Bioactive Compounds from Cuphea glutinosa Cham. & Schltdl.: Promising Effects on Antihypertensive Action

Abstract

Cuphea glutinosa Cham. & Schltdl. (Lythraceae) is popularly used as antimicrobial, diuretic, anti-inflammatory, and antihypertensive. This species features a great diversity of flavonoids, predominantly quercetin glycosides. This study aimed to investigate the chemical composition, and acute antihypertensive effect in vivo from C. glutinosa leaf extracts on N(G)-nitro-L-Arginine methyl ester (L-NAME) induced hypertensive rats. The leaf extraction was conducted by exhaustive maceration and infusion. The chromatographic analyses were performed by ultra-fast liquid chromatography and photodiode array detection (UFLC-PDA). The assessment of antihypertensive activity in vivo was performed through the monitoring of the hemodynamic parameters. C. glutinosa shows a predominance of quercetin glycosides, majority the miquelianin. The extracts displayed an antihypertensive effect in rats in doses of 2.5 to 50 mg/kg. These results suggest that the C. glutinosa extracts present antihypertensive potential by the presence of flavonoids and interactions among the phytoconstituents. This research demonstrates that the extracts of C. glutinosa are flavonoid-rich and ratify the folk medicine usage of C. glutinosa as hypotensive and antihypertensive agents, in order to contribute to evidence-based traditional medicines.

Keywords:
Cuphea glutinosa; UFLC-PDA; miquelianin; hemodynamic parameters; antihypertensive action

GRAPHICAL ABSTRACT

HIGHLIGHTS

Bioactive compounds from Cuphea glutinosa: promising antihypertensive effect

Antihypertensive effect, in vivo, of Cuphea glutinosa in hypertensive rats

Acute antihypertensive effect, in vivo, from Cuphea glutinosa leaves

Cuphea glutinosa antihypertensive effect as a therapeutic alternative

INTRODUCTION

Hypertension is a serious disease that presents relevant cardiovascular risk factors, besides being responsible for the high mortality rate globally [¹-²]. Blood pressure (BP) can be affected by many factors, such as lifestyle and genetic traits, therefore new alternatives are needed to treat hypertension [³]. Moreover, many drugs used to treat BP still cause several side effects, leading to the search for other alternatives with fewer adverse effects. In this context, research indicates that natural products, such as herbal medicinal, are among the most important sources of new future drugs [⁴].

Cuphea genus (Lythraceae) includes around 260 species, mainly spread in North and South America [⁵-⁶]. Studies performed on phytochemicals are reported in Cuphea species, where a great diversity of flavonoid structures in these plant extracts observed predominantly quercetin glycosides. Cuphea glutinosa is popularly known in Brazil as "sete-sangrias" and is employed commonly in folk medicine as anti-inflammatory, diuretic, and antihypertensive [⁷-¹¹]. Additionally, Santos and coauthors [¹⁰] reported the presence of phenolic compounds, primarily flavonoids (quercetin derivatives), besides potential in the angiotensin-converting enzyme (ACE)-inhibition from C. glutinosa extracts. In addition, according to Teixeira and coauthors [¹²] in a review of studies on the popular use of medicinal plants, the C. glutinosa was assigned to antihypertensive effects.

Natural products have been used substantially in drug discovery efforts. On the other hand, there are still many resources to probe in natural product research. Therefore, the present study aims to investigate the chemical composition, and, in vivo the acute antihypertensive effect of leaf extracts from Cuphea glutinosa Cham. & Schltdl. on N(G)-nitro-L-Arginine methyl ester (L-NAME) induced hypertensive rats.

MATERIAL AND METHODS

Chemicals and reagents

N(G)-nitro-L-Arginine methyl ester (L-NAME) and urethane were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile and methanol were purchased from Tedia (Fairfield, OH, USA). Formic acid was purchased from Merck (Darmstadt, Germany). Purified water was obtained using the Milli-Q Plus® system from Millipore (Milford, MA, USA).

