4 - Rajiv Gandhi University of Health Sciences



EFFECT OF MORINGA OLEIFERA (Lam.) LEAVES ON STRESS MODULATED SEXUAL BEHAVIOUR IN MALE RATS

M.Pharm Dissertation Protocol

Submitted to the

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA.

By

SREEHARI. Y

B. Pharm.

Under the Guidance of

Mr. BHEEMACHARI

Assistant Professor,

Department of Pharmacology,

N.E.T Pharmacy College, Raichur

DEPARTMENT OF PHARMACOLOGY

N.E.T. PHARMACY COLLEGE

RAICHUR – 584103

2008-2009

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA.

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR

DISSERTATION

|1. |Name of the candidate |Sreehari. Y |

| |and Address |S/o Y.Bheemsen Rao |

| | |O.R.T – 172,SPM new colony, |

| | |Sirpur Kaghaznagar, dist- Adilabad. |

| | |Andhrapradesh. 504296 |

|2. |Name of the Institution: |N.E.T. Pharmacy College, |

| | |Raichur- 584103 |

|3. |Course of study and Subject: |Master of Pharmacy in Pharmacology |

|4. |Date of admission to the Course: |14th June 2008 |

|5. |Title of the topic: |

| | |

| |EFFECT OF MORINGA OLEIFERA (Lam.) LEAVES ON STRESS MODULATED SEXUAL BEHAVIOUR IN MALE RATS |

|6 |Brief resume of the intended work: |

| |6.1: Need for the study: ENCLOSURE -I |

| |6.2:Review of the literature ENCLOSURE -II |

| |6.3:Main objective of the study ENCLOSURE -III |

|7 |Material and methods: |

| |7.1: Source of the data ENCLOSURE -IV |

| |7.2: Methods of the collection of the data ENCLOSURE - V |

| |7.3: Does the study require any investigations or interventions to be conducted on patients, other human or animals? If so,|

| |please describe briefly. YES (Rats) |

| |7.4: Has ethical clearance been obtained from your institute in case of 7.3? |

| |YES: IAEC NO. : 576/2002/bc/IAEC/CPCSEA |

|8 |List of References ENCLOSURE-VI |

| | | |

|9. |Signature of the candidate: | |

| | |(Sreehari.Y) |

|10. |Remarks of the guide: | The work proposed in this synopsis can be carried out in our institute.|

|11. |Name and designation of | |

| |11.1 Guide |Mr. BHEEMACHARI |

| | |Assistant Professor & HOD |

| | |Department of Pharmacology, |

| | |N.E.T. PHARMACY COLLEGE, RAICHUR. |

| |11.2 Signature | |

| | | |

| |11.3 Co-guide |------- |

| | | |

| |11.4 Signature | |

| | |---------- |

| |11.5 Head of the department | |

| | | |

| | | |

| | | |

| |11.6 Signature |Mr. BHEEMACHARI |

| | |Assistant Professor & HOD |

| | |Department of Pharmacology, |

| | |N.E.T. PHARMACY COLLEGE, RAICHUR. |

| | | |

| | | |

| | | |

| | | |

| | | |

| | | |

|12. |12.1 Remarks of the Chairman |Prof. H. DODDAYYA |

| |and Principal |Principal, |

| | |N.E.T. pharmacy college, |

| | |Raichur, 584103. |

| |12.2 Signature | |

| | | |

| | | |

| | | |

ENCLOSURE –I

6. Brief resume of the intended work:

6.1: Need for the study

The art of making love, achieving ecstasy, and enjoying sexual pleasure have been described in all ancient scripts as a divine act. That still holds the same values in modern society. Sexual dysfunction may affect men of every age, race, and background. The emotions, uncertainities and life style disturbances like stressful working area, nutrient deficient food coincide with this condition and often have a significant effect on a man's self-esteem, as well as, his relationship with his partner.1

In males, stress induces suppression of testosterone secretion, spermatogenesis and libido. These altered male reproductive functions also involve reduced sperm counts, sperm concentration, sperm morphology, semen quality and sperm motility.2

