Liver diseases are a large public healthy problem in the ...



A Study on Hepato And Nephro protective activity of

Spilanthes radicans

M. PHARM DISSERTATION PROTOCOL

SUBMITTED TO THE

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE

BY

NITISH JOSHI B.Pharm.

UNDER THE GUIDANCE OF

DR. SHIVAKUMAR SWAMY M.Pharm., Ph.D

H.O.D & PRINCIPAL

MALLIGE COLLEGE OF PHARMACY,BANGALORE

[pic]

MALLIGE COLLEGE OF PHARMACY

#71 SILVEPURA,BANGALORE - 90

Rajiv Gandhi University of Health Sciences,

Karnataka, Bangalore.

Annexure – II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

|01 |Name and Address of the Candidate |Nitish Joshi |

| | |S/O Mr.Prakash Chandra Joshi. |

| | |Ranikhet Road,Ramnagar. |

| | |Nainital,Uttarakhand |

|02 |Name of the Institution |Mallige College Of Pharmacy |

| | |#71 Silvepura, |

| | |Chikkabanavara Post |

| | |Bangalore 90 |

|03 |Course of the Study Branch |M.Pharm ,(Pharmacology). |

|04 |Date of Admission to course | 05.07.2010 |

|05 |Title of the Topic |A Study on Hepato And Nephroprotective activity of Spilanthes radicans |

|06 |Brief resume of the intended work |Enclosure – I |

| |6.1. Need for the Study | |

| |6.2. Review of the Literature |Enclosure – II |

| |6.3. Objective of the Study |Enclosure – III |

|07 |Materials and Methods | |

| |7.1. Source of data |Enclosure – IV |

| |7.2. Methods of collection of data |Enclosure – V |

| |7.3. Does the study require any |Enclosure – VI |

| |Investigations on animals? | |

| |If yes give details | |

| |7.4. Has ethical clearance been | Yes |

| |obtained form your institution | |

| |in case of 7.3. | |

|08 |List of References |Enclosure – VII |

|09 |Signature of the Candidate |(Nitish Joshi) |

|10 |Remarks of the Guide | |

| | |The present research work is original and not published in any of the |

| | |journals with best of my knowledge upon extensive literature review. This |

| | |work will be carried out in the Pharmacology laboratory by Mr.Nitish Joshi|

| | |under my supervision. |

|11 |Name and Designation of | |

| |(in Block Letters) |Dr. SHIVAKUMAR SWAMY |

| |11.1. Guide |M. Pharm., Ph. D. |

| | | |

| | |Principal & HOD |

| | |Mallige College Of Pharmacy, |

| |11.2.Signature |Bangalore, Karnataka. |

| | | |

| | | |

| |11.3.Co-Guide (if any) | |

| | |Mr.Prashanth Baganal Asst. Professor |

| |11.4.Signature | |

| | | |

| | | |

| | | |

| | | |

| | | |

| |11.5. Head of the Department | |

| | | |

| | |Dr. SHIVAKUMAR SWAMY |

| | |M. Pharm., Ph. D. |

| | |Principal & HOD |

| |11.6.Signature |Mallige College Of Pharmacy, |

| | |Bangalore, Karnataka |

| | | |

| | | |

| | |The present study is permitted to perform in the Pharmacology laboratory |

|12 |Remarks of the Principal |of our institution and the study protocol has been approved by IAEC. |

| | | |

| | |(Dr. Shivakumar Swamy) |

| |12.1. Signature | |

ENCLOSURE: I

06. Brief resume of the intended work :

6.1. Need for the study :

INTRODUCTION:

Hepato and nephro diseases are large public health problems in the world1. Towards these pathologies which may be appear with multiple and diversified causes and appearances, modern medicine does not find complete curative treatments for disease related to liver, kidney and other organ related disorders2.

Liver is one of the largest organ in human body and is the chief site

for intense metabolism and excretion. So it has a surprising role in the maintenance, performance and regulating homeostasis of the body. It is involved with almost all the biochemical pathways to growth, fight against disease, nutrient supply, energy provision and reproduction3. But it is continuously and variedly exposed to environmental toxins and abused by poor drug habits ,alcohol and prescribed over the counter drugs which can eventually lead to various liver ailment like hepatitis, cirrhosis and alcoholic liver diseases4,5.