Plant material

The plant material was collected in August 2013, at Uruguaiana city, Rio Grande do Sul state, Brazil (S 29°39'3.0'' W 56º48'24.3''). The species was identified as Cuphea glutinosa Cham & Schltdl by Dr. Neves P.O. and Dr. Schneider A. of the Herbarium of the Federal University of Pampa and issued a voucher specimen herbarium number 168.

Extracts and samples preparation

The leaves were dried at 40°C for 5 days. Exhaustive maceration was performed using particle size ≤180 µm; 40% ethanol as solvent; plant:solvent ratio 1:10 (w/v); for 10 days with two solvent renovations. The infusion was performed using particle size ≤180 µm; 100% distilled water as solvent; plant:solvent ratio 1:20 (w/v) and to a temperature of 80°C. After extraction, the samples were filtered and lyophilized [⁷]. The two different techniques used permit observing which one extracts the higher concentration of active compounds and compares them.

For the in vivo assay, the lyophilized extracts were dissolved in 0.9% saline solution in a concentration of 500 mg/mL and filtered through a 0.22 µm filter for administration to animals. All samples of the lyophilized extracts were administered in cumulative doses ranging from 2.5 to 50 mg/kg.

Instrumental analysis

The maceration (40% ethanol) and infusion (100% water) extract chromatographic analyses, at a concentration of 10 mg/mL, were carried out in UFLC-PDA Prominence^®, equipped with LC‐20 AD pumps, SIL‐20 AC HT autosampler, degasser DGU‐20A3, a CTO‐20 AC column oven, and SPD‐M20A PDA detector (Shimadzu, Kyoto, Japan). For the equipment control and data analyses, the LC Solution V. 1.24 SP1 software was used.

The chromatographic conditions used were described according to Yang and coauthors [¹³] adapted to UFLC, using a reverse-phase system, in the conditions as follows: fast C18 analytical column Shim-pack XR-ODS column (50 x 2 mm, 2.1 µm); mobile phase consisted of a mixture of water containing 0.1% formic acid (pH 3.0 adjusted with triethylamine) as solvent A, and acetonitrile:methanol (4:1, v/v) as solvent B, prepared daily and filtered through a 0.22 μm membrane filter (Millipore), and at a flow rate of 0.2 mL/min. The column oven was operated at 25 ± 1°C, an injection volume of 5.0 µL, and detection at 340 nm. The gradient elution employed was 0-2.29 min 0% (B), 2.30-7.29 min 5% (B), 7.30-9.59 min 10% (B), 10.00-12.29 min 15% (B), 12.30-14.59 min 20% (B), 15.00-17.29 min 25% (B), 17.30-19.59 min 25% (B), 20.00-27.29 min 60% (B), 27.30-29.59 min 60% (B), 30.00-35.00 min 0% (B).

For identification of the major compound, the previously isolated compound was used for ESI-MS/MS analysis. The analysis conditions were according to Santos and coauthors [¹⁰,¹⁴], using an Ultra-High-Performance Liquid Chromatograph Nexera X2 (UHPLC, Shimadzu, Japan), on a micrOTOF-Q III (Bruker Daltonics, Bremen, Germany) detector, equipped with an ESI interface operating in positive ion mode.

Animals

Male Wistar rats with four months, weighing approximately 350 g, from the Federal University of Santa Maria were used. The animals were kept in boxes and under standard conditions: 22ºC, 12 h light-dark cycle, water, and commercial feed ad libitum. The experimental protocol began only after it had been approved by the Ethical Committee for Animal Use at Federal University of Pampa (Protocol 006/2013), and was performed according to the Brazilian Laws for the Scientific Use of Animals and International Guidelines for Use and Care of Laboratory Animals.

In vivo antihypertensive activity (Hemodynamic parameters)

The determination of blood pressure was by hemodynamic parameters measuring. The rats were anesthetized with urethane (1.2 g/kg, intraperitoneally (i.p.)), which was evaluated by the response to painful stimuli and supplemented when necessary [¹⁵]. After they were submitted to surgery of catheterization of the carotid artery (to monitor the hemodynamic parameters) with polyethylene catheters (PE10 Clay-Adams) completed with 50 IU/mL of heparinized saline. This catheter was linked to a pressure transducer coupled to an analog-digital converter (Biopac Systems MP150, Inc.; CA).