On the other hand chronic stress by immobilization has been reported to produce an increase in circulating levels of adrenocorticotrophin (ACTH), as well as a general inhibitory effect on pituitary-gonadal function, both in males and females. In males, chronic stress induces low circulating levels of testosterone (T), prolactin and follicle stimulating hormone (FSH), although testosterone is known to be critical for an adequate expression of male sexual behavior.2

Erectile dysfunction is considered as one of the most important public health problem, since it affects larger percentage of men. Despite the increasing availability of effective conventional medical treatments, plant-derived and herbal remedies continue to provide a popular alternative for men seeking to improve their sexual life. 3

Sexual dysfunction is a serious medical and social symptom that occurs in 10–52 % of men and 25–63 % of women. In men aged 40–70 years, an estimated 34.8 % have moderate to complete erectile dysfunction.4 It has been recently estimated that more than 152 million men worldwide experienced erectile dysfunction (ED) in 1995, and that this number will rise by 170 million, to approximately 322 million by the year 2025. Treatment of ED usually involves the psychotherapeutic approach. Pharmacotherapy involves locally acting vasoactive drugs such as Papaverin and Alprostadil, and first-line oral therapy for ED includes phosphodiesterase type 5 (PDE-5) inhibitors such as Sildenafil, Vardenafil, and Tadalafil, which inhibit hydrolysis of the second messenger cyclic Guanosine monophosphate (cGMP), whose production is promoted by nitric oxide (NO) release within the penile smooth cells.4

Central stimulants like apomorphine and herbal drugs with aphrodisiac activity are also involved in treatment of ED. Surgical interventions are also used, including insertion of penile prostheses. The available drugs and treatments have limited efficacy, unpleasant side effects such as headache, flushing, dyspepsia, nasal congestion and color visual disturbances with PDE-5 inhibitors as well as contraindications in certain disease conditions.4

Hence, there is an increasing demand for the alternative therapies, particularly herbal therapies that are believed to be effective, safe and economical. In this context, in the present study an attempt is proposed to evaluate the effect of Moringa oleifera (Lam.) leaves extract on stress-induced changes in sexual behavior.

ENCLOSURE –II

6.2: Review of Literature

Literature survey reveals that, various forms of physical and psychological stress are believed to reduce sexual functions several studies have examined the relationship between stress and sexual behavior in male rats, with induced stresses such as restraint and electrical shock. These reports show that chronic psychological and physical stresses induce erectile dysfunction, which results from neurotransmission including the median preoptic area, and a reduced blood flow in genital organs. Based on these findings, we suspect that stress affects sexual function in both men and women. Hence, alternative therapies are gaining prime importance, particularly herbal drugs.5

Moringa oleifera (Lam.) Family: Moringaceae is a small or middle-sized tree, about 10 m in height, cultivated throughout India. It is a multipurpose tree, used as vegetable, spice, a source of cooking and cosmetic oil and as a medicinal plant. It is known as Drumstick in English, Saragvo in Gujrati, Soanjna in Hindi, Sajna in Bengali, Nugge in Kannada, Sigru in malyalam, Shevga in Marathi, Shobhanjana in Sanskrit, Munaga in Telugu and Murungai in Tamil.6

It is reported to contain alkaloids, flavonoids, anthocyanins, proanthocyanidins and cinnamates.7 It is used in abortion , diabetes and as an antipyretic , anthelmentic and antiherpes simplex virus type I (HSV-I) .All parts of the tree are considered to possess medicinal properties and used in the treatment of ascites, rheumatism, and venomous bites and as cardiac and circulatory stimulant. The root is laxative, expectorant, diuretic, and good for inflammations, throat, bronchitis, piles, cures stomatitis, urinary discharges and obstinate asthma . The root bark is useful in heart complaints, eye diseases, inflammation, dyspepsia, and enlargement of spleen. The root and bark are abortifacient . The leaves are anthelmintic, aphrodisiac, cures hallucinations, dry tumors, hiccough and asthma. Dried powder of leaf extract produces abortifacient activity in rats .The flowers cure inflammations and muscle diseases. The fruit cures biliousness, pain, Leucoderma and tumor. The seed cures eye diseases and head complaints. Oil is useful in leprous ulcers and as external application for rheumatism . The roots and seeds are prescribed for the treatment of snakebites and scorpion stings . Seeds extracts have been proposed as an eco-friendly alternative, due to their traditional use for the clarification of drinking water.7

