A Comprehensive Study on Anti-hypertensive properties of Punica granatum (Pomegranate), Cynara scolymus (Artichoke), Coscinium fenestratum (Yellow vine) in Phytopharmacological, Molecular Biology Researches

Sagar Singh Jough¹, Rakesh Kumar Saini², Aamna Parveen³

¹Department of Pharmacology, Ch. Sughar Singh Pharmacy College, Etawah, U.P., India.

²Department of Pharmaceutical Chemistry, Ch. Sughar Singh Pharmacy College, Etawah, U.P., India.

³Department of Pharmaceutical Chemistry, Institute of Pharmacy, CSJM University, Kanpur, U.P., India.

*Corresponding Author E-mail: ssingh99756@gmail.com

ABSTRACT:

Hypertension is a turning into a significant danger to the world. In the hunt of lead atoms from plant beginning as a substitute for poisonous engineered drugs, 26 Indian restorative plants and nourishments were screened for their ACE (Angiotensin Converting Enzyme) inhibitory movement. IC50 (half restraint of ACE) estimations of hydroalcoholic unrefined concentrates and division were dictated by a colorimetric technique. Dynamic parts were additionally screened to decide the compound energy, mode, explicitness and instrument of restraint. Normalization was finished by deciding aggregate phenolics and flavonoids as gallic corrosive and quercetin counterparts/mg of concentrate individually. Among 26 unrefined concentrates, Cynara scolymus extricate indicated the best action, IC50 esteem 356.62μg/mL. Pro restraint coming about because of protein precipitation was most noteworthy in Coscinium fenestratum. Lineweaver-Burk plots uncovered a serious method of restraint for Punica granatum ethyl acetic acid derivation part. Divisions of  Cynara scolymus  were seen as vague inhibitors of ACE. Coscinium fenestratum parts restrained the ACE by Zn2+ particle chelation. Further, in the quest for sheltered and powerful lead atoms from normal sources, (MP) Mucuna pruriens L. (Fabaceae) seeds were used for investigating the antihypertensive potential. Generally it is utilized as diuretic and Hypotensive. Bioassay-guided divisions were used for the separation of dynamic mixes by segment chromatography. IC50 esteem, protein energy and restraint system were resolved. In vivo time and portion subordinate hypotensive examination followed by changes in the MAP (Mean blood vessel pressure) actuated by angiotensin I (3 nmol/kg), angiotensin II (3nmol/kg), and to bradykinin (10nmol/kg) in anesthetized rodents was finished. Plasma and tissue ACE exercises were additionally decided. Phytochemical examination by spectroscopic methods uncovered the nearness of realized mixes like genistein, ursolic corrosive and L-DOPA from the ethyl acetic acid derivation and water part separately. In vitro examination uncovered MP ethyl acetic acid derivation portion (MPEA) and genistein as the most dynamic part (IC50 156.45μg/mL) and compound (IC50 253.81μM) individually. Lineweaver-Burk plots uncovered a non-serious method of hindrance. Expert protein precipitation was the recommended instrument for restraint. The concentrate indicated a time and portion subordinate decline in the MAP.

KEYWORDS: Hypertension, ACE, Cynara scolymus, Coscinium fenestratum, Punica granatum.

INTRODUCTION:

Hypertension is normally eluded as hypertension, is that the commonest reason for visiting a specialist, representing more than twenty million visits per year. Individuals of any age are influenced by hypertension, from kids to the old, with the expanding occurrences in old, particularly in individuals of 50 years or more seasoned. Hypertension or hypertension is viewed as the most well-known cardiovascular issue that tortures right around one billion individuals worldwide and is a main source of horribleness and mortality.¹

It has been determined that by 2020, incessant ailments will represent very nearly 75% of all passing’s worldwide and that 71% of passing’s because of cardiovascular malady and 75% of passing’s because of stroke will happen in creating nations.² In the year 2000, about 26.4% of the total populace endured hypertension, and it is expanding at an disturbing rate with a forecast of 60% in 2025.³ Since hypertension does now not show any indications, it is hard to analyze. As a final product, hypertension is generally not, at this point appropriately oversaw.⁴

Hypertension is a significant hazard factor for some cardiovascular ailments, for example, arteriosclerosis, congestive cardiovascular breakdown, coronary illness, end-stage renal maladies, myocardial dead tissue and stroke.⁵ What's more, it additionally prompts irregular renal capacity coming about in renal disappointment.