Hepatotoxicity implies chemical-driven liver damage. The liver plays a central role in transforming and clearing chemicals and is susceptible to the toxicity from these agents. Certain medicinal agents, when taken in overdoses and sometimes even when introduced within therapeutic ranges, may injure the organ. Other chemical agents, such as those used in laboratories and industries, natural chemicals (e.g. microcystins) and herbal remedies can also induce hepatotoxicity. Chemicals that cause liver injury are called hepatotoxins.More than 900 drugs have been implicated in causing liver injury6 and it is the most common reason for a drug to be withdrawn from the market. Drug induced liver injury is responsible for 5% of all hospital admissions and 50% of all acute liver failures.7,8

Kidney being one of the vital organs of human body performing the function of detoxification needs protection for healthy life. Many herbal drugs have been investigated for various ailments, however, very few herbs have been considered for nephroprotection it is often associated with a reduction in proteinuria and the amelioration of dyslipidaemia, but not with changes in systemic blood pressure 9.

Nephrotoxicity is a poisonous effect of some substances, both toxic chemicals and medication, on the kidneys. There are various forms of toxicity.10 Nephrotoxicity should not be confused with the fact that some medications have a predominantly renal excretion and need their dose adjusted for the decreased renal function (e.g. heparin).The kidney is highly susceptible to toxicants for two reasons. A high volume of blood flows through it and it filtrates large amounts of toxins which can concentrate in the kidney tubules. Nephrotoxicity is toxicity to the kidneys. It can result in systemic toxicity causing:

• decreased ability to excrete body wastes

• inability to maintain body fluid and electrolyte balance

• decreased synthesis of essential hormones (e.g., erythropoietin)11

But there are not many drugs available for the treatment of liver and renal disorders12,13.The addition of newer drugs, besides contributing to better therapeutic potential, has added another dimension to the causation of renal disease. The abuse or indiscriminate use of drugs, coupled with easy availability, has taken its toll on the kidneys. Drug-related causation was implicated in 18.3% of acute renal failure cases reported in a one year survey.14 In India, a study reported 20% incidence of drug-induced acute renal failure with a large proportion (40%) being secondary to aminoglycoside usage.15 Drugs continue to be taken off the market due to late discovery of hepatotoxicity and nephrotoxicity . Due to its unique metabolism and close relationship with the gastrointestinal tract, the liver is susceptible to injury from drugs and other substances. 75% of blood coming to the liver arrives directly from gastrointestinal organs and then spleen via portal veins which bring drugs and xenobiotics in concentrated form. Several mechanisms are responsible for either inducing hepatic injury or worsening the damage process. Many chemicals damage mitochondria, an intracellular organelle that produce energy.Its dysfunction releases excessive amount of oxidants which in turn injures hepatic cells. Activation of some enzymes in the cytochrome P-450 system such as CYP2E1 also lead to oxidative stress.16 Injury to hepatocyte and bile duct cells lead to accumulation of bile acid inside liver. This promotes further liver damage.17

Most of the commonly used drugs are known to cause liver damage . Acetaminophen (Paracetamol) is usually well tolerated in prescribed dose but overdose is the most common cause of drug induced liver disease and acute liver failure worldwide 18. Although individual analgesics rarely induce liver damage due to their widespread use, NSAIDs have emerged as a major group of drugs exhibiting hepatotoxicity19. Isoniazid (INH) is one of the most commonly used drug for tuberculosis and is associated with mild elevation of liver enzymes in up to 20% of patients and severe hepatotoxicity in 1-2% of patients20.Some of the drugs withdrawn for hepatotoxicity includes Troglitazone, bromfenac, trovafloxacin, ebrotidine, nimesulide , nefazodone, ximelagatran and pemoline. 21,22

Drugs which are known to cause nephrotoxicity includes antibiotics like Aminoglycosides (10-15% Incidence of Acute Tubular Necrosis) occurs in 10-20% patients on 7 day course.Sulfonamides,Amphotericin B (Incidence 80-90%, esp. with deoxycholic acid formulation ) Foscarnet and Quinolones (e.g. Ciprofloxacin, Levofloxacin) . AntiHyperlipidemics which includes Statin Drugs (Rhabdomyolysis) and Gemfibrozil (associated with Acute Renal Failure due to Rhabdomyolysis) ,NSAIDs and drugs of abuse like Cocaine ,Heroin, Methamphetamine, and Methadone.23

