Antihypertensive effect of patulitrin and other constituents from Tagetes patula L. (French marigold) in acute L-NAME induced hypertensive rats

Abstract The work is aimed to evaluate the blood pressure reducing effect of constituents from methanol extract and associated constituents of Tagetes patula flowers in normotensive and L-NAME induced hypertensive rats. The HPLC analysis of methanol extract of Tagetes patula flowers (JFM) resulted in the quantitative identification and percent comparison of four phenolic constituents, protocatechuic acid (PA), methyl protocatechuate (MPA), patulitrin (TRIN) and patuletin (PAT). All the extracts, fractions and compounds examined showed significant blood pressure lowering activity. Patulitrin (TRIN) which has emerged as the major constituent (15.33%) of T. patula flowers showed significant 30% and 68% fall in blood pressure in normotensive and L-NAME induced hypertensive rats respectively. The patuletin (PAT), which is an aglycone of TRIN displayed high percentage (84%) of antihypertensive activity. Further, comprehensive and advanced studies on these constituents may result in preparation of an effective blood pressure lowering medicine with active precious rare flavonoids, patuletin and patulitrin. Graphical Abstract


Introduction
Hypertension is a chronic medical condition that leads to several life-threatening diseases.More than a quarter of adult world population suffers hypertension and every year it causes approximately 13% of global death.Nitric oxide (NO) has a pronounced effect on vascular smooth muscle tone and blood pressure.Various endotheliumdependent agonists, such as acetylcholine and bradykinin stimulate endothelial nitric oxide synthase (eNOS) by increasing intracellular calcium levels in endothelial cells which binds to calmodulin and results in nitric oxide release (Garland et al. 2011).Disruption of endothelial cells causing lack of synthesis and low availability of NO seems to involve in various cardiovascular diseases including atherosclerosis, coronary heart diseases and hypertension (Katzung 2012).Although several remedies are available, but due to associated adverse effects and several other limitations, it is feared that number of hypertensive population will be enormously increased in near feature (Loizzo et al. 2008).A safe and effective new drug is therefore, direly needed.
Plants have been the source of food and medicine since antiquity and even today nearly 80% of population, particularly in third world countries, relies on phytomedicine ranging from common cold to cancer (Ali et al. 2011;Azhar et al. 2019;Zheng et al. 2021).Although man knew the healing power of plants from very beginning of its civilization but it is only 15-20% of plants which have so far been investigated chemically or pharmacologically and major part of the flora is still required to be explored.Scientists all over the world have focused their attention on this issue and they are constantly working to explore indigenous plants for their pharmacologically active constituents (Ali et al. 2021).Keeping this in view, our research group has also isolated several constituents from indigenous medicinal plants, such as, rhamnosyl oxy benzyl carbamate and thiocarbamates from leaves of Moringa oleifera (Faizi et al. 1994), opuntiol glucoside from cladodes of Opuntia dillenii (Saleem et al. 2005), aloe emodin from leaves of Aloe barbadensis (Saleem et al. 2001), and malic acid, citric acid and derivatives from roots of Tagetes patula (Saleem et al. 2004) as potent hypotensive agents.In continuation of this work, research embodied here describes the blood pressure lowering constituents from flowers of Tagetes patula.

