EVALUATION OFCARDIOPROTECTIVE ACTIVITY OF ARGYREIA NERVOSAROOT EXTRACT ON ISOPRENALINE INDUCED MYOCARDIAL INJURY IN RATS

A.BRIEF RESUME OF THE INTENDED WORK:

Need of study:

Medicinal herbs and plant extracts are now generally considered as effective medicines to be respected, appreciated and they play a major role in modern pharmacy. World Health Organization estimated that about 80% of the world's population relies on herbs for their primary healthcare needs. There has been an explosion of scientific information concerning plants, crude plant extracts and various substances from plants as medical agents during the last 20 to 30 years. Although herbal medicine has existed since the dawn of time, our knowledge of how plants actually affect human physiology remains largely unexplored. Numbers of plants are claiming various medicinal uses and many researches are going on in this view. One such plant,Argyreia nervosa, which has various medicinal properties, is widely used in Ayurveda, the ancient traditional medicinal system in India. In this review a comprehensive account of the morphology, phytochemical constituents and pharmacological activities are included in view of the many recent findings of this plant.

Argyreia nervosa invites attention of the researchers worldwide for its pharmacological activities ranging from aphrodisiac to nematicidal activities1-14. Argyreia nervosa, belongs to family Convolvulaceae is a climbing shrub with woody tomentose stem, found mainly in Deccan, Karnataka and East slopes of the West Ghats at an altitude of 900 meters15. It is commonly known as Elephant creeper and in Samudra-sok Hindi16. Traditionally, leaves are used by Rajasthani tribe to prevent conception17. Seeds of Argyreia nervosa found to possess hypotension, spamolytic18 and anti-inflammatory activity19. Chemical analysis revealed thepresence of triterpenoids, flavanoids, steroids and lipids20. Roots of Argyreia nervosa proved the immunomodulatory activity against the myelosuppressive effects induced by Cyclophosphamide21. 24R-ergost-5-en-11-oxo-3 beta-ol alpha-D glucopyranoside xylose was isolated from seeds of Argyreia nervosa known as Argyreioside22.

Many polyherbal formulations containing this plant parts are available in the market. However, less information is available regarding the clinical study, toxicity study, phyto-analytical studies of this plant. Several phyto-chemical studies have reported but still it needs progress. With the availability of primary information, further studies can be carried out such as clinical evaluation, phyto-analytical studies, toxicity evaluation. The plant is pre-clinically evaluated to some extent; if these claims are scientifically and clinically evaluated then it can provide good remedies and help mankind in various ailments23.

Cardiovascular Diseases (CVD) remain one of the leading causes of deaths despite several advancements in the medical interventions. Among these, the ischemic heart diseases, acute myocardial infarction (AMI) in particular, is one of the most alarming values. AMI, which arises out of a lot risk-factors working in concert, gives rise to a lot of unfavorable biochemical outcomes. The end-result of which is the ultimate morbidity of the patient or even death. The synthetic drugs that constitute the current pharmacological armamentarium are themselves effective in managing the condition but not without setbacks. These hunches have accelerated the need for natural products, which may be used as dietary supplement to prevent the development of an acute myocardial infarction24.

As indicated above, Argyreia nervosa have been exploited in the treatment of variety of cardiovascular ailments in folklore, however, there is dearth of report that confirms the ethno pharmacological claims of cardio protective efficacy of the seeds of the plant using modern scientific techniques in experimental animals. Thus the present study is also based on the anti-inflammatory and antioxidant properties of which are useful in Argyreia nervosaameliorating pathophysiology of myocardial infarction and hence this study is designed to evaluate the cardio protective potential ofArgyreia nervosasweetin isoprenaline induced cardiac dysfunction and metabolic derangement in rats.

Review of literature

Argyreia nervosa is widely distributed throughout India. The plant appears to have a broad spectrum of activity on several ailments. Various parts of the plant have been explored for central nervous depressant activity, nootropic activity, aphrodisiac activity, anticonvulsant activity, immunomodulatory activity, antioxidant activity, analgesic activity, anti-inflammatory activity, hypoglycemic activity, hepatoprotective activity, antibacterial activity, antifungal activity and many other activities. It is reported to contain flavonoids, sterol glycoside, flavonoids and essential oil. The pharmacological studies reported in the present review confirm the therapeutic value ofArgyreia nervosa25.