The WHO estimates that 80% of people living in developing countries rely almost exclusively on traditional medicine for their primary health care needs. Medicinal plants form the back bone of traditional medicine and hence more than 3300 million people utilize medicinal plants on a regular basis. Demand for medicinal plants is increasing due to growing recognisation of natural products being non toxic, having no side effects. Moreover in these societies herbal remedies have become more popular in the treatment of minor ailments on account of the increasing cost of personal health maintenance.24 However, only a small proportion of hepatoprotective plants as well as formulations used in traditional medicine are pharmacologically evaluated for their safety and efficacy25. Some of the medicinal plants like Spilanthes ciliate are used in the management of liver disorders have been scientifically investigated and reported to possess measurable hepatoprotective effects26 against various experimental animal models.Some plants of Asteraceae family like Calendula officinalis is used for hepato and renoprotection27 .However, still more numbers of medicinal plants are needed to be screened for their hepatoprotective and nephroprotective efficacy.Hence, searching the safe and potent remedies from the herbal origin for the treatment of hepatic and nephro disorders has become most fascinating and desired area of research for the pharmacologists.

Spilanthes radicans , also biosynonymed as Acmella radicans is an indigenous herb of the family Asteraceae.28 It is an important medicinal plant with rich source of therapeutic constituents. The genus Spilanthes (Asteraceae) comprises 30 species and 9 additional intraspecific taxa that are mainly distributed in the tropical and subtropical regions around the world (Jansen, 1985). In particular, this species is famous as a folklore remedy for toothache and for throat and gum infections, earning it the English nickname, the “toothache plant”. Spilanthes contains a number of biologically active compounds (Prachayasittikul et al., 2009), of which the most studied have been the alkylamides, which this plant possesses in abundance29 (Nakatani et al., 1992).Alkylamides from other plants have shown such activity by inhibiting Human Cytochrome P-4502E1 Activity 30.

It has been found that the other species of Spilanthes radicans like Spilanthes ciliate have antihepatotoxic activity, Spilanthes acmella Murr. (para-cress or toothache plant) is a medicinal plant of the Compositae family 31, which is known in Thai as Phak-Kratt Huawaen and has long been used as a traditional medicine for toothache, headache and treatment of asthma, rheumatism, fever, sore throat and haemorrhoids 31,33. Its root decoction has been used as a laxative and diuretic drug 32,34.

Literature survey reveals that the alkamides from the species of other plants like Herba Echinaceae Purpureae35 are extensively used for a variety of condition like hepatotoxicity and cytotoxicity.The presence of alkamides in Spilanthes radicans has already been proved , however the hepatoprotective and nephroprotective activity of Spilanthes radicans is not investigated scientifically. The present study is planned to evaluate the hepatoprotective and nephroprotective activity of this plant in animal models.

Rajesh et al. studied the toxicity evaluation of Spilanthes acmella having the same chemical constituents and the maximum no-lethal dose was found to be 5000mg/kg body weight; hence 1/10th of the dose was taken as effective dose (500mg/kg body weight) for the ethanol extract of Spilanthes acmella, Murr. leaves for diuretic activity36.

ENCLOSURE: II

6.2 Review of literature

Spilanthes radicans also known as (Adormemuela,Indianfood,quiebramuelas) biosynonymed as Acmella radicans37 is an indigenous herb of the family Asteraceae.38

➢ Palani et al. conducted a study on in vivo analysis of nephro & hepato protective effects and antioxidant activity of Madhuca longifolia against acetaminophen-induced toxicity & oxidative stress39 .

➢ Gopi et al. studied acetaminophen induced hepato and nephrotoxicity by silymarin and Terminlia chebula 40.

➢ Ramírez et al. studied the presence of alkamides in Acmella radicans41.

➢ Suja et al conducted a study on antihepatotoxic activity of Spilanthes ciliate 42.

➢ Pandey conducted a study on antimalarial activity of three species of Spilanthes (Akarkara) against malaria (Anopheles stephensi Liston,Anopheles culicifacies, species C) and filaria vector43.

➢ Li-chen et al. conducted a study on Anti-inflammatory Effect of Spilanthol from Spilanthes acmella on Murine Macrophage by Down-Regulating LPS-Induced Inflammatory Mediators44.

➢ Ratnasooriya et al. conducted a study on Diuretic activity of Spilanthes acmella flowers in rats45.

➢ Morikawa studied that Eighty percent (80%) aqueous acetone extract from fruit of Piper chaba (Piperaceae) was found to have a hepatoprotective effect on D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced liver injury in mice. Among the isolates, several amide constituents inhibited D-GalN/tumor necrosis factor-alpha (TNF-alpha)-induced death of hepatocytes and its mode of action was suggested to depend on the reduced sensitivity of hepatocytes to TNF-alpha46.