After stabilization of 30 minutes, to induce hypertension L-NAME (nitric oxide (NO) synthesis inhibitor) was administered intravenously at a dose of 30mg/kg. For the appraisal of the extract efficiency on hemodynamic parameters, an ascendant curve was performed in extract cumulative concentrations administered i.p., with a volume of 0.2mL of saline, where each dose was administered 30 minutes after the other.

Systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate (HR) were recorded throughout 3 hours. The select doses were established based on a preliminary curve with doses ranging from 1 to 100 mg/kg. Infusion extract was administered at doses of 10, 20, and 50 mg/kg, and maceration was administered at doses of 2.5, 5, 10, and 20 mg/kg. Animals were divided into 4 groups (𝑛 = 6): Control (normotensives) - Animals that received only the vehicle (0.9% NaCl); Control + L-NAME - Animals that received L-Name and vehicle (0.9% NaCl); L-NAME + Infusion - Animals that received L-Name and extract from the infusion of C. glutinosa leaves, and L-NAME + Maceration - Animals that received L-Name and extract from the maceration of C. glutinosa leaves.

Statistical Analysis

All values were expressed as the mean ± standard deviation (SD), and results were analyzed by one-way ANOVA for repeated measures followed by Tukey’s test. The significance level was set at 𝑝 < 0.05.

RESULTS AND DISCUSSION

Instrumental analysis

The major compound of C. glutinosa is miquelianin, a flavonol derivative of quercetin linked to glucuronic acid [⁷,⁸,¹⁰,¹⁶]. This compound shows up in 18.65 minutes and exhibit more abundance for maceration extract with a greater peak area. According to the literature and observed UV profile (PDA-detection), it is possible to suggest in Figures 1A and 1B the compounds following: isoquercetin (1), kaempferol-3-glucoside (3), miquelianin (4), quercetin-3-arabinoside (5), and quercitrin (6) for the maceration and infusion extracts.

In addition, the maceration extract composition showed quercetin-acetyl-glucuronide (2) and quercetin (7) compounds, which were described in previous studies for the C. glutinosa species [⁷,¹⁰]. Therefore, this composition difference may be related to solvent polarity because flavonoids have more affinity for polar solvents [¹⁷]. Additionally, Santos and coauthors [⁸] reported that ethanol extracts showed a higher concentration of miquelianin from C. glutinosa extracts, with a significant yield.

Extraction time and temperature, solvent, and composition of a sample are known as the important parameters to be evaluated, and the extraction efficiency can be affected by the chemical nature of phytochemicals, besides the yield of extraction depends on the solvent as the varying polarity. Therefore, differences observed in the concentration of the compounds might be ascribed to the different extraction methods, and solvents used [¹⁸,¹⁹].

Results showed that the solvent mixture employed in the extraction procedure had a major role in the concentration of phenolic compounds. These results indicate that the ethanol and water mixture enhances the concentration of the phenolic compounds present in the sample. According to Do and coauthors [¹⁸], the combined use of water and organic solvent may facilitate the extraction of chemicals that are soluble in water and/or organic solvent. This may be the reason why the concentration of phytochemicals in the extract obtained by maceration (40% ethanol).

Figure 1
Representative chromatogram from extracts at a concentration of 10 mg/mL, obtained in the analysis by UFLC-DAD (detection at 340 nm, and injection volume of 5.0 μL), and mass spectrum. A C. glutinosa infusion extract (100% water), and B C. glutinosa maceration extract (40% ethanol). Isoquercetin (1), quercetin-acetyl-glucuronide (2), kaempferol-3-glucoside (3), miquelianin (4), quercetin-3-arabinoside (5), quercitrin (6), and quercetin (7). C Mass spectrum, chemical structure, and fragmentation were suggested through analysis of the major compound from C. glutinosa using UHPLC-MS in positive ionization mode. (1) miquelianin, (2) quercetin ([M+H]+), and (3) glucuronic acid (fragment m/z 176).