History of hypertension:

To comprehend the pathophysiology, causes and treatment of hypertension it has been read for quite a long while. Side effects of "vatarakta" was depicted by an antiquated Indian specialist Sushruta (600 BC) who is additionally the writer of the book "Sushruta Samhita" "Heart beat infection" is the old name of hypertension, authentic records show that different methods used to treat this sickness were needle therapy, phlebotomy and seeping by the parasite.⁶

Classification of hypertension:

Blood pressure is of two types, systolic blood pressure and diastolic blood pressure.⁷ Systolic blood pressure is the blood pressure in vessels during a heartbeat. Diastolic blood pressure is the pressure between heartbeats. Hypertension is further sub-classified into hypertension stage I, hypertension stage II and isolated systolic hypertension (Table 1.1).

Resistant hypertension is a condition when medication does not reduce the blood pressure to the normal level 120/80mmHg.

Types of hypertension:

Hypertension can be divided into two groups, primary (essential) hypertension and secondary hypertension. Primary (essential) hypertension effect 90 to 95% of patients and is the most prevalent type of hypertension, the etiology primary hypertension is unknown on the other hand secondary hypertension occurs due to known reason, and affects only 5 to 10% of the total hypertensive population.⁸ The optimal blood pressure for a healthy individual above 18 years of age is defined as a mean systolic blood pressure < 120 mmHg and a mean diastolic blood pressure < 80 mmHg. Blood pressure values greater than these could further be categorized according to the level of severity.

Table 1: Classification of blood pressure for adults age 18 years or older

Category	Systolic BP (mm Hg)	Diastolic BP (mm Hg)
Optimal	< 120	< 80
Normal	120-129	80-84
High Normal	130-139	85-89
Hypertension
Stage 1 (mild)	140-159	90-99
Stage 2 (moderate)	160-179	100-109
Stage 3 (severe)	180-209	110-119
Stage 4 (very severe)	> 210	> 120

Pathophysiology of hypertension:

Cardiac output and peripheral resistance all the two factors which determine the blood pressure. Mechanisms leading to secondary hypertension are clear but the mechanism for the essential hypertension is still ambiguous. The mechanism associated with essential hypertension can be explained by many theories which are base on the total peripheral resistance.

When the Kidneys unable to excrete sodium, it results in the secretion of atrial natriuretic factor (ANF) to promote excess salt secretion resulting into the rise of total peripheral resistance as a side effect.⁹

Risk of hypertension increases due to the vasoconstriction caused by overactive RAS which results in increased blood volume through retention of sodium and water.¹⁰

Hypertension is also developed and maintained due to over-activity of SNS by increasing the stress responses.¹¹

Renin-angiotensin system (RAS):

The renin-angiotensin-aldosterone system (RAAS) is a hormonal cascade that functions in the homeostatic control of arterial pressure, tissue perfusion, and extracellular volume.

Dysregulation of the RAAS plays an important role in the pathogenesis of cardiovascular and renal disorders. RAS plays a vital role in the homeostatic control of arterial pressure, tissue perfusion and extracellular volume. It functions as an unusual endocrine axis, in which the active hormone, angiotensin (Ang) II, is formed by the sequential proteolytic cleavage of its precursors.¹²

RAS cascade is initiated by the regulated secretion of renin from juxtaglomerular cells of the nephron, a functional unit of the kidney. Renin, a preprohormone is activated by the proteolytic removal of a 43-amino acid pro-segment peptide from its Nterminus.¹² The secretion of active renin is mainly regulated by four interdependent factors:

1. A renal baroreceptor mechanism in the afferent arteriole that senses changes in renal Perfusion pressure.

2. Changes in the delivery of NaCl (sensed as changes in Cl- ion concentration) to the distal Tubule.

3. Sympathetic nerve stimulation via β1-adrenergic receptors, and Negative feedback by a direct action of Ang II on the juxtaglomerular cells.¹³

Angiotensin converting enzyme:

ACE is a zinc metalloprotease (dipeptidyl carboxypeptidase/exopeptidase/kininase II), which assumes a basic job in the control of pulse by ideals of its interest in the RAS.¹⁴ It is found in a wide assortment of mammalian tissues, essentially as a layer bound ectoenzyme and is confined on the plasma films of different cell types, including vascular endothelial cells, microvillar brush fringe epithelial cells (e.g., renal proximal tubule cells) and neuroepithelial cells.^15,16 Pro additionally exists in a solvent structure in plasma, however this structure may basically reflect turnover and freedom of film bound ACE. The layer bound ACE is physiologically significant as opposed to plasma ACE since it represents the quick transformation of Ang I to Ang II.^17,18

Fig. 1.1: Renin-angiotensin system

Angiotensin converting enzyme 2 (ACE2):

ACE2, a homologue of human ACE has been shown to be an essential regulator of cardiac function.¹⁹ It is also a zinc metallopeptidase with significant structural homology to ACE (42% sequence homology) and does not convert Ang I to Ang II.²⁰ In contrast to ACE, ACE2 is highly tissue-specific and is restricted to heart, kidney and testis.²¹ The vasoconstrictor actions of Ang II may be opposed by the formation of a hexapeptide, Ang (1-7). ACE2 converts Ang II into Ang (1-7) with a higher catalytic efficiency than any other known Ang (1-7) peptide forming enzyme. The reported potential involvement of ACE2 in both Ang II degradation and Ang (1-7) peptide production add another arm of complexity to the RAS.²² ACE2 is a critical enzyme that can play a balancing role in the control of vasoactive and growth promoting activities of the RAS.²³

Natural ACE inhibitors:

Different types of natural food derived compounds have been investigated on their ACE inhibitory properties. Food protein derivatives are a major group of compounds investigated as potential ACE inhibitors.

Fig. 1.2: Goat milk being utilized for lead findings as ACE Inhibitors                                    Fig. 1.3: Artichoke (C.scolymus)

Food proteins can be divided into three categories as animal-derived, plant-derived and microorganism-derived peptides. Secondary metabolites produced in plants are another group of natural compounds which are identified as potential ACE inhibitors.

These sources include the following plants and animals: goat milk, soybean, mung bean, sunflower, rice, corn, wheat, buckwheat, broccoli, mushroom, garlic, spinach, and grapes. Some terpenoids and polyphenolic compounds including flavonoids, hydrolysable tannins, xanthones, procyanidins, caffeolyquinic acid derivatives are found to be effective as natural ACE inhibitors.²⁴

Cynara scolymus:-

C. scolymus or artichoke has been utilized restoratively since the fourth century BCE. It is utilized alone or in mix with other restorative plants (Gentiana lutea, Curcuma longa, Mentha piperita, Achillea millefolium, Foeniculum vulgare, Helichrysum arenarium) as covered tablet or case in various nations. In Australia, 1–2 covered tablets or cases (300–600 mg), is utilized three times each day for stomach related grumblings, dyspepsia, improvement of lipid digestion, post treatment after hepatitis, sub-intense or incessant maladies of biliary tract or after consideration of cholecystectomy (careful evacuation of the gallbladder).²⁵ Artichoke contains the bioactive agents apigenin and luteolin. The total antioxidant capacity of artichoke flower heads is one of the highest reported for vegetables. Cynarine is a chemical constituent in Cynara. The majority of the cynarine found in artichoke is located in the pulp of the leaves, though dried leaves and stems of artichoke also contain it.