➢ Virgilijus et al. concluded that Echinacea purpurea decreases cadmium-induced liver necrosis and mitotic activity of hepatic cells and increases the apoptotic activity of liver cells47.

➢ Barnes et al. assessed that several components of genus Echinaceae particularly the alkamides and caffeic acid derivative,have clear pharmacologic activity48.

➢ Rodeir et al. suggest that the alkamide content of Heliopsis longipes ,Mangifera indica L , Thalassia testudinum inhibit the major human P450 enzymes involved in drug metabolism and could induce potential herbal-drug interactions49.

➢ Cabral-De-Anda studied that the extracts of Acmella radicans proved to inhibited the growth of Mycobacterium tuberculosis in up to 80%, suggesting that medicinal plants are an important source of potential active compounds with antifimic activity50.

CHEMICAL COMPOSITION51:

The composition of the essential oil of the fresh whole Acmella radicans (Jacq.) (Asteraceae) plant and two root-extracts from southern India were investigated by gas chromatogramphic–spectroscopic (GC-FID and GC-MS) and olfactive methods to identify those volatiles responsible for the characteristic, pleasant aroma of these samples. The essential oil was found to be rich in 2- tridecanone (30.1%),1-pentadecene (25.2%), trans-β-caryophyllene (12.8%), elemol (4.6%), guaiol (3.1%) and some further caryophyllene derivatives, the petroleum ether extract was rich in 1-pentadecene (54.1%), 2-tridecanone (10.3%),2-pentadecanone (6.7%), 2-pentadecanol (4.1%), trans-β-caryophyllene (3.6%) and 1-tridecene (3.0%); and the petroleum ether–methanol–ethyl acetate extract was rich in 2-tridecanone (58.2%), 1-pentadecene (15.00%) and palmitic acid(16.6%).

Besides these we found six alkamides in this plant.

➢ N-isobutyl-(2E,6Z,8E)-decatrienamide (affinin)

➢ N -(2-methylbutyl) -(2E,6Z,8E)-decatrienamide

➢ N-(2-methylbutyl)- (2E,4Z,8E,10E)-dodecatetraenamide

➢ N-(2-phenylethyl)- (2Z,4E)-octadienamide

➢ N-(2-phenylethyl)-nona-2E-ene-6,8-diynamide and

➢ 3-pheny-N-(2-phenylethyl)-2-propenamide.

The profiles of these six alkamides in A. radicans vary with the time and developmental stage.52

ENCLOSURE: III

6.3 Objectives of the study :

The present research work is an attempt to establish the possible preventive and curative hepatoprotective and nephroprotective efficacy using 70% alcoholic extract of leaves and roots of Spilanthes radicans in rats with the following objectives.

1. To prepare 70% alcoholic extract of leaves and root of Spilanthes radicans.

2. To analyse the crude extract for the presence of phytoconstituents.

3. To evaluate the hepato and nephroprotective activity in animal models.

ENCLOSURE: IV

7. Materials and methods

7.1 Source of data:

The work is aimed to generate data from experiments to be conducted at pharmacology laboratory of our institution. Albino rats and mice will be used for this purpose.

The experiments, which involves the following steps:

1. Collection and identification of the roots and leaves of Spilanthes radicans.

2. Extraction of leaves and root of Spilanthes radicans with 70% alcohol.

3. Conducting qualitative chemical tests for the presence of phytoconstituents.

4. Evaluation of the crude extract for the hepato and nephroprotective activity

in rats.

ENCLOSURE: V

Method of collection of data:

1) Collection of raw material:

For this study, the leaves and root of Spilanthes radicans will be collected from the surrounding gardens of FRLHT (Foundation for Revitalisation of Local Health Traditions) Jarakabande Kaval, Post Attur,via Yehalanka , Bangalore (560106). The sample will be identified by the Botanist. Fresh leaves and root will be cleaned and shade dried at room temperature.

2)Extraction with 70% alcohol:

Then the powdered materials will be extracted with 70% alcohol by Soxhlet’s extraction method53. Thereafter, the extracts will be concentrated using rotary flash evaporator and percentage yield of the same will be recorded.

3) Test for phytoconstituents:

The crude extracts thus obtained will be subjected to preliminary phytochemical screening following the standard procedures described in the literature.