Hawaiian Baby Wood rose (Argyreia nervosa) is a perennial climbing vine, also known as Elephant Creeper and Woolly Morning Glory. This plant is a rare example of a plant whose hallucinogenic properties have only recently been discovered by non-Hawaiians. While its cousins in the Convolvulaceae family, such as the Rivea corymbosa (Ololiuhqui) and Ipomoea violacea (Morning Glory), were used in shamanic rituals of Latin America for centuries, the Hawaiian Baby Wood rose was not traditionally recognized as a hallucinogen. Its properties were first brought to attention in the 1960s, despite the fact that the chemical composition of its root is nearly identical to those of the two species mentioned above25.

Argyreia nervosa is a popular Indian medicinal plant, which has long been used in traditional Ayurvedic Indian medicine for various diseases. This plant is pharmacologically studied for nootropic, aphrodisiac, immunomodulatory, hepatoprotective, antioxidant, anti-inflammatory, anti-hyperglycemic, anti-diarrheal, anti-microbial, anti-viral, nematicidal, anti-ulcer, anti-convulsant, analgesic and central nervous depressant activities. A wide range of phytochemical constituents have been isolated from this plant. A comprehensive account of the morphology, phytochemical constituents and pharmacological activities reported are included in view of the many recent findings of importance on this plant25.

Such medicinal herbal plants has given their potential to produce significant therapeutic effect, they can be useful as drug or supplement in the treatment/management of various diseases. Herbal drugs or medicinal plants, their extracts and their isolated compound(s) have demonstrated spectrum of biological activities. Such have been used and continued to be used as medicine in folklore or food supplement for various disorders. Ethno pharmacological studies on such herbs/medicinally important plants continue to interest investigators throughout the world25.

The alcoholic extract of the root exhibited statistically significant anti-inflammatory26 activity against granuloma formation technique in albino rats which comparable to acetylsalicylic acid. The extract did not show much activity against formalin induced arthritis in rats.

Pharmacologically it helps in healing of wounds and increases blood circulation to the effective part. It strengthens the nervous system and promotes memory, Its antiinflamatory26 in action and is cardiac supporter. It helps in digestion and also helpful in throat related problems. It increases sperm count and decreases the inflammation in uterus. It is also helpful in resolving urine related problems and also effective in diabetes.

The hexane extract of the root yielded tetradecanyl palminate, 5, 8- oxidotetraco san-10-one27 and two novel aryl esters characterized as stigmasteryl para-hydroxy cinnamate and hexadecanyl p-hydroxy cinnamate along with scopoletin28, coumarin glucoside29 and scopoletin30 as important phyto constituents. The other constituents isolated were caffeic acid and ethyl caffeate31, another study also revealed the presence of ergoline alkaloids in the seeds. The ergolines were indicated to be of clavine type32. The free amino acids reported in the seeds were glutamic acid, glycine, isoleucine, leucine, lysine, phenylalanine, tyrosine, praline, amino butyric acid33, 34.

In Unani system of medicine, its roots are known to possess aphrodisiac and diuretic properties, and have been used to treat gonorrhea; while its leaves are anti-phlogestic, emollient, local stimulant, rubifacient and vesicant. Internally, the extract ofA. nervosaleaves has been used to cure the boils and swellings. Externally, it is applied on eczema, etches, ringworm infections and skin diseases. Its seeds are used as hypotensive, spasmolytic and tonic35.50% ethanolic extract of the seeds in a preliminary biological screening showed antispasmodic activity in the isolated guinea pig ileum36and antibacterial activity againstStaphylococcus aureus. The alcoholic extract of the root exhibited statistically significant anti-inflammatory activity against granuloma technique in albino rats37.The seed-oil showed antifungal activity againstAspergillius flavusandAlternaria solani38.A paste of its tubers is applied externally in abscesses of stomach.A. nervosaBurm. Has been reported for its anticonvulsant activity39 and nootropic activity40.

The isoprenaline induced myocardial infarction in rodents offers a relevant model to study the effect of natural products. The model is simple in execution and the biochemical, histological and electrocardiographic changes closely mimic that seen in case of an AMI. The model has so far been widely used by many researchers to study the effect of drugs in AMI. Natural products, which include crude herbals, extracts, herbo-mineral formulations, polyherbal formulations, etc., have been used for the treatment of ischemic heart diseases in traditional systems of medicine. A range of natural products have been tried for activity against acute myocardial infarction with considerable success. The colossal collections of chemicals present in the natural products possess antioxidant, anti-inflammatory26 and other properties that prove useful in ameliorating the pathophysiology of acute myocardial infarction. Besides, these natural products may also augment the conventional treatment and offer better management of the condition with fewer side effects41, 42.

Objective of Study:

The objective of the present study is to evaluate the cardio protective activity of alcoholic extract of Argyreia nervosa root onisoprenaline induced myocardial injured rats.