Coscinium fenestratum:

Coscinium fenestratum, or yellow vine as it is sometimes referred to in English, is a flowering woody climber, native to South Asia and Mainland Southeast Asia. It is rare and critically endangered in many of its habitats.

Coscinium fenestratum is a member of the family Menispermaceae and the genus Coscinium. The plant is known by many different names, such as Tree turmeric, False calumba, Colombo weed, Weniwel, Daru Haridra (in Sanskrit), Mara Manjal (in Tamil and Malayalam), Haem herb (in Thai), Voer Romiet (in Khmer)²⁶. The primary bioactive ingredient in Coscinium fenestratum is berberine, but also palmatine and jatrorrhizine.

Fig. 1.4: Coscinium fenestratum    Fig. 1.5: Punica granatum

Punica granatum:

Punica granatum (Pg), commonly known as pomegranate (Pg), is a member of the monogeneric family, Punicaceae, and is mainly found in Iran which is considered to be its primary centre of origin. Pg and its chemical components possess various pharmacological and toxicological properties including antioxidant, anti-inflammatory (by inhibiting pro-inflammatory cytokines), anti-cancer and anti-angiogenesis activities. They also show inhibitory effects on invasion/motility, cell cycle, apoptosis, and vital enzymes such as cyclooxygenase (COX), lipooxygenase (LOX), cytochrome P450 (CYP450), phospholipase A2 (PLA2), ornithine decarboxylase (ODC), carbonic anhydrase (CA), 17beta-hydroxysteroid dehydrogenase (17β-HSDs) and serine protease (SP). Furthermore, they can stimulate cell differentiation and possess anti-mutagenic effects.²⁷

PROCESS OF EXTRACTION:

Extraction was completed utilizing diagnostic evaluation solvents. 5 liter Round base carafe was taken in which 1 kg powder of plant material was moved. An adequate amount approx 2.0 liter of hydroalcoholic dissolvable was included until the coarse particles of the plant material were totally doused. The compartment was delicately shaken for 36 h at room temperature with discontinuous shaking. The concentrate got was separated utilizing Whatman channel paper and the buildup was again separated with a new dissolvable for another 12 h. The concentrate was separated and both the filtrates were pooled together. The dissolvable was evacuated utilizing Buchi type turning evaporator and the concentrate was exposed to freeze-drying in a lyophilizer till dry powder was gotten for additional utilization.

Quantitative assurance of all out phenolic substance:

All out phenolic substance were resolved for the concentrates which were seen as dynamic. Assurance of complete phenolic mixes was performed on hydroalcoholic extricates by the Folin-Ciocalteu strategy. Each example was blended in with 1ml Folin-Ciocalteu reagent and 0.8 ml of 7.5% Na2CO3. This technique includes the decrease of Folin-Ciocalteu reagent by phenolic mixes present in the concentrate, with attending arrangement of a blue complex decided at 765 nm by UV-noticeable spectrophotometer (UV-1700 Shimadzu, Japan) after 90min at room temperature. Gallic corrosive was utilized for building the standard bend and the mean of three readings was utilized to decide the allout phenolic content communicated as μg of gallic corrosive counterparts/mg of concentrate (GAEs).²⁸

Quantitative determination of total flavonoids contents:

Determination of all out flavonoids was finished by the aluminum chloride colorimetric technique. 0.6mL of 2% aluminum chloride arrangement was blended in with 0.6 mL weakened standard quercetin arrangements or concentrates. The arrangement was brooded for 60 min at room temperature after legitimate blending. The volume of 6% aluminum chloride was subbed by a similar volume of refined water in the clear. Response blends absorbance was estimated against clear at 420nm frequency with an UV-Vis spectrophotometer (UV-1700 Shimadzu, Japan). Fixation was dictated by examination with the standard adjustment bend (2-200μg/mL) of quercetin. The centralization of all out flavonoid content in the test tests was determined from the adjustment plot (Y = 0.0001x + 0.001, R2 = 0.994) and communicated as μg quercetin counterparts/mg of concentrate (QEs). All the measurements were completed in triplicate.^29,30