4) Determination of acute toxicity:

The maximum no-lethal dose of Spilanthes acmella having the same chemical constituents was found to be 5000mg/kg body weight hence 1/10th of the dose was taken as effective dose (500mg/kg body weight) for the ethanol extract of Spilanthes acmella, Murr. leaves for diuretic activity54.

5) Screening of hepato and nehroprotective activity55.

Hepatoprotective and nephroprotective activity of 70% alcoholic extract of leaves and root of Spilanthes radicans will be evaluated for the possible preventive and curative effects on CCl4 and paracetamol induced liver injury in rats. Liver protective efficacy of the test extracts will be assessed by measuring the biochemical markers level such as SGPT, SGOT, ALP, ACP and Bilirubin (Total and Direct) . Further, histopathological examination of the liver and kidney sections will be determined.

CCl4 induced hepatotoxicity56:Sixteen adult male wistar rats were equilibrated for seven days, randomly divided into four groups of four rats each and housed in separate cages. They were fasted for 12hrs prior to the experiment. CCl4 was dissolved in corn oil in the ratio of 3:1 (v/v) and administered to the rats. Rats in group I (control) were intraperitoneally injected (i.p.) with 5ml/kg corn oil. Rats in groups II, III and IV were injected (i.p.) with 5ml/kg CCl4 (single dose). After 48hrs, extract of Spilanthes radicans leaf was administered to groups III and IV respectively.The rats were sacrificed after 5 days, liver is collected and histopathologic studies are made.

In the serum the following parameters are determined :

• Total bilirubin

• Total bile acids

Paracetamol induced hepato and nephrotoxicity57:A total of 24 animals were equally divided into 4 groups of six each. Group – I served as normal control received 0.5% (CMC) carboxy methyl cellulose solution (1 ml/kg) once daily for 3 days. Group – II served as paracetamol control, administered with paracetamol (3gm/kg) as single dose on day 3. Group – III received Spilanthes radicans extract (200 mg/kg) once daily for 3 days. Group – IV served as reference control, received Silymarin (25mg/kg) once daily for 3 days. Group III and IV received paracetamol (3gm/kg) as single dose on day 3, thirty minutes after the administration of Spilanthes radicans and Silymarin respectively. All the test drugs and paracetamol were administered orally by suspending in 0.5% CMC solution. After 48 hrs. of paracetamol feeding, the blood was collected by direct cardiac puncture under light ether anesthesia and liver protective efficacy of the test extracts will be assessed by measuring the biochemical markers level such as SGPT, SGOT, ALP, ACP and Bilirubin (Total and Direct). Further, histopathological examination of the liver and kidney sections will be determined.

Cisplatin induced nephrotoxicity58: Five groups (n=6) were used to study the effect of extract against Cisplatin induced renal toxicity in rats. Group I was administered with equivalent volumes of vehicle (distilled water) for 10 days, which served as normal control. Group II, III, IV and V were administered cisplatin 5 mg/ kg of body weight single dose, i.p. (intraperitoneal). The blood was withdrawn on 5th day from group second and on 15th day from group third to check the persistence of renal injury. IV group served as curative regimen, which was treated with extract of Spilanthes radicans and animals of group V were treated with cystone (standard drug) 500 mg/kg along with cisplatin 5mg/kg for 5 days. After 2 weeks of treatment, animals were anaesthetized by anesthetic ether and sacrificed. Blood was then collected by cardiac puncture and kidneys were dissected out immediately and transferred into 10% formalin for its histopathological studies 59,60.

6) Statistical analysis :

The data obtained from the above findings will be subjected to statistical analysis using one-way ANOVA followed by Turkey Kramer Multiple Comparison Test to assess the statistical significance of the results.

7) Work plan details :

Total duration for the completion of proposed research work may be ten months

1. Collection of plant materials including authentication - One month.

2. Duration of experimentation on animals including - Five months.

preparation of crude extracts

3. Literature collection - Two months.

4. Dissertation writing and communication of research - Two months.

papers.

ENCLOSURE-VI

3. Does the study require any investigation or interventions to be conducted on patients or other humans and animals? if so please describe briefly.

The proposed study requires the investigation on albino rats of either sex (Wistar Strain) weighing 150 - 200 gm for the hepatoprotective and nephroprotective activity. Whereas albino mice of Swiss Strain will be utilized for the acute toxicity study.

4. Has ethical clearance been obtained from your institution in case of 7.3?

The present study protocol is approved from Institutional Animal Ethics Committee .

ENCLOSURE VII

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