Evaluation of Annona Squamosa Leaf Extract for its Adaptogenic and Antidepressant Activity
M.PHARMA DISSERTATION PROTOCOL SUBMITTED TO
RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES, KARNATAKA,
BANGALORE-560041
By
Mr. NAGARAJ B. Pharma.,
Under the Guidance of
Mrs. N. C. Nagalakshmi M. Pharma.,
Assistant Professor
MALLIGECOLLEGE OF PHARMACY,
MalligeCollege of Pharmacy,
#71 Silvepura, Bangalore-560090
Rajiv Gandhi University of Health Sciences,
Karnataka, Bangalore.
Annexure – II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
01 / Name and Address of the Candidate / Nagarajs/o Anilkumar Kolkur,
Ganeshwadi, CHITGUPPA,
Dt.Bidar, State:Karnataka INDIA.
02 / Name of the Institution / MalligeCollege of Pharmacy
#71 Silvepura, Chikkabanavara Post,
Bangalore-560090
03 / Course of the Study Branch / M.Pharma (Pharmacology)
04 / Date of Admission to course / 11-11-2010
05 / Title of the Topic / Evaluation of Annona squamosaLeaf Extract for its Adaptogenic and Antidepressant Activity.
06 / Brief resume of the intended work
6.1. Need for the Study / Enclosure – I
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 Investigations on animals?
If yes give details / Enclosure – VI
7.4. Has ethical clearance been obtained from your institution in case of 7.3. / Yes
08 / List of References / Enclosure – VII
09 / Signature of the Candidate / (Nagaraj)
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.Nagaraj under my supervision.
11 / 11.1Name and Designation of Guide / Mrs. N. C. Nagalakshmi M. Pharma.,
Assistant Professor
MalligeCollege of Pharmacy,
Bangalore, Karnataka.
11.2.Signature
11.3.Co-Guide
11.4.Signature
11.5.Head of the Department / Dr. Shivakumar SwamyM. Pharma., Ph.D.,
HOD & Principal,
MalligeCollege of Pharmacy,
Bangalore, Karnataka.
11.6.Signature
12 / Remarks of the Principal
12.1. Signature / The present study is permitted to perform in the Pharmacology laboratory of our institution and the study protocol has to be approved by IAEC.
(Dr. Shiva Kumar Swamy)
Enclosure I
06. Brief resume of the intended work:
Stress is an internationally recognized phenomenon fortified by advancement of industrialization in a demanding civilization. Thus, every individual is likely to face stressful situations in day-to-day life. Stress represents a reaction of the body to a stimulus that tends to alter its normal physiological equilibrium or homeostasis and has been defined as a nonspecific response of the body to any demand imposed on it. Several plants have been investigated for their adaptogenic and rejuvenating properties in traditional medicine. The drugs of plant origin are gaining increasing popularity due to their minimum toxicity and more effectiveness1.
Stress is a common phenomenon that is experienced by every individual. When stress becomes extreme, it is harmful for the body and hence, needs to be treated. Stress is involved in the pathogenesis of a variety of diseases that includes psychiatric disorders such as depression and anxiety, immuno-suppression, endocrine disorders including diabetes mellitus, male impotence, cognitive dysfunction, peptic ulcer, hepato and nephro dysfunction, hypertension, and ulcerative colitis2, 3.
General adaptation syndrome (GAS)
Physiologists define stress as how the body reacts to a stressor, real or imagined a stimulus that causes stress. Acute stressors affect an organism in the short term; chronic stressors over the longer term.
Alarm is the first stage. When the threat or stressor is identified or realized, the body's stress response is a state of alarm. During this stage adrenaline will be produced in order to bring about the fight-or-flight response. There is also some activation of the Hypothalamic-Pituitary-Adrenal (HPA) axis, producing cortisol.
Resistance is the second stage. If the stressor persists, it becomes necessary to attempt some means of coping with the stress. Although the body begins to adapt to the stress or demands of the environment, the body cannot keep this up indefinitely, so its resources are gradually depleted.
Exhaustion is the third and final stage in the GAS model. At this point, all of the body's resources are eventually depleted and the body is unable to maintain normal function. The initial autonomic nervous system symptoms may reappear (sweating, raised heart rate etc.). If stage three is extended, long term damage may result as the body, and the immune system is exhausted and function is impaired resulting in decompensation. The result can manifest itself in obvious illnesses such as ulcers, depression, and diabetes, trouble with the digestive system or even cardiovascular problems, along with other mental illnesses.
