Hypertension, a chronic condition characterized by a systolic blood pressure (SBP) of ≥ 140 mmHg and/or a diastolic blood pressure (DBP) of ≥ 90 mmHg [1], significantly increases the risk of heart attack, stroke, kidney failure, and blindness, and causes premature death [2]. Uncontrolled blood pressure is one of the most critical human health concerns worldwide, not just in high-income regions like central and Eastern Europe, but also in low-income countries of South Asia and sub-Saharan Africa [3, 4]. Hypertension treatment follows guidelines from the Joint National Committee 8, which recommends angiotensin-converting enzyme inhibitors or angiotensin receptor blockers as first-line therapy, and diuretics or calcium channel blockers as an alternative [5].

Several traditional medicinal herbs and natural products, such as ma huang, St. John’s wort, yohimbine, garlic, and licorice, are being investigated as alternatives for hypertension management [6, 7]. An elegant study by Kamyab et al. [8] found that numerous plants are used for empirical hypertension therapy worldwide, including plants from the Asteraceae, Liliaceae, Apiaceae, Rosaceae, and Apocynaceae families. Because of their advantages over standard antihypertensive drugs, such as fewer unwanted side effects, cost-effectiveness, and the easier availability of medicinal plants, herbal remedies are being incorporated into the clinical treatment of hypertension through evidence-based medicine [9].

Cucumber (Cucumis sativus L. [C. sativus]), which has been used empirically for cardiovascular disease therapy, belongs to the family Cucurbitaceae and contains cucurbitacin as the major metabolite. A previous study reported that cucurbitacin B from Cucumis melo L. has hypotensive effects [10]. Lagenaria siceraria (Molina) Standl. Fruit, which belongs to the Cucurbitaceae family, has also been reported to have hypotensive effects [11]. In vitro analysis indicates that the aqueous fraction of cucumber inhibits angiotensin II (Ang II)-induced oxidative stress, which may have hypotensive effects [12]. Cucumis sativus (C. sativus) has also been studied for its moderate diuretic activity [13]. However, evidence supporting cucumber use for hypertension management is lacking, and its effects in an Ang II-induced model of hypertension have not been evaluated. Moreover, using herbs in combination with standard antihypertensive agents may positively or negatively impact the effect of standard medication [14]. This study investigated the dose-dependent antihypertensive effects of C. sativus alone or when combined with losartan in an Ang II-induced rat model of hypertension.

1. Plant material sourcing and processing

C. sativus (CS) plant samples were obtained from Desa Cimekar, Bandung, West Java, and identified at Herbarium Bandungense School of Life Sciences and Technology, Bandung Institute of Technology (coordinate: –6.931720432222963, 107.77177718067999). Concentrated CS fruit juice was prepared through the following steps: (a) the fruits were washed, (b) the clean fruits were juiced using a juicer without adding water, (c) the juice was filtered, (d) the filtrate was freeze-dried using an EYELA^® freeze dryer FD-5N, (e) the freeze-dried juice was stored at –20℃. The plant concentrate yield was 3% (w/w).

2. Drugs and chemicals

Losartan potassium (Los) was purchased from PT Kalbe Farma. The human Ang II used to induce hypertension was purchased from Sigma–Aldrich (product no. A9525, CAS no. 4474-91-3). All other chemicals used for experimental purposes were of analytical grade.

3. Preliminary phytochemical screening

Phytochemicals are bioactive plant constituents, such as flavonoids, saponins, and terpenoids. To determine the presence of bioactive components, a preliminary phytochemical screening was performed as previously reported [15].

4. Determination of calcium, potassium, and zinc

One gram of CS was burned to ashes in a muffle furnace at 500℃. The ash was then put in a volumetric flask, mixed with 0.1 N HNO₃ to a final volume of 25 mL, and then filtered using Whatman paper no. 42 before further examination. A calibration curve for calcium, potassium, and zinc determination was generated using calcium carbonate, potassium chloride, and zinc oxide as standards, respectively, using atomic absorbance spectrometry (Agilent^® Spectr-AA variant 55B, 422.7 nm). At least three readings were obtained and the results were presented as average values.

5. Ethics approval

Ethical approval for the use of laboratory animals was granted by the animal research ethics committee of Bandung Institute of Technology (ethics certificate No. 03/KEPHP-ITB/10-2019).