Moreover, ESI-MS/MS analysis was performed to characterize the major compound of C. glutinosa isolated. The mass spectrum, chemical structure, and fragmentation of this compound, miquelianin named, can be seen in Figure 1C, with m/z 479 and the fragment at m/z 303 after the elimination of glucuronic acid (m/z 176). Studies by Santos and coauthors [⁷,¹⁰,¹⁶] describe the mass break of the glucuronic acid bound to quercetin and report the majority presence of flavonoids, mainly quercetin derivatives in the Cuphea species.

Furthermore, Santos and coauthors [⁸] optimized, and validated an ultra-performance liquid chromatographic method for the quantification of miquelianin in C. glutinosa leaves, using analytical standard miquelianin which showed the same UV maximum values of the sample and, the addition of standard in sample solution resulted in increment only in the miquelianin peak area.

In vivo antihypertensive activity (Hemodynamic parameters)

Primarily, the animals demonstrated a BP increase of around 30% after the L-NAME injection, and after 15 minutes, it reached the value that remained during the experiment period. The acute effect of extracts on the hemodynamic parameters can be observed in Figures 2A and 2B. Therefore, C. glutinosa extracts showed a BP decrease was significant at concentrations of 20 and 50 mg/kg for infusion and only 2.5 and 5 mg/kg for maceration. This significant decrease in the mean BP ranged from 20.54%, 20.37%, 19.06%, and 15.65% at the doses of 2.5 mg/kg (maceration), 50 mg/kg (infusion), 5.0 mg/kg (maceration), and 20 mg/kg (infusion) respectively.

Besides, according to Figure 1B, the compounds intensity is much higher for maceration extract. Therefore, it suggests that the dose reached a saturation point, as the dose increment did not improve the response, and no significant difference in hemodynamic parameters following the administration of 10 and 20 mg/kg i.p.

Flavonoid-rich extracts can reduce elevated blood pressure and can be an agent in the treatment for inhibiting ACE activity [²⁰]. The flavonoids are considered competitive-inhibitors type, meaning that they can compete with the substrate in enzyme active site binding [²¹]. Thus, the compounds could compete for this binding, justifying a difference in the higher concentrations, which suggests active sites binding saturation.

Among flavonoids from apple peel extract, quercetin-3-O-glucuronic acid was the most effective inhibitor of ACE, which showed competitive enzyme inhibition [²⁰]. Moreover, Galindo and coauthors [²²] also described that quercetin-3-glucuronide and isorhamnetin-3-glucuronide are the main plasma metabolites of quercetin exerted an antihypertensive effect (in vivo), and doses low of quercetin-3-glucuronide significantly reduced blood pressure.

Figure 2
Effect of C. glutinosa in hemodynamic parameters. Register of the systolic and diastolic blood pressure with different administration of extract doses, and heart rate during the register. *𝑝 < 0.05; **𝑝 < 0.01 (𝑛 = 6), where Control: animals that received only the vehicle (0.9% NaCl); A: infusion extract (10, 20 and 50 mg/kg), and B: maceration extract (2.5, 5, 10 and 20 mg/kg).

Additionally, among the compounds present in the extract for maceration extract there is quercetin, a known compound with a hypotensive and antihypertensive effect, whose hypertensive rat models demonstrated to induce a reduction in BP [²³]. Also, studies with conjugated metabolites of quercetin, including quercetin-3-glucuronide, showed involvement in the antihypertensive response [²²]. Therefore, quercetin-3-glucuronide or the interaction of quercetin derivatives could be related to the mechanism antihypertensive effect of the C. glutinosa extracts.

Furthermore, some flavonoids quercetin derivatives displayed structural features fundamental for ACE-inhibition, regulating the BP effectively [²⁴]. A current study reported that some crude extracts of Cuphea species and miquelianin showed ACE-inhibiting activity, where C. glutinosa and miquelianin displayed a rate of inhibition percentages of 32.14% and 31.66%, respectively, relative to Captopril-positive control that presented an ACE inhibition of 49.14% in the same concentration. In addition, the ACE inhibitory effect induced by the crude extracts can be due to the presence of miquelianin and the synergism among flavonoids [¹⁰].