An adaptogen increases the power of resistance against physical, chemical or biological noxious agents; it has a normalizing influence on body. The mode of action of adaptogen is basically associated with the stress system. Adoptogens increase the capacity of stress system to respond to external signals at the higher level of the equilibrium of activating and deactivating mediators of stress response. The plant adoptogen is defined as “Smooth pro-stressors which reduce reactivity of host defense systems and decrease damaging effects of various stressors due to increased basal level of mediators involved in the stress response”3.
Mental depression is one of the common chronic illnesses that affect the mood, thought, physical health and behavior of an individual. In the recent years it is recognized as a major health problem. Although the mechanism provoking depression has not been clearly elucidated, the main trigger is known to be exposure tochronic stress4.
Depression is a mental disorder and is recognized to be symptomatically, psychologically and biologically heterogeneous. The disorder is characterized by apathy, loss of energy, retardation of thinking and activity, as well as profound feelings of gloominess, despair and suicidal ideation.
According to the World Health report, approximately 450 million people suffer from a mental or behavioral disorder. This accounts to 12.3% of the global burden of disease, and will be expected to rise 15% by 2020. Psychiatric illness is also often associated with suicide and there are between 10 and 20 million suicide attempts every year.
In spite of the availability of various classes of antidepressant drugs, depression continues to be a major medical problem. Various plants are being used in complementary and alternative medicines for management of stress and mood disorders, because of their minimum toxicity and more effectiveness5.
6.1. Need for the study:
The family (Annonaceae), is a large family which comprising about 130 genera over 2000 species; the most important genera having a largest number of species of Annona, with 120 species, from genera, the species of Annona squamosa commonly known as custard apple is cultivated throughout India. The plant is traditionally used for the treatment of epilepsy, dysentery, cardiac problem, worm infestation, constipation, hemorrhage, antibacterial, dysuria, fever, and ulcer. It also has antifertility6and antitumor7. Phytochemical, pharmacological, antibacterial andantiovulatory have been studied with seed extract8. The aqueous leaf extract has also beenreported to ameliorate hyper thyroidism9. Ethanolic extract of leaves and stem are reportedto have an anti cancerous activity10. It is used as an insecticidal agent and has beeninvestigated by several workers11-12. Free radical scavenging activity13, hypoglycemic anti-diabetic effect14and hepato protective activity of Annona squamosa was reported in theleaf extract15. Aporphine alkaloids16, flavonoids13, glycoside17, terpine derivative and novel diazepine, squamoline13 were isolated from this plant.
Perusal of the literature survey indicates that there are no literatures or studies were done on the Adaptogenic and Antidepressant activity of leaf extact ofAnnona squamosa. The uses of herbal drugs are being more popular.This study is designed to evaluate the Adaptogenic and Antidepressant activity of leaf extract of Annona squamosa.
Enclosure II
6.2 Review of literature:
Annona squamosa also known as (Custard apple, Sugar apple, Sweet-sop, Seetaphal) is an indigenous herb of the family Annonaceae.
Smita Sharma et al. Conducted anti-inflammatory and anti-depressant activity of leaves and rhizome extract of Annona squmosa and Curccuma longa.The polyherbal formulation showed the significant anti-inflammatory activity comparable to the standard drug Indomethacin against carrageenan induced rat paw edema method. The polyherbal formulation reduced the inflammation induced by carrageenan by 49.3% and 61.73% on oral administration at 100 mg/ kg and 200 mg/kg respectively as compared to the control treated group an analgesic activity comparable to the standard drug tramadol HCl. Antidepressant activity comparable to the standard drug Fluoxetine HCl.
SharmaMC et al. In vitro studies of the using some medicinal herbal leaves against antidepressant, analgesic activity, and anti-inflammatory activity. The polyherbal formulation showed the significant anti-inflammatory activity comparable to the standard drug Indomethacin against carrageenan induced rat paw edema method. The polyherbal formulation reduced the inflammation induced by carrageenan by 53.0% and 47.0% on oral administration at 100 mg/ kg and 200 mg/kg respectively as compared to the control treated group an analgesic activity comparable to the standard drug tramadol HCl.
Mujeeb Mohd et al.Antidiabetic activity of the aqueous extract of Annona squamosa in streptozotocin induced- hyperglycemic rats.The aqueous extract of roots of Annona squamosa L. at a dose of 250 mg/kg and 500 mg/kg body weightrespectively was tested for antidiabetic activity in Streptozotocin (STZ) - induced hyperglycaemic rats. Theblood glucose levels were measured at 0, 2h, 4h and 6h after the treatment. The aqueous extract reduced theblood glucose in STZ- induced diabetic rats from 285.52 to 208.81 mg/dl, 6h after oral administration ofextract (P<0.01). The antidiabetic activity of aq. Extract of Annona squamosa was compared withglibenclamide, an oral hypoglycaemic agent (3mg/kg).
