocholestrolemic and lipid lowering properties of saponins [40] and methylxanthines [41].
Literature has shown that terpenes have potential to decrease the activity of HMG-COA reductase thus limiting the synthesis of cholesterol [42], as Salvadiol; a terpenoid is active constituent of S. bucharica [7]. Hence, the aforementioned ndings suggest that antihyperlipidemic effect of extract might be linked with the presence of terpenes.
To provide the further basis for antihyperlipidemic effect of SB, vascular reactivity to various concentrations of acetylcholine (10 -9 -10 -4 mol/L) was studied in rat thoracic aorta. Aqueous methanol extract obtained from aerial parts of Salvia bucharica potentially prevented the loss of endothelium functional integrity that was signi cantly compromised in fructose treated rat thoracic aorta. As endothelial dysfunction endorses dyslipidemia, atherosclerosis and hypertension, so, in the light of present ndings, it can therefore be deduced, that Salvia bucharica produced antihperlipidemic effect which might be attributed to multiple possible pathways including anticipation of endothelial dysfunction.
Additionally, it is popularly known that chronic administration of fructose culminates in fat deposition in hepatocytes and increased liver weight [43]. Hence, to evaluate the possible protective effect of Salvia bucharica, histopathological studies of liver tissues were performed. The result presented less alteration in hepatic vein and sinosidal spaces were examined in extract treated groups, when compared to fructose treated group re ecting protective role of Salvia bucharica on hepatocytes. Moreover, observation of hepatocytes clearly demonstrated that size of hepatocytes and central vein in Salvia bucharica treated rats was equivalent to normal rats and was also comparable to standard treated group.
It is evident from recorded results that atherogenic index (AI) and coronary risk index (CRI) was signi cantly ameliorated in both models (lipofundin and fructose induced hyperlipidemia) by treatment of aqueous methanol extract of Salvia bucharica. Atherogenic index being an important indicator of coronary risk is of great consideration [44].
Reactive oxygen species are involved in oxidation of low density lipoprotein, major contributor in endothelial dysfunction [5].The signi cant nding of present work is the antioxidant effect of S. bucharica. The results of DPPH radical scavenging assay predicted that S. bucharica showed more pronounced effect as compared to ascorbic acid (Fig 7). Hence, it could be proposed from aforementioned ndings that antihyperlipidemic effect of Salvia bucharica might be attributed to the presence of terpenes and free radical scavenging potential of plant.
To assess safety pro le aqueous methanol extract of S. bucharica was administered in mice, ndings of acute toxicity study evinced that LD 50 of extract is above 5000 mg/kg, which makes the extract a candidate of class 5 that is lowest toxic. Further results of subacute toxicity study showed no toxic effects thus referring the extract to be quite probably safe. Aqueous methanol extract obtained from aerial parts of Salvia bucharica M. Pop possesses antihyperlipidemic activity in the experimental animals at the dose of 500 mg/kg. It signi cantly reduced lipid pro le (p<0.05) in the lipofundin and fructose induced hyperlipidemia. Furthermore, in fructose fed rats, studies on the isolated aorta showed the vascular protective activity and histopathological examination displayed that this extract has hepatoprotective potential. Toxicity studies demonstrated that this plant extract is safer upto 5000 mg/kg and no sign of morbidity shown. However the results are too much encouraging but further research work is required for scienti c validation and to isolate the compound for further studies.

Conclusion
It is concluded that Salvia bucharica has shown dose dependent antihyperlipidemic activity .In addition, Studies performed on the isolated thoracic aorta and histolopathological studies on liver demonstrated that Salvia bucharica has endothelial dependent vasoprotective and hepatoprotective activity respectively as compared to fructose fed rats. Furthermore, the plant has been found to be safe in toxicity studies.     Free radical scavenging activity of Salvia bucharica against DPPH minimumstandardsofreportingcheklist.pdf