IN VITRO ASSAYS:

Angiotensin I-Converting-Enzyme (ACE) inhibitory activity:

The technique used here is a spectrophotometric strategy dependent on the generally utilized technique presented by Cushman and Cheung (1971). This technique is straightforward, touchy, and quick, requiring no dissolvable extraction and can, accordingly, be utilized for high-throughput screening of ACE inhibitors. Expert inhibitory action was tested by estimating the arrival of HA from the substrate HHL. The examine blend contained 125μL of a 0.05 Molar (M) sodium borate cradle (pH 8.2), containing 0.3 M NaCl, 50μL of 5mM HHL (1mL stock was set up by dissolving 2.15mg of HHL in 1mL arrangement) and 25μL of ACE (2.5 Milli units (mU)), which was pre-hatched with various example convergences of the plant inhibitor (10mL stock arrangement of plant inhibitor was readied including 10mg of plant inhibitor in 10ml of dissolvable). The response was halted after brooding at 37°C for 30 min by the expansion of 0.2mL of 1 M HCl. Pyridine (0.4 mL) was included trailed by 0.2mL of BSC (the request for expansion of reagents is basic) The arrangement was gradually blended utilizing a vortex blender and cooled on ice. The yellow shading created was estimated at 410 nm utilizing spectrophotometer (UV-1700 Shimadzu, Japan)^31,32. Schematic graph of the measure method is appeared in Fig. 3.3. One unit of ACE movement is characterized as the measure of catalyst, which discharges 1μmol of HA per min at 37^◦C and pH 8.2.

IN VIVO ASSAYS:

Direct blood pressure estimation in anesthetized rodents:

For the estimation of circulatory strain surgery was done in all gatherings of creatures as indicated by the strategy quickly normotensive male and female Wistar rodents were anesthetized with ketamine (100mg/kg) and xylazine (20mg/kg), given by the intramuscular course, and enhanced at 1hr span. The surgery was done at the neck of the creatures to open the left carotid conduit which was cannulated and associated utilizing a transducer coupled to an iWorx recording framework, and its application program LabScribe2 Data Recording and Analysis Software, permitting the chronicle of the mean blood vessel circulatory strain (MAP). After the surgery, 15 min span was given for adjustment of the circulatory strain, before the infusion of any medication.³³

Time and dose-dependent hypotensive effects of MPHA in normotensive rats:

In the first part of this experiment two groups were created, group-1 as test group of animals received orally (100, 200, 300mg/kg) of MPHA and group-2 as control group of animals received normal saline (NS) at 1, 2, 4 and 10 hrs at a constant volume of 100μL/100 g body weight before the animals were prepared for MAP measurement, as described above.³⁴

Administration of MPHA, MPEA, Ursolic acid, Genistein and L-DOPA under MAP measurement:

After the optimization of dose and time of action of the crude extract, the change in MAP for MPHA, MPEA, and isolated compounds were determined in the next (second) part of the experiments. In the second part of experiment six groups were created, the test groups of animals received a single oral dose of MPHA (100mg/kg) group-1, MPEA (50mg/kg) group- 2, ursolic acid (10mg/kg) group-3, genistein(10mg/kg) group-4 and L-DOPA (25mg/kg) group-5, while the control group (group-6) of animals (CT) received NS only. The MAP was measured in these groups after 1.0 h of drug treatments. Genistein treated animal (group-4) showing the best activity was further evaluated for its intravenous pressoric effects. Group-4 animals were treated with genistein, at doses of 10, 20, 40mg/kg (i.v.) at an interval of 10-15 min between each injection. The vehicle was given to the control animals.

