Ameliorative effects of Saussurea lappa against hypertension and anxiety in animal models

Preparation of extract

The fully dried roots were procured from the local market and ground into a coarse powder. This powder was then sequentially extracted with 70% ethanol using a Soxhlet apparatus for 3–4 days. The resulting extract was collected and concentrated using a rotary evaporator, operating at the boiling point of ethanol (68°C) and a rotational speed of 89 RPM, for 40–50 min.^[9]

Estimation of extracted plant material

The resultant plant extract was subsequently desiccated using a heated water bath, and the percentage yield for the plant was determined through the following equation, yielding a value of 14.72% w/w.^[9]

The equation utilised to compute the percentage yield is provided here as:

Percentage yield = Theoretical yield/Practical Yield × 100

The percentage yield of S. lappa = weight of leaves extract (g)/weight of dried leaves powder (g) × 100

% yield = 3.24/22 × 100.

The extract yield derived from S. lappa was determined to be 14.72% w/w.

Phytochemical screening

The coarsely powdered roots of S. lappa were extracted using various solvents such as chloroform, methanol, hexane, dichloromethane, ethanol and water for phytochemical examination. The objective of this process was to identify the most effective solvent for further extraction using a Soxhlet apparatus. The ideal solvent would be the one in which the maximum number of phytoconstituents could be detected. To achieve this, 0.5 g of the powder was soaked in 5 mL of each solvent (chloroform, methanol, hexane, dichloromethane, ethanol and water) for 24 h. The mixtures were then filtered and subjected to various phytochemical tests [Table 1].^[10-12]

Choice of solvent

Ethanol was chosen for extraction because it is a popular solvent for plant extraction as it can efficiently extract both polar and non-polar compounds, is safe, easy to handle and widely available. It dissolves various compounds needed for research, such as alkaloids, glycosides, tannins and terpenoids, which have potent biological activities.^[7,11]

Experimental animals

24 Swiss albino mice (20–40 g) of either sex were randomly chosen for the anti-anxiety and 24 Wistar albino rats (150–200 g) of either sex were randomly chosen for the anti-hypertensive activity and assembled into 4 groups (n = 6). They were acclimatised and housed in an animal house with a 12 h:12 h light-dull cycle at 27 ± 2°C temperature and 45–55% relative humidity. Sustenance and water are very important. This study did not involve human participants; therefore, approval from the Institutional Review Board (IRB) was not required. Creatures were arbitrarily partitioned into four groups, for example control, standard, low section of test and high section of test. Gathering 1 was considered as control and treated with superior water.^[13,14] Gathering 2 treated as standard, gatherings 3 treated as test drug (S. lappa), which is low dose (200 mg/kg) and gatherings 4 treated as test drug (S. lappa), which is high dose (400 mg/kg.^[3]

Elevated plus maze model

The below context illustrates the average count of entries and the duration of time spent in both open and closed arms subsequent to drug administration. The findings demonstrated a notable increase in the number of entries and time spent in the open arms among the groups treated with the extract, comparable to those treated with traditional anxiolytic medications. The administration of S. lappa ethanolic extract (SLEE), at doses of 200 mg/kg and 400 mg/kg, significantly augmented the count of entries and the duration of time mice spent in the open arms.^[15] Conversely, mice treated with the standard medication diazepam exhibited a considerably higher (P < 0.001) total number of entries in the open arms and longer periods spent in the open arms, while allocating less time in the closed arms.^[16,17]

The mean duration of time spent in the open arms showed a significant increase in the groups receiving the extract, as compared to the control group treated with the vehicle [Table 2]. In the control group, the average duration was 128.5 ± 6.71 s, whereas in the groups treated with S. lappa at doses of 200 mg/kg and 400 mg/kg, the durations were 186.8 ± 29.2 s and 238.1 ± 16.3 s, respectively [Figure 1]. Similarly, the average number of entries into the open arms was higher in the extract-treated groups compared to their respective control groups. In the control group, the average number of entries was 4.83 ± 2.31, while in the groups treated with S. lappa at doses of 200 mg/kg and 400 mg/kg, the numbers were 11.8 ± 3.97 and 14.3 ± 3.50, respectively [Figure 2].