Therefore, Cuphea glutinosa Cham. & Schltdl has antihypertensive property could be a therapeutic benefit alternative because ACE-inhibitors reduce BP and decrease cardiovascular events in high-risk populations by connecting a zinc molecule to the active site of ACE, such as the flavonoids [²⁵]. However, the mechanism underlying the antihypertensive effect from the data found in this study is difficult to categorize. Thus, future research will be necessary to verify the inhibition mechanism of these samples.

CONCLUSION

The present study is the first that describes the antihypertensive action in vivo of C. glutinosa extracts, which is extremely important because the incidence of hypertension, considered one of the most relevant cardiovascular risk factors, has been increasing the search for new therapeutic alternatives, mainly of natural origin. Moreover, these results corroborate with the popular usage of this species as hypotensive and antihypertensive agents in order to contribute to evidence-based traditional medicine. However, despite the promising results, further investigations are necessary to ascertain the exact action mechanism of the observed antihypertensive effect.

Acknowledgments

This study was supported partly by the Research Support of the State of Rio Grande do Sul (FAPERGS); and Coordination for the Improvement of Higher Education Personnel (CAPES) - Finance Code 001.

REFERENCES

• ¹
  Baharvand-Ahmadi B, Asadi-Samani M. A mini-review on the most important effective medicinal plants to treat hypertension in ethnobotanical evidence of Iran. J. Nephropharmacol. 2016;6:3-8.
• ²
  Bhagani S, Kapil V, Lobo MD. Hypertension. Medicine 2018;46:509-15.
• ³
  Dlamini Z, Hull R, Makhafola TJ, Mbele M. Regulation of alternative splicing in obesity-induced hypertension. Diabetes Metab. Syndr. Obes. 2019;12:1597-615.
• ⁴
  Atanasov AG, Waltenberger B, Wenzig EMP, Linder T, Wawrosch C, Uhrin P, et al. Discovery and resupply of pharmacologically active plant-derived natural products: A review. Biotechnol. Adv. 2015;33:1582-614.
• ⁵
  Graham A, Freudenstein JV, Luker M. Phylogenetic study of Cuphea (Lythraceae) based on morphology and nuclear rDNA ITS sequences. Syst. Bot. 2006;31:764-78.
• ⁶
  Zago AM, Manfron MP, Morel AF, Zanetti GD. Morfonatomia do caule de Cuphea glutinosa Cham. & Schltdl. (Lythraceae). [Morpho-anatomy of the stem of Cuphea glutinosa Cham & Schltdl. (Lythraceae)]. Rev. Bras. Farmacogn. 2009;19:720-6.
• ⁷
  Santos MC, Farias LS, Merlugo L, Oliveira TV, Barbosa FS, Fuentefria AM, et al. UPLC-MS for identification of quercetin derivatives in Cuphea glutinosa Cham. & Schltdl (Lythraceae) and evaluation of antifungal potential, Curr. Pharm. Anal. 2018;14:586-94.
• ⁸
  Santos MC, Kotez M, Mendez ASL, Henriques AT. Ultrasound-assisted extraction optimization and validation of ultra-performance liquid chromatographic method for the quantification of miquelianin in Cuphea glutinosa leaves. Talanta 2020;216:120988.
• ⁹
  Santos MS, Soares KD, Beltrame BM, Bordignon SAL, Apel MA, Mendez ASL, et al. Cuphea spp.: antichemotactic study for a potential anti-inflammatory drug. Nat. Prod. Res. 2020;35:6058-61.
• ¹⁰
  Santos MC, Toson NSB, Pimentel MCB, Bordignon SAL, Mendez ASL, Henriques AT. Polyphenols composition from leaves of Cuphea spp. and inhibitor potential, in vitro, of angiotensin I-converting enzyme (ACE). J. Ethnopharmacol. 2020;255:112781.
• ¹¹