Influence of MPEA and isolated compounds on vascular responses to bradykinin, Ang-I and Ang-II:

30 min after the surgical procedure, the baseline was obtained after MAP stabilization, at this time the rats were injected with Ang-I, or Ang-II, both at doses of 3 nmol/kg (i.v.), or bradykinin (BK; 10nmol/kg) (Lima-Landman et al., 2007; Montenegro et al., 2009). MAP was measured after the administration of these drugs. Now different groups of animals received a single 200μl intravenous bolus of MPEA (50mg/kg), ursolic acid (10 mg/kg), genistein(10mg/kg) and L-DOPA (25mg/kg), captopril (10nmol/kg), losartan (1mg/kg), 5 min after the treatment of plant inhibitors, the injections of Angio I, Angio II, and BK at the same dose described above were repeated, and effects obtained were compared with initial dosing of the drugs done before.

Plasma and tissue ACE activity assay:

After conducting all the in vivo experiments, thoracotomies were done in all rats after anesthetizing them with urethane (1000mg/kg, i.p.). To determine the serum ACE inhibition activity, thoracic aorta was cut for the collection of blood in chilled tubes containing a solution of heparin. At the same time, thoracic aortas were collected over liquid nitrogen and stored at −80°C for the determination of tissue ACE. Plasma ACE inhibitory activity was determined in the animals orally treated, 1.0 h prior, genistein(10mg/kg and 20mg/kg) control (vehicle) and captopril (20mg/kg) as a standard by the method of Montenegro et al 2009. For tissue ACE activity, all extractions were done on the organs of individual animals. 50-100mg (wet weight) of the aorta was minced and homogenized with the use of a glass homogenizer in 3.0ml of extraction buffer (10mM HEPES, pH 7.4, containing 125mM NaCl solution).³⁵

DISCUSSION:

Hypertension is a becoming a major threat to the world. In the search of lead molecules from plant origin as a substitute for toxic synthetic drugs, 26 Indian medicinal plants and foods were screened for their ACE (Angiotensin Converting Enzyme) inhibitory activity. Active fractions were further screened to determine the enzyme kinetics, mode,specificity and mechanism of inhibition. Standardization was done by determining total phenolics and flavonoids as gallic acid & quercetin equivalents/mg of extract respectively.

Among 26 crude extracts, Cynara scolymus extract showed the best activity, IC50 value 356.62μg/mL. ACE inhibition resulting from protein precipitation was highest in Cosciniumfenestratum. Lineweaver-Burk plots revealed a competitive mode of inhibition for Punica granatum ethyl acetate fraction. Fractions of  Cynara scolymus were found to be non-specific inhibitors of ACE.

Coscinium fenestratum fractions inhibited the ACE by Zn2+ ion chelation. Punica granatum seed bioassay-guided fractions were utilized for the isolation of active compounds by column chromatography. Spectroscopic analysis of Punica granatum seed revealed the presence of known compounds like genistein, ursolic acid and L-DOPA from the ethyl acetate and water fraction respectively.

CONCLUSION:

Our study revealed utilization of Indian medicinal plants as a potential source of angiotensin converting enzyme inhibitors. Among the assayed plants, the fraction of many plants produced significant ACE inhibition. Enzyme kinetics and mechanism of inhibition was determined by various parameters. Results from the in vitro ACE inhibitory activity of the tested fractions were utilized for in vivo development of potential antihypertensive agents after further investigations of active plant for compound isolation and evaluating activity using animal models. It also revealed that the fractions and compounds of Punica granatum seeds have potential antihypertensive activity via ACE inhibition. The experimental evidence related to ACE inhibitory effect of the Punica granatum seed and its isolated compounds proves the usefulness in the treatment of hypertension and established the rationale for its therapeutic uses in Ayurveda.

ACKNOWLEDGEMENT:

I would like to acknowledge Dr. Rakesh Kumar, Director, CSSGI, Etawah, for his guidance.

I would like to thank Ms. Aamna Parveen, M.Pharm (Research Scholar) CSJM University for completing this study.

Finally I would like to thank our family, friends and god for their cooperation and help.

CONFLICT OF INTEREST:

The author declare no conflict of interest.

REFERENCES:

1. Carlsson, Axel C., et al. "Cardiovascular and total mortality in men and women with different blood pressure levels–A 26-year follow-up." Blood Pressure. 18.3 (2009): 105-110.