Crude ethanolic extract of dried seeds, hot water infusion of flowers, leaves, roots, seeds and bark, crude methanolic extract of the roots is reported to produce anti-inflammatory activity. Oil from dried seeds, methanol and ethanol extract of free dried leaves is reported to produce antioxidant activity. Defatted and shell free seeds, fresh leaves juice, roots and bark is reported to produce antimicrobial activity. Aqueous extract of stem bark, ethanolic extract of leaves, ethanolic and aqueous extracts of whole pod and their parts, namely, coat, pulp and seed are reported to treat cardiac disorders. Leaves and fruits is reported to produce antihyperlipidemic activity. Paste of leaves, ethanolic extract of seeds is reported to produce anticancer activity. Aqueous and ethanolic extract of roots and flower, ethanolic extract of leaves is reported to produce antihepatotoxic activity. Leaves is reported to produce antihypertensive activity.7

Though, literature quotes the usefulness of leaves in treatment of sexual dysfunction, there is no authentic scientific data reported about the same. Hence the present work is proposed with the aim of evaluation of leaves of Moringa oleifera for its effect on stress modulated sexual behaviour in various validated animal models.

ENCLOSURE –III

6.3 Main objective of study:

The main objective of the proposed work is to evaluate the effect of Moringa oleifera (Lam.) leaves extract on normal sexual behavior and stress modulated sexual behavior in rats.

.

ENCLOSURE – IV

7. Materials and Methods:

7.1: Source of the data:

Whole work is planned to generate data from laboratory based experimental animal models as described in various National/International journals and experimental pharmacology books and through e- publishing and Helinet of RGUHS, Bangalore.

ENCLOSURE – V

7.2: Methods of collection of the data (including sampling procedure if any):

Data will be generated from experimental animal studies and are to be subjected for statistical analysis by ANOVA followed by Dunnet’s‘t’ test and p-values lower than 0.05 will be considered as statistically significant.

The whole study is divided into two phases for the sake of convenience.

In phase-I:

1. Preparation of various extracts of leaves of Moringa oleifera (Lam.)

The plant Moringa oleifera (Lam.) will be identified and authenticated by renowned botanist. From the authenticated plant the leaves will be collected. Further the leaf powder of Moringa oleifera (Lam.) has to be successively extracted with 70% ethanol and 30% water using Soxhlet apparatus. The dried extract will be subjected to oral toxicity study by following up and down method as per OECD guidelines No. 425 to obtain LD50 value.8 Three doses i.e. 1/20, 1/10 and 1/5th of the LD50 value will be selected and considered as low, medium and high doses respectively.

2. Preliminary phytochemical Screening

The preliminary phytochemical investigations will be carried out with Moringa oleifera (Lam.) leaf extracts for qualitative identification of phytochemical constituents like alkaloids, glycosides, phytosterols, fats and oils, etc.

Tests will be carried out by following standard methods. All the chemicals and reagents used in the tests will be of analytical grade.

In phase II:

1. Evaluation of Aphrodisiac activity 9, 10, 11

Male Albino rats weighing between (150-200 g) will be divided into following six groups of six rats each.

Group 1: Normal control (receives vehicle) p.o.

Group 2: Standard drug (testosterone 15mg/kg) p.o.

Group 3: Standard drug (sildenafil citrate 0.7mg/kg i.p.) p.o.

Group 4: Low dose of hydro alcoholic extract of Moringa oleifera ( Lam.) p.o.

Group 5: Medium dose of hydro alcoholic extract of Moringa oleifera (Lam.) p.o.

Group 6: High dose of hydro alcoholic extract of Moringa oleifera (Lam.) p.o.

All the treatments will be given for fourteen consecutive days. For the evaluation of aphrodisiac activity, on the 14th day these rats will be individually placed in cages, 3 h following the administration of treatment and will be given 15 min adaptation periods. A receptive female rat will be taken for each male, that had been brought into oestrus (oestradiol benzoate 12 μg in olive oil injected subcutaneously 56 hours prior