6. Animals

The animal facilities and protocols complied with the standards for the care and use of experimental animals. Adult male Wistar rats weighing 225 ± 25 g were housed in standard laboratory conditions under a 12:12-hour light–dark cycle, with free air circulation, at 25 ± 2℃ and 65 ± 10% humidity. Husk replacement was done every 2-3 days. The rats were fed standard animal feed with free access to water. The animals were acclimatized to laboratory conditions for seven days and fasted with free access to water for 12 hours before each experiment.

7. Evaluation of the antihypertensive activity of CS

The antihypertensive activity of CS was evaluated as previously described [16, 17]. Briefly, Ang II was used to induce blood pressure (BP) elevation in rats. For this, the animals (four per group) were randomly assigned to the following groups: 1) Group 1 (normal control), which received the vehicle (0.5% Na CMC solution), 2) Group 2, which received the vehicle and Ang II, 3) Groups 3-6, which received CS at 9, 18, 27, and 36 mg/kg, respectively, along with Ang II, 4) Groups 7 and 8, which received Los at 2.25 mg/kg in combination with CS at 9 and 18 mg/kg, respectively, along with Ang II, and 5) Groups 9 and 10, which were treated with Los at 2.25 and 4.5 mg/kg, respectively, along with Ang II. CS and losartan treatments lasted for four weeks. Ang II induction and BP measurements were done on the 28^th day of the experiment.

All rats, except the normal control (Group 1), underwent hypertension induction via intraperitoneal Ang II (100 µg/kg) injection 15 minutes after administering CS or Los. SBP and DBP readings were taken on conscious rats 15 minutes before induction, immediately after induction (0), and 15, 30, 60, 90, and 120 minutes after induction, using a CODA^® Noninvasive Blood Pressure System (Kent Scientific Corporation, USA) according to the manufacturer’s instructions. Five measurements were taken per rat and expressed as average readings.

8. Statistical analyses

Data are presented as mean ± standard deviation. Differences between multiple groups were compared using one-way analysis of variance (ANOVA) followed by the LSD test, using SPSS version 27.0. p < 0.05 indicated statistically significant differences.

1. Phytochemical screening

Flavonoids, saponins, and terpenoids were detected in CS (Table 1).

Table 1

The result of phytochemical screening of C. sativus.

Phytochemical components	Result	Observation
Flavonoid	+	Pink solution
Saponin	+	Foam stable for 10 minutes
Terpenoid	+	Orange solution

2. Calcium, potassium, and zinc content

Calcium, potassium, and zinc levels in CS were 16.65 ± 0.09 µg, 2.01 ± 0.17 mg, and 0.30 ± 0.03 µg per gram of dried fruit, respectively.

3. The effect of CS fruit on BP

Intraperitoneally injecting male Wistar rats with Ang II (100 µg/kg) increased their BP when compared with the normal controls, with the optimal increase occurring within 30 minutes of induction (SBP: 117.5 ± 2.4 vs 155.3 ± 5.9 mmHg in the normal control vs Ang II groups; DBP: 75.0 ± 0.8 vs 102.3 ± 2.8 mmHg in the normal control vs Ang II groups; p < 0.05, Fig. 1). The BP returned to baseline 120 minutes after induction.

Figure 1. Effects of systemic angiotensin II injection on blood pressure. Angiotensin II was administered at 100 ug/kg i.p. at 15 minutes after administration of C. sativus, losartan, or sodium-CMC (vehicle). (A) systolic blood pressure; (B) diastolic blood pressure. The red arrow represents time of angiotensin II injection, the same time for recording at time zero. *p < 0.05 C. sativus or losartan + Ang II vs. Ang II. #p < 0.05 Ang II vs. normal control.

BP monitoring revealed that CS (9, 18, 27, and 36 mg/kg) significantly lowered the Ang II-induced BP elevation (Fig. 2A, B). Thirty minutes after hypertension induction, the average SBP change (Δ), when compared with time zero in the groups that received CS + Ang II (CS doses: 9, 18, 27, and 36 mg/kg), were 28.8, 24.8, 22.8, and 11.5 mmHg, respectively (Fig. 2A), whereas the DBP changes were 18.3, 15.8, 15.5, and 9.0 mmHg, respectively (Fig. 2B). From 30 minutes after induction to the end of the treatment period, all groups treated with CS + Ang II exhibited a significant (p < 0.05) progressive, dose-dependent, SBP and DBP decline when compared with the Ang II group.