Tagetes patula linn
(Asteraceae) known in vernacular as Jafri, is native to Central America and distributed throughout world as an ornamental and medicinal plant (Chadha 1976).Its flowers are mainly used in flavor and fragrance industry as well as in garlands and free form for cultural and religious purposes.The herb is considered diuretic, sedative and digestive.Its leaves are useful in kidney troubles and muscular pains while roots and seeds are purgative (Chadha 1976).Flowers are hepatoprotective (Vasilenko et al. 1990) and also use in cardiovascular drug preparation (Grigorescu et al. 1986).The reported phytochemicals from different parts of Tagetes patula are ceramide, benzofurans, carotenoides, flavonoids, thiophenes, terpenes and terpenoids (Faizi et al. 2008;Faizi Dar et al. 2011;Faizi, Fayyaz et al. 2011;Bano et al. 2019).The plant has been reported to possess mainly flavonoids like patuletin and patulitrin, quercetin, quercetagetin, and quercetagetin-glucosides which are responsible for its antioxidant (Chkhikvishvili et al. 2016), analgesic (Faizi Dar et al. 2011), antimicrobial (Faizi et al. 2008), anti-neoplastic, anti-inflammatory (Jabeen et al. 2016) and anti-thrombogenic activities (Narayana et al. 2001).The presence of various polyphenolic compounds in T. patula caused the pronounced cardio-protective effect in nitric oxide deficient hypertension (Curin and Andriantsitohaina 2005;Perez-Vizcaino et al. 2006).
The plant is considered diuretic, sedative and digestive.Its leaves are useful in kidney troubles and muscular pains while roots and seeds are purgative (Chadha 1976).Flowers are hepatoprotective (Vasilenko et al. 1990) and also used in cardiovascular drug preparation (Grigorescu et al. 1986).The plant has been reported to possess mainly carotenes, flavonoids like patuletin and patulitrin as the major compounds while quercetin, quercetagetin, quercetagetin-glucosides which are present in minor quantity in T. patula, responsible for its antioxidant (Chkhikvishvili et al. 2016), analgesic (Faizi Dar et al. 2011), antimicrobial (Faizi et al. 2008), anticancer (Faizi Dar et al. 2011;Kashif et al. 2015),anti-neoplastic and anti-thrombogenic activities (Narayana et al. 2001).Thiophene α-terthienyl of T. patula is known for nematicidal, insecticidal, antiviral and cytotoxic activities (Faizi Dar et al. 2011).The presence of various polyphenolic compounds in T. patula caused the pronounced cardio-protective effect in nitric oxide deficient hypertension (Curin and Andriantsitohaina 2005;Perez-Vizcaino et al. 2006) however, T patula was not evaluated in this respect.This is the first report of phenolic constituents (Figure 1) of T. patula flowers showing blood pressure reducing activities.Earlier, root extract of T. patula was investigated for its hypotensive constituents (Saleem et al. 2004).N G -Nitro-L-arginine-methyl ester (L-NAME) is a nitric oxide synthase inhibitor and L-NAME induced hypertension is a well-established model of experimental hypertension.L-NAME is reported to elevate blood pressure, decrease NO signaling and reduce femoral artery diameter and lead to endothelial dysfunction in animals (Paulis et al. 2008).The present study intended to evaluate the pharmacological effect of T. patula flowers in normotensive as well as in L-NAME induced hypertensive rats.

Extraction and isolation
Chromatograms of external standards PA, MPA, TRIN, PAT (Figure 1) were taken at 270 nm, showing retention time including 8. 19, 22.209, 28.385 and 43.096 min.respectively (Figure S2a). Figure S2b showed the presence of all four standards in JFM extract.The quantitative HPLC analysis of JFM-EA, JFM-But and JFM-Aq revealed TRIN as the major constituent of extract and phases examined (Table S1).This is the first report indicating presence of TRIN as major chemical component of T. patula flowers.