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Figure 1:

Impact of Saussurea lappa (200 and 400 mg/kg) as evaluated by the elevated plus maze model. The graphical representation demonstrated a noteworthy rise in the average duration of time spent in the open arms within the groups receiving the extract, in comparison to the control group treated with the vehicle. In the control group, the average duration was 128.5 ± 6.71 s, whereas in the groups treated with S. lappa at doses of 200 mg/kg and 400 mg/kg, the durations were 186.8 ± 29.2 s and 238.1 ± 16.3 s, respectively. Data are expressed as mean ± SEM (n = X). *P < 0.05, **P < 0.01, ***P < 0.001 compared with control.

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Figure 2:

Impact of Saussurea lappa (200 and 400 mg/kg) as assessed by the elevated plus maze model. The graphical representation exhibited that the extract-treated groups demonstrated a greater average number of entries into the open arms compared to their respective control groups. In the control group, the average number of entries was 4.83 ± 2.31, while in the groups treated with S. lappa at doses of 200 mg/kg and 400 mg/kg, the numbers were 11.8 ± 3.97 and 14.3 ± 3.50, respectively. Data are expressed as mean ± SEM (n = X). ***P < 0.01, ***P < 0.001 compared with control.

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Anti-hypertensive activity

Numbers below display the measurements of several parameters in rats subjected to different treatments: Control – high salt diet (HSD), atenolol (standard) and both doses of S. lappa root extract. The findings in Figure 3 indicate that salt-induced procedure significantly elevated mean arterial blood pressure (MABP), diastolic blood pressure (DBP) and systolic blood pressure (SBP) compared to the vehicle group. In contrast, the treatments with atenolol, SLEE 200 mg/kg and SLEE 400 mg/kg significantly reduced MABP, DBP and SBP levels as compared to the salt group.^[15,18,19] In addition, the HSD group’s heart rate (HR) was substantially higher than the control group. HR did not change significantly between the atenolol, both SLEE treatment groups and the high salt-induced group.^[20,21] These findings indicate that in rats with high salt induced hypertension, therapy with both atenolol and SLEE has been shown to lower blood pressure levels.^[11,22] The provided findings [Table 3] demonstrated that the SLEE 400 mg/kg dose was significantly more effective in treating hypertension than the 200 mg/kg dose and had an impact equivalent to the standard medicine atenolol. These data clearly showed that the root extract of S. lappa has considerable anti-hypertensive properties.^[23]

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Figure 3:

Impact of Saussurea lappa (200 and 400 mg/kg) as evaluated by the BIOPAC apparatus model. The graphical representation demonstrated a noteworthy decrease in blood pressure parameters (MAP, SBP, DBP) and heart rate (HR) in the groups receiving the extract, in comparison to the control group treated with the vehicle (HSD). Atenolol (10 mg/kg) served as the standard drug. Data are expressed as mean ± SEM (n = X). **p<0.01, ***p<0.001, ****p<0.0001 compared with control.

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Histopathological examination of heart

The consumption of a diet high been associated with hypertension, metabolic disorders and cognitive decline.^[24,25] These dietary factors can lead to dysregulated lipid metabolism, insulin resistance and increased production of reactive oxygen species within the heart. In the context of hypertension, the elevated blood pressure exerts increased workload on the heart, leading to hypertrophy of cardiac myocytes. This pathological cardiac remodelling can result in structural changes [Figure 4] such as cardiomyocyte hypertrophy, interstitial fibrosis and alterations in the extracellular matrix.^[26] The chronic inflammatory state triggered by high fructose intake further exacerbates cardiac injury. Inflammation within the heart can be initiated by various factors, including oxidative stress, immune cell infiltration and the release of pro-inflammatory cytokines.^[27]

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Figure 4:

Histopathology of rat heart. This figure provides a clear visual representation of the observed differences among the groups. With (a) standard drug (Atenolol), (b) the one with high salt diet, (c-d) both the doses of Saussurea lappa extract. The groups subjected to the extract treatment display intermediate outcomes, positioned between the normal control and disease control groups. Histologically, most of the cardiac tissue appeared normal. Cardiac myocytes and endocardium appeared normal. Few congested vessels are seen in fructose induced group. Overall, no inflammation or any degenerative lesion were seen.

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