Figure 2. Attenuation effects of C. sativus (9, 18, 27, 36 mg/kg) and losartan on angiotensin II-induced elevation of blood pressure in rats. C. sativus, losartan, or sodium-CMC (vehicle) was administered 15 minutes prior to induction of angiotensin II (100 ug/kg, i.p). Time of angiotensin II injection was set for recording at time zero. (A) present change of systolic blood pressure from the time of observation to time zero; (B) present change of diastolic blood pressure from the time of observation to time zero. *p < 0.05 C. sativus or losartan + Ang II vs. Ang II. #p < 0.05 Ang II vs. normal control.

4. The effect of losartan-CS cotreatment on BP

The effects of various doses of CS, Los, and their combination on BP are shown in Figs. 3, 4. Thirty minutes after hypertension induction, the average SBP Δs when compared with time zero in the groups that received CS at 9 and 18 mg/kg, Los at 2.25 mg/kg, a combination of CS (9 mg/kg) + Los (2.25 mg/kg), and Los at 4.5 mg/kg along with Ang II, were 28.8, 24.8, 17.0, 13.8, and 6.5 mmHg, respectively. At 120 minutes of treatment, the SBP Δs were 6.3, 6.0, 0.8, 1.5, and 0.3, respectively (Fig. 3A). After the same treatments, DBP Δs were 18.3, 15.8, 13.8, 13.3, and 7.8 mmHg after 30 minutes and 6.0, 4.3, 3.0, 2.5 and 0.5 mmHg after 120 minutes (Fig. 3B). The BP Δs in the groups that received half the CS dose + Los was comparable to that of the full CS dose.

Figure 3. Effects of C. sativus (9 and 18 mg/kg), losartan (2.25 and 4.5 mg/kg), and combination C. sativus (9 mg/kg) + losartan (2.25 mg/kg) on angiotensin II-induced hypertensive rats. C. sativus, losartan, or their combination was administered 15 minutes prior to induction of angiotensin II (100 ug/kg, i.p). The red arrow represents time of angiotensin II injection and was set for recording at time zero. (A) systolic blood pressure; (B) diastolic blood pressure.

Figure 4. Effects of C. sativus (18 and 36 mg/kg), losartan (2.25 and 4.5 mg/kg), and combination C. sativus 18 mg/kg + losartan 2.25 mg/kg on angiotensin II-induced hypertensive rats. C. sativus, losartan, or their combination was administered 15 minutes prior to induction of angiotensin II (100 ug/kg, i.p). The red arrow represents time of angiotensin II injection and was set for recording at time zero. (A) systolic blood pressure; (B) diastolic blood pressure. *p < 0.05 combination of C. sativus 18 mg/kg + losartan 2.25 mg/kg + Ang II vs. C.sativus 36 mg/kg + Ang II.

At 30 minutes after hypertension induction, the average SBP Δ when compared with time zero in the groups that received CS at 18 and 36 mg/kg, Los at 2.25 mg/kg, a combination of CS at 18 mg/kg + Los at 2.25 mg/kg, and Los at 4.5 mg/kg along with Ang II, were 24.8, 11.5, 17.0, 8.8, and 6.5 mmHg, respectively (Fig. 4). At 120 minutes, the SBP Δs were 6.0, 2.0, 0.8, 0.5, and 0.3 mmHg, respectively (Fig. 4A). In the same conditions, DBP Δs at 30 and 120 minutes after hypertension induction were 15.8, 9.0, 13.8, 9.0, and 7.8 mmHg and 4.3, 0.8, 3.0, 1.0, and 0.5 mmHg, respectively (Fig. 3B). The BP Δ in rats that received half the dose of the herb-drug combination (CS 18 mg/kg + Los 2.25 mg/kg along with Ang II) was significantly different when compared with treatment using the full CS dose (36 mg/kg) + Ang II induction (p < 0.05) but insignificantly different when compared with the full Los dose 4.5 mg/kg + Ang II.