Hypotensive and antihypertensive study
Methanol extract (JFM) of Tagetes patula flowers and its fractions, JFM-But, PA and MPA showed dose dependent hypotensive effect at 3 and 30 mg/kg, respectively in normotensive rats (Table S2).JFM-Aq and TRIN, however, displayed comparable effects at log doses (Table S2).
TRIN which possesses antibacterial activity (Faizi et al. 2008) showed comparable hypotensive effect at 3 and 30 mg/kg (Table S2).Effect appears to be more pronounced in antihypertensive model where it displayed gradual decrease in blood pressure from 11.17% (at 0.15 min) to 68.00% (at 15 min) (Table S3).
PAT--the aglycone of TRIN which is antioxidant, analgesic (Faizi Dar et al. 2011) anti-inflammatory (Jabeen et al. 2016), anticancer (Kashif et al. 2015) and antibacterial (Faizi et al. 2008) agent of T. patula, displayed significant hypotensive action at the dose of 3 mg/kg (Table S2).PAT seems to be the most significant antihypertensive agent as it caused regular decrease in MABP from 33.37% (at 0.15 min) to 83.85% (at 15 min) at the dose of 3 mg/kg.Except PAT, all of the substances examined at the dose of 30 mg/kg (Table S3).TRIN and PAT belong to flavonoids, bearing benzopyran nucleus, which is significant class of pharmacologically active natural products (Narayana et al. 2001).As far as our knowledge is concerned, this is the first report of hypotensive activity of PAT and TRIN.However, among other flavonoids, quercetin has been reported as strong aortic muscle relaxant (Morales and Lozoya 1994) while luteolin, eriodictyol and naringenin have inhibitory effect on tonic-I and tonic-II phases associated with inhibition of PKC and calcium influx (De Rojas et al. 1996).Since benzopyranes are lipid soluble and glucosidation generally increases compound's water solubility, that's why TRIN was solubilized in saline but its aglycone (PAT) had solubility problem at higher dose.Comparable hypotensive effect of TRIN and PAT designate that flavonoid aglycone may be responsible for the activity.However, sugar moiety in TRIN might have positive effect on pharmacokinetic properties.
PA, which is a potent antibacterial (Liu et al. 2008) and anti-mutagenic agent (Stagos et al. 2006) and its methyl ester MPA which possesses significant antioxidant (Faizi Dar et al. 2011) and nematicidal activities (Faizi, Fayyaz et al. 2011) displayed dose dependent blood pressure reducing effect that returns to initial level in less than a minute in normotensive rats (Table S2) and gradually within 15 min in hypertensive model of rats (Table S3).JFM-PE and its constituent (α-terthienyl), JFM-EA and PAT (at 30 mg/kg) could not be examined due to solubility problem.
The intravenous administration of L-NAME (20 mg/kg) produced significant rise in MABP (43.05%,Table S3).JFM, JFM-But, PAT and TRIN exhibited constant and highly significant decrease in MABP till fifteen minutes after drug injection.Antihypertensive effect caused by JFM-Aq, PA and MPA returned gradually to initial level in observed period of time (Table S3).

Mechanism of action of hypotensive study
All the tested substances except JFM-But when examined on rats pretreated with atropine sulphate (10 −3 M), failed to produce hypotensive action indicating the involvement of muscarinic receptors to lower the blood pressure.Furthermore, highly significant antihypertensive effect of all substances including JFM-But showed a relationship between muscarinic receptor and endothelium derived relaxant factor (EDRF).Acetylcholine stimulated relaxation is achieved in response to EDRF, leading to calcium (Ca +2 ) efflux which is responsible to release nitric oxide (NO) by the activation of endothelium nitric oxide synthase (eNOS).Nitric oxide then, stimulates cGMP which triggers protein kinase G (PKG) and produces an overall reduction in Ca-influx and promotes Ca-dependent muscle relaxation (Katzung 2012).

Supporting information
Plant material, extraction and isolation.The HPLC analysis for the identification and comparison of extract components.The mass and 1D-NMR spectral data of commercial and isolated compounds.Hypotensive and antihypertensive study and statistical analysis.

Conclusion
Methanol extract of flowers (JFM), its butanol phase (JFM-But), and their four pure constituents exclusively, TRIN and PAT have emerged as blood pressure reducing agents of T. patula flowers in both healthy and disease rat models.The presence of bio privileged flavonoids and major active moieties have been found to inhibit endothelial dysfunction induced by nitric oxide impairment in experimental animal models.TRIN also appears to be the major glucoside of Tagetes patula flowers in present studies.Further biological and toxicological studies in different animal models are required to turn these findings into safe, effective and less expensive antihypertensive drug.