Specific Objectives:

  • To collect and authenticate roots of Argyreia nervosa.
  • To prepare and carryout phytochemical evaluation of alcoholic extract of Argyreia nervosa root.
  • To arrive at the therapeutic dose range after acute toxicity studies following WHO guidelines.
  • To explore the cardio protective activity of extract in isoprenaline induced acute myocardial damage in rats.
  • To study the possible role of extract in isoprenaline induced chronic myocardial dysfunction and metabolic derangement in rats.

Note: Cardio protective efficacy will be ascertained on the basis of estimation of biochemical parameters (Lactate dehydrogenase- LDH, Creatinine phophokinase-CK MB) and antioxidant profile (Superoxide dismutase-SOD and catalase) in serum and heart tissue homogenate based. These evaluations will be supported by Histopathological studies.

B. Methodology

Source of Data:

Data will be obtained from laboratory based studies by using Sprague dawley rats of either sex weighing between 150-200 grams maintained at room temperature having free access to food (std pellet diet), tap water ad libitum. These studies will be carried out in intact animal that will be supported by biochemical data and histopathological studies.

Method of Collection of Data:

Chemicals and reagents will be procured from standard companies. Isoprenaline induced myocardial damage will be used as model to evaluate cardio protective efficacy. Suitable biochemical and histological investigation will be carried out in animal model. The data collected will be based on animal experimentation as per the parameters studied under each animal model.

PREPARATION OF EXTRACT43:

Argyreia nervosa roots will be shade dried and powdered, And then drug will be sent for soxhlet’s extraction with 70% ethanol. The extract will be filtered with watman no.1 filter paper and then solvent will be evaporated at reduced pressure by using rotavapour apparatus to get a viscous mass, which will then stored at 40 C until used. The expected % yield of the extract to be obtained is 3.9%. For animal use, the extract will be dissolved in 2% gum acacia.

Experimental protocol

The cardio protective role of Argyreia nervosa will be determined in isoprenaline induced myocardial damage in rats. The Sprague dawley rats of either sex will be divided into following groups consisting of six animals each:

  • Group-I- Normal Control
  • Group II- Isoprenaline (ISO) control
  • Group-III- Argyreia nervosa (AN- 30 days oral treatment low dose)
  • Group-IV- Argyreia nervosa (AN- 30 days oral treatment high dose)
  • Group-V- Carvedilol (2mg/kg, orally for ten days) 44.

Isoprenaline induced chronic myocardial damage in rats41:

During 30 days of AN, ten days of Carvedilol in their respective groups, animals in all groups except group I will receive isoprenaline 3mg/kg/day subcutaneously. During treatment, haemodynamic parameters such as body weight, physiological changes and electrocardiographic observations will be noted. At the end of treatment, blood samples will be collected and serum will be separated. Heart tissue homogenate will be prepared in 0.25 M sucrose (10%) and both serum and heart tissue homogenate will be studied for CK-MB, LDH, SOD, Catalase, TBARS etc. Microscopic slides will be prepared to study histopathological and electron microscopic changes.

Isoprenaline (ISO) induced acute myocardial necrosis in rats42:

At the end of treatment of animals as mentioned in experimental protocol, blood samples will be collected and serum will be separated. Influence of chronic therapy of Argyreia nervosa in presence and absence of Carvedilol will be determined by estimating LDH, CK-MB and TBARS in serum. Subsequently, ISO (150 mg/kg, s.c) will be administered for two consecutive days. Symptoms and mortality in each group will be recorded and compared with those of the rats given ISO alone. After anesthetizing the rat with a combination of ketamine hydrochloride (75mg/kg, i.p) and xylazine (8.0mg/kg, i.p), leads will be attached to the dermal layer of both the front paws and hind legs and recording will be made on polygraph with the help of electrodes ECG system (subcutaneous lead II method). Blood samples will be collected and separated serum will be evaluated again for estimating LDH, CK-MB and TBARS. Forty-eight hours after the first ISO administration, the rats will be sacrificed and autopsied. Three excised hearts will be homogenized to prepare heart tissue homogenate (HTH) using sucrose (0.25 M). The endogenous biological markers such as LDH, CK-MB and antioxidants (Superoxide dismutase and catalase) will be determined in heart tissue homogenate. Microscopic slides of remaining three hearts will be prepared for studying histopathological and electron microscopic changes.

Statistical analysis

The statistical significance will be assessed using one-way analysis of variance (ANOVA) followed by Dunnet comparison test. The values will be expressed as mean ± SEM and p < 0.05 will be considered significant.

Acute toxicity studies:

Acute toxicity studies for cold water extract of Argyreia nervosa root. Will be conducted as per WHO guidelines using Sprague dawley rats.

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