2. Murray, B. A., and R. J. FitzGerald. "Angiotensin converting enzyme inhibitory peptides derived from food proteins: biochemistry, bioactivity and production." Current Pharmaceutical Design. 13.8 (2007): 773-791.

3. Virani Paras, Virani Kinjal. A Review on New Antihypertensive Agent: Irbesartan. Asian J. Res. Pharm. Sci. 6(1): Jan.-Mar., 2016; Page 34-36.

4. Vasudevan N J. Flax Seeds – An Overview in Hypertension. Int. J. Adv. Nur. Management. 2017; 5(3): 273-278.

5. Vasan, Ramachandran S., et al. "Impact of high-normal blood pressure on the risk of cardiovascular disease." New England Journal of Medicine. 345.18 (2001): 1291-1297.

6. Indu Sharma, Bharat Parashar, Hitesh Kumar Dhamija, Ritu sharma. An Ayurvedic Arena for Hypertension Treatment. Asian J. Pharm. Res. 2(2): April-June 2012; Page 54-58.

7. Swaty Chapagai, G Rachel Andrews, Nitish Naik. A Study to Assess the Knowledge and Quality of Life of Pacemaker Patients with a view to Develop an Information Booklet. Asian J. Nur. Edu. and Research.2017; 7(1): 108-114.

8. Garach Bhavikkumar D. The rationale of use of combination therapy in hypertensive patients. Research J. Pharmacology and Pharmacodynamics. 2013; 5(1): 19-26.

9. Atlas, Steven A., and John H. Laragh. "Atrial natriuretic peptide: a new factor in hormonal control of blood pressure and electrolyte homeostasis." Annual Review of Medicine. 37.1 (1986): 397-414.

10. Moderation of the Plasma Chemistry and Hematological Indices of Normal and Salt-loaded Rats by an Aqueous Extract of the Rhizomes of Sansevieria Liberica: Implications for the Management of Hypertension and Associated Complications. Asian J. Pharm. Res. 3(3): July-Sept. 2013; Page 134-140.

11. Esler, Murray. "The sympathetic system and hypertension." American Journal of Hypertension. 13.S4 (2000): 99S-105.

12. Atlas, Steven A. "The renin-angiotensin aldosterone system: pathophysiological role and pharmacologic inhibition." Journal of Managed Care Pharmacy.  (2007): 9-20.

13. Brown, Morris J. "Direct renin inhibition—a new way of targeting the renin system." Journal of Renin-Angiotensin-Aldosterone System 7.2 suppl (2006): S7-S11.

14. Dhananjay Sangle, Amit Naik, Amol Ghorpade, Vikram Ghatkar, Suvarna Ghuge, Dipti Ingle, Rishikesh Ingle. Cost Effectiveness Analysis Study between Atenolol and Amlodipine in Essential Hypertension. Research J. Pharm. and Tech. 6(9): September 2013; Page 1001-1003.

15. Erdos EG. “The angiotensin I converting enzyme.” Fed Proc. 36.5 (1977): 1760-1765.

16. Skeggs, Leonard T., Joseph R. Kahn, and Norman P. Shumway. "The preparation and function of the hypertensin-converting enzyme." Journal of Experimental Medicine. 103.3   (1956): 295-299.

17. Shi, Lijun, et al. "Angiotensin-converting enzymes and drug discovery in cardiovascular diseases." Drug Discovery Today. 15.9 (2010): 332-341.

18. Carey, Robert M., and Helmy M. Siragy. "Newly recognized components of the reninangiotensin system: potential roles in cardiovascular and renal regulation." Endocrine Reviews. 24.3 (2003): 261-271.

19. Crackower, Michael A., et al. "Angiotensin-converting enzyme 2 is an essential regulator of heart function." Nature. 417.6891 (2002): 822-828.

20. Carey, Robert M., and Helmy M. Siragy. "Newly recognized components of the reninangiotensin system: potential roles in cardiovascular and renal regulation." Endocrine Reviews. 24.3 (2003): 261-271.