  Santos MC, Mendez ASL, Henriques AT. Cuphea genus: A systematic review on the traditional uses, phytochemistry, pharmacology, and toxicology. Curr. Tradit. Med. 2024;10:e220823220154 . https://dx.doi.org/10.2174/2215083810666230822100119
  » https://dx.doi.org/10.2174/2215083810666230822100119
• ¹²
  Teixeira K, dos Santos P, Zanette VC, DalBó S, Amaral PA. Medicinal plants that can cause changes in blood pressure and interactions with antihypertensive agents. Am. J. Ethnomed. 2017;4:1-8.
• ¹³
  Yang B, Kortesniemi M, Liu P, Karonen M, Salminen JP. Analysis of hydrolyzable tannins and other phenolic compounds in Emblic leafflower (Phyllanthus emblica L.) fruits by high performance liquid chromatography−electrospray ionization mass spectrometry. J. Agric. Food Chem. 2012;60:8672-83.
• ¹⁴
  Santos MC, Henriques AT, Mendez ASL. Analytical methods of phytochemicals from the Cuphea genus - a review. Drug Anal. Res. 2021;5:04-10.
• ¹⁵
  Chaswal M, Das S, Prasad J, Katyal A, Fahim M. Cardiac autonomic function in acutely nitric oxide deficient hypertensive rats: role of the sympathetic nervous system and oxidative stress. Can. J. Physiol. Pharmacol. 2011;24:865-74.
• ¹⁶
  Santos MC, Soares KD, Beltrame BM, Toson NBS, Pimentel MCB, Bordignon SAL, et al. Polyphenolic composition and in vitro antihypertensive and anti-inflammatory effects of Cuphea lindmaniana and Cuphea urbaniana. Chem. Biodivers. 2021;18:e2100041.
• ¹⁷
  Ferreira O, Pinho SP. Solubility of flavonoids in pure solvents. Ind. Eng. Chem. Res. 2012;51:6586-90.
• ¹⁸
  Do QD, Angkawijaya AE, Tran-Nguyen PL, Huynh LH, Soetaredjo FE, Ismadji S, et al. Effect of extraction solvent on total phenol content, total flavonoid content, and antioxidant activity of Limnophila aromatica. J. Food Drug Anal. 2014;22:296-302.
• ¹⁹
  Monteiro M, Santos RA, Iglesias P, Couto A, Serra CR, Gouvinhas I, et al. Effect of extraction method and solvent system on the phenolic content and antioxidant activity of selected macro- and microalgae extracts. J. Appl. Phycol. 2020;32:349-62.
• ²⁰
  Kim YA, Korystova AF, Kublik LN, Levitman MK, Shaposhnikova VV, Korystov YN. Flavonoids decrease the radiation-induced increase in the activity of the angiotensin-converting enzyme in rat aorta. Eur. J. Pharmacol. 2018;837:33-7.
• ²¹
  Balasuriya N, Rupasinghe HPV. Antihypertensive properties of flavonoid-rich apple peel extract. Food Chem. 2012;135:2320-5.
• ²²
  Galindo P, Rodriguez-Gómez I, González-Manzano S, Dueñas M, Jiménez R, Menéndez C, et al. Glucuronidated quercetin lowers blood pressure in spontaneously hypertensive rats via deconjugation. PloS one. 2012;7:e32673.
• ²³
  Perez-Vizcaino F, Duarte J, Jimenez R, Santos-Buelga C, Osuna A. Antihypertensive effects of the flavonoid quercetin. Pharmacol. Rep. 2009;61:67-75.
• ²⁴
  Hussain F, Jahan N, Rahman K, Sultana B, Jamil S. Identification of hypotensive biofunctional compounds of Coriandrum sativum and evaluation of their angiotensin-converting enzyme (ACE) inhibition potential. Oxid. Med. Cell. Longev. 2018;2018:1-11.
• ²⁵
  Larson A, Symons JD, Jalili T. Quercetin: A Treatment for hypertension?-A Review of efficacy and mechanisms. Pharmaceuticals 2012;3:237-50.

• Funding:

  This research received no external funding.

Edited by

Editor-in-Chief:

Paulo Vitor Farago

Associate Editor:

Jane Manfron Budel

Publication Dates

• Publication in this collection
  11 Oct 2024
• Date of issue
  2024

History

• Received
  19 Sept 2023
• Accepted
  20 July 2024