ChandrashekarC et al.Isolation, characterizations and free radical scavenging activity of Annona squamosa leaf.The chemical compound isolated was analyzed by IR, LC-MS and the compound was confirmed flavones type compound on the basis of spectral data. The in vitro antioxidant activity of isolated compound (AS-1) was evaluated by free radical scavenging activity of different concentrations (10μg, 50 μg, and 100 μg) using 1, 1-diphenyl-2 picryl hydrazil method (DPPH). The results of assay were then compared with synthetic antioxidant Butylated hydroxyl anisole (BHA).The isolated compound exhibit (9.62, 24.28, and 45.62%) significant free radical scavenging activity.
Chemical composition:
Annona squamosa consist of steroids, alkaloids, saponins, terpenes, tannins, phenolic substances, carbohydrates, volatile oil and mucilage. Quantitative estimations revealed the presence of 2% of volatile oil in fresh leaves and 15.26% of total mucilage content in the dried leaf powder21.
Twelve compounds were isolated from Annona squamosa. Their structures were identified as liriodenine , moupinamide , -kauran-16 alpha-ol-19-oic acid , 16 beta, 17-dihydroxy-kauran-19-oic acid , anonaine , 16 alpha, 17-dihydroxy-kauran-19-oic acid , -isokaur-15-en-17,19-dioic acid , squamosamide , 16 alpha-methoxy-kauran-19-oic acid , sachanoic acid,-kauran-19-al-17-oic acid , daucosterol22.
Enclosure III
6.3 Objectives of the study:
The present study is undertaken to evaluate the possible Adaptogenic and Antidepressant efficacy using aqueous and ethanolic extract of leaves of Annona squamosa in rats and mice with the following objectives:
- Collection, Authentication and Extraction (ethanolic and aqueous) of Leaves of Annona squamosa.
- Qualitative estimation of phytochemical constituents.
- Standardization of the therapeutic dose and confirmation of any acute toxicity of ethanolic and aqueous extracts.
- To evaluate the Adaptogenic activity on different animal models like,
- Anoxia Stress Tolerance
- Swimming Endurance Test in Rats
- Cold Resistant Stress
- To evaluate the Antidepressant Activity in Different Animal Models.
- Tail suspension test (TST)
- Forced Swim Test (FST)
- Elevated Plus-Maze.
Enclosure IV
07. 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:
- Collection and authentication of the leaves of Annona squamosa.
- Extraction of leaves of Annona squamosa with aqueous and ethanol.
- Conducting qualitative chemical tests for phytochemical screening.
- Evaluation of the crude leaves extract for the Adaptogenic and Antidepressant activity in rats and mice.
Enclosure V
7.2Method of collection of data:
1) Collection of raw material:
The leaves of Annona squamosa will be collected from the surrounding gardens of Tarabanahalli. The sample will be identified and authenticated by the botanist. Fresh leaves will be cleaned and shade dried at room temperature.
2) Extraction with Water and Ethanol:
Powder will be extracted with ethanol and water by Soxhlet’s extraction method13. Thereafter, the extracts will be concentrated using rotary flash evaporator and percentage yield of the same will be recorded. Finally the concentrated crude extract will be used for the study.
3) Test for phytoconstituents:
The crude extracts thus obtained will be subjected to preliminary phytochemical screening following the standard procedures described in the literature15.
4) Determination of acute toxicity:
Swiss albino mice of either sex (25-30g) are selected for the test. Acute toxicity studies will be carried as per OECD guidelines. 10 miceare randomly divided into two groups of five miceeach. The toxicological effects will be assessed on the basis of mortality and behavioral changes, occurs during 48 hours23.
5) Screening of Adaptogenic activity:
Anoxia Stress Tolerance24:
Albino mice of either sex weighing between 18-22 g are divided into 7 groups of six in each. Hermetic vessel of 500 ml air capacity is used for this test. Each animal is kept in the hermetic vessel and the time to show the first sign of convulsion is noted, it is immediately removed from the vessel and resuscitated if needed. After one week of drug treatment the animals are once again exposed to the anoxia stress. Similarly the animals are also observed at the end of 2nd and 3rd week with the same treatment and the time duration for anoxia stress tolerance is noted.