21. Ferrario, Carlos M., and Jasmina Varagic. "The ANG-(1–7)/ACE2/mas axis in the regulation of nephron function." American Journal of Physiology-Renal Physiology 298.6 (2010): F1297-F1305.

22. Verma, Amrisha, et al. "ACE2 and Ang-(1-7) confer protection against development of diabetic retinopathy." Molecular Therapy 20.1 (2012): 28-36.

23. Paras Virani, Rajanit Sojitra, Bhadresh Savaj, Hasumati Raj, Vineet Jain. Simultaneous Estimation of Irbesartan and Atorvastatin by First Order Derivative Spectroscopic Method in their Synthetic Mixture Use in Hypertension Condition. Asian J. Pharm. Tech. 2015; Vol. 5: Issue 1, Pg 1-7.

24. Kang, Dae Gill, et al. "Angiotensin converting enzyme inhibitory phenylpropanoid glycosides from Clerodendron trichotomum." Journal of Ethno Pharmacology 89.1 (2003): 151- 154.

25. HMPC, Assessment report on Cynara scolymus L., folium, in: European Medicines Agency (Ed.) EMA/HMPC/150209/2009, London E14 4HB, United Kingdom, 2011.

26. Agusta, A., 2003. Coscinium fenestratum (Gaertner) colebr. In lemens, RMHJ and bunyapraphatsara, N. Plant Resour. South East Asia: Med. Poisonous Plants. 3: 139-140.

27. Facciola S. Cornucopia: a Source Book of Edible Plants. Vista, California: Kampong Publications; 1990. p. 166.

28. Dewanto, Veronica, Xianzhong Wu, and Rui Hai Liu. "Processed sweet corn has higher antioxidant activity." Journal of Agricultural and Food Chemistry. 50.17 (2002): 4959- 4964.

29. Diksha Sharma, Dinesh Kumar Mehta, Karun Bhatti, Rina Das, Ram Mohan Chidurala. Amlodipine and Atenolol: Combination Therapy Versus Monotherapy In Reducing Blood Pressure - A Focus On Safety And Efficacy. Research J. Pharm. and Tech. 2020; 13(6): 3007-3013.

30. Jimsheena, V. K., and Lalitha R. Gowda. "Colorimetric, high-throughput assay for screening angiotensin I-converting enzyme inhibitors." Analytical Chemistry. 81.22 (2009): 9388-9394.

31. Mohan Krishna Lokireddy, Jayachandra Reddy P, M. Srinivasa Murthy. New Spectrophotometric Method for Assay of Amphotericin B in Bulk and Its Pharmaceutical Formulations. Res. J. Pharm. Dosage Form. and Tech. 6(1): Jan.-Mar. 2014; Page 15-17.

32. Priyanto, Anugerah Dany, et al. "Screening, discovery, and characterization of angiotensin-Iconverting enzyme inhibitory peptides derived from proteolytic hydrolysate of bitter melon seed proteins." Journal of Proteomics.128 (2015): 424-435.

33. de Souza, Priscila, et al. "Hypotensive mechanism of the extracts and artemetin isolated from Achillea millefolium L.(Asteraceae) in rats." Phytomedicine. 18.10 (2011): 819-825.

34. Gallagher, Patricia E., et al. "Estrogen regulation of angiotensin-converting enzyme  mRNA." Hypertension 33.1 (1999): 323-328.

35. Fujita, Hiroyuki, Tomohide Yamagami, and Kazunori Ohshima. "Effects of an ACEinhibitory agent, katsuobushi oligopeptide, in the spontaneously hypertensive rat and in borderline and mildly hypertensive subjects." Nutrition Research. 21.8 (2001): 1149-1158.

Received on 29.06.2020            Modified on 20.10.2020

Accepted on 13.01.2021      ©Asian Pharma Press All Right Reserved

Asian J. Res. Pharm. Sci. 2021; 11(2):126-132.

DOI: 10.52711/2231-5659.2021-11-2-6