In this study, we find that Ang II significantly elevates SBP and DBP in rats when compared with the normal control group (p < 0.05), indicating that Ang II successfully induced hypertension. Ang II plays an important role in maintaining equilibrium and the homeostatic regulation of BP. In addition to its vasoconstrictor activity, Ang II is suggested to increase peripheral vascular resistance through increased sympathetic nervous system activity, increased prostaglandin concentration (which modulates vasoconstriction), and decreased nitric oxide (NO) bioactivity via reactive oxygen species formation (which have oxidant activity and induce oxidative stress, causing vasoconstriction) [18-22].

Losartan, an anti-hypertension medication, blocks angiotensin II receptor type 1 in the renin–angiotensin system [23]. Here, we find that pretreatment with losartan significantly diminished the effect of Ang II on BP.

Cucumis sativus (C. sativus) has for a long time been widely used not only as a vegetable but also in traditional medicine. There is compelling evidence that C. sativus has numerous pharmacological activities, such as antibacterial, anticancer, antioxidant, and analgesic effects [24-26]. This study investigated the antihypertensive effects of C. sativus in a rat model of hypertension. Our findings indicate that pretreatment with C. sativus reduced Ang II-induced BP elevation dose-dependently. Throughout the experiments, the SBPs and DBPs of the rats that received the vehicle and Ang II (Group 2) remained significantly higher (p < 0.05) when compared with the C. sativus-treated groups (Groups 3-6). Significant differences in BPs (when compared with Group 2) were observed from 30 minutes after induction, onwards. However, longer observation was not feasible because, after two hours of induction, the BP fell to levels similar to those of the normal control group (Group 1).

The antihypertensive effects of C. sativus can be attributed to its possession of major compounds and its mineral content. Phytochemical analysis showed that our C. sativus sample contained major compounds, including flavonoids, saponins, and terpenoids, which is consistent with previous reports that flavonoids like kaempferol, quercetin, and cucurbitacin have been isolated from this plant [27]. Flavonoids are been reported to have biological effects, including antioxidant, vasorelaxant, and antihypertensive activities [28, 29]. Kaempferol mediates an antihypertensive effect by inducing endothelium-dependent vasorelaxation via the NO–cGMP–PKG signaling pathway, causing vasodilation [30]. Quercetin, the prototypical, abundant dietary flavonoid, has been shown to significantly reduce BP in clinical trials [31]. Cucurbitacin is structurally a terpene and is found in many plants of the Cucurbitaceae family. It exerts hypotensive effects through the suppression of Ang II-induced SBP increase and induction of acetylcholine-mediated vasodilatation in mesenteric arteries [10]. The triterpene, saponin, is suggested to exert antihypertensive activity by activating calcium-activated potassium channels in vascular smooth cells, reducing calcium influx and vasodilation [32].

C. sativus also has a high mineral content and our findings show that its fruit contains calcium, potassium, and zinc. Consistent with this finding, Niyi et al. [33] found that cucumber contains several minerals, including Ca, Cu, Fe, K, Mg, Na, and Zn. Calcium can reduce intracellular calcium by inhibiting parathormone, thereby reducing BP. Potassium can increase sodium excretion, lower renin secretion, facilitate arterial vasodilation, and decrease response to endogenous vasoconstrictors. Magnesium is reported to exert strong vasodilator activity via its ability to decrease vascular smooth muscle contractility [34]. Zinc contributes to nitric oxide synthase activity, which is critical for NO synthesis [35, 36]. NO can diffuse into adjacent smooth muscles and bind to the heme moiety of cytosolic guanylate cyclase, thereby elevating guanosine monophosphate levels, which promotes vasodilatation [37, 38]

The significantly stronger BP-lowering effect observed in the group treated with half the C. sativus-losartan combination dose when compared with the group treated with the full C. sativus dose, and its comparability with the effect of full dose losartan, suggests that the effects of the combination were additive. Additive effects occur when the combined effects of compounds are similar to the sum of the compounds’ independent effects [39].

Here, we show that the C. sativus fruit dose-dependently prevents BP elevation. Our data indicate that when administered together with losartan, C. sativus can have an additive BP-lowering effect. Mechanistically, the blood pressure-lowering effect is likely to be mediated by the action of C. sativus’ active ingredients on the renin–angiotensin–aldosterone pathway, which warrants further study.

The authors would like to thank Kusnandar Anggadiredja for critically reading the manuscript and all members of the Pharmacology lab for fruitful discussions. We highly appreciate the kind assistance of staff at the School of Pharmacy ITB in conducting the research.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.