Research Design
Group -IControl treated with vehicle
Group-IINegative Control
Group -IIIStandard drug (Withania somnifera-100mg/kg)
Group -IVAnnona squamosa alcoholic extract dose -1
Group - VAnnona squamosa alcoholic extract dose -2
Group-VIAnnona squamosa aqueous extract dose -1
Group-VIIAnnona squamosa aqueous extract dose -2
Swimming Endurance Test in Rats24:
Rats of either sex (160-200 g) are divided into 7 groups of six in each are used for the study. Stress is exerted in rats by keeping them in cylindrical vessels (length 48 cm and width 30 cm) filled with water to a height of 25 cm and the total swimming time for individual rat is noted, the rats are allowed to swim daily till exhausted . Extracts are given to rats once daily for a period of 7 days and on 8th day mean swimming time for each group is calculated, blood is collected through retro orbital plexus under light ether anesthesia to estimate biochemical parameters like blood glucose, triglycerides, cholesterol, BUN and blood cell count (RBC, WBC, DLC). Animals are sacrificed by cervical dislocation and the weights of organs such as liver, adrenals, spleen are to be recorded after washing with alcohol.
Research Design
Group -IControl treated with vehicle
Group-IINegative Control
Group -IIIStandard drug (Withania somnifera-100mg/kg)
Group -IVAnnona squamosa alcoholic extract dose -1
Group - VAnnona squamosa alcoholic extract dose -2
Group-VIAnnona squamosa aqueous extract dose -1
Group-VIIAnnona squamosa aqueous extract dose -2
Cold Resistant Stress24:
Rats are divided into 7 groups of 6 animals in each are subjected to cold stress by exposing them to 4 ± 100C daily for 2 hours. The extracts are administered daily once to respective groups. This procedure is repeated for a period of 10 days. Blood is collected through orbital plexus under light ether anesthesia to estimate biochemical parameters like blood glucose, triglycerides, cholesterol, BUN and blood cell count (RBC, WBC, DLC). Animals are sacrificed by cervical dislocation and the weights of organs such as liver, adrenals, spleen are to be recorded after washing with alcohol.
Research Design
Group -IControl treated with vehicle
Group-IINegative Control
Group -IIIStandard drug (Withania somnifera-100mg/kg)
Group -IVAnnona squamosa alcoholic extract dose -1
Group - VAnnona squamosa alcoholic extract dose -2
Group-VIAnnona squamosa aqueous extract dose -1
Group-VIIAnnona squamosa aqueous extract dose -2
Screening of Antidepressant Activity:
Tail Suspension Test (TST)25:
The mice are hung by the tail on a plastic string 75 cm above the surface with the help of an adhesive tape. The duration of immobility is observed for a period of 8 minutes. The duration of immobility is recorded during the last 6 minutes of the observation period. Mice are considered to be immobile only when they hung passively and are completely motionless.
Research Design
Group -IControl treated with vehicle
Group-IINegative Control
Group -IIIStandard drug (Imipramine-10mg/kg)
Group -IVAnnona squamosa alcoholic extract dose -1
Group - VAnnona squamosa alcoholic extract dose -2
Group-VIAnnona squamosa aqueous extract dose -1
Group-VIIAnnona squamosa aqueous extract dose -2
Forced Swim Test (FST)25:
Each mouse is placed individually in 5 litre glass beakers, filled with water upto a height of 15 cm and are observed for duration of 6 minutes. The duration of immobility is recorded during the last 4 minutes of the observation period. The mouse is considered immobile when it floated motionlessly or made only those moments necessary to keep its head above the water surface. The water is changed after each test.
Research Design
Group -IControl treated with vehicle
Group-IINegative Control
Group -IIIStandard drug (Imipramine-10mg/kg)
Group -IVAnnona squamosa alcoholic extract dose -1
Group - VAnnona squamosa alcoholic extract dose -2
Group-VIAnnona squamosa aqueous extract dose -1
Group-VIIAnnona squamosa aqueous extract dose -2
Elevated Plus-Maze26:
The elevated plus-maze comprised two open (30 cm×5 cm×0.25 cm) and two enclosed (30 cm×5 cm×15 cm) arms that radiated from a central platform (5 cm×5 cm) to form a plus sign. The maze is constructed of black painted wood. A slight raised edge on the open arms (0.25 cm)provided additional grip for the animals. The plus-maze is elevated to a height of 40 cm above floor level by a single central support. The experiment is conducted during the dark phase of the light cycle (9:00–14:00 h). The trial is started by placing an animal on the central platform of the maze facing an open arm. The number of entries into, and the time spent in, each of the two types of arm, are counted during a 5 min test period. The percentage open arm entries and percentage open arm time are used as indices of anxiety. A mouse is considered to have entered an arm when all four paws are on the arm. The apparatus is cleaned thoroughly between trials with damp and dry towels. All behavioral recordings will be carried out with the observer unaware of the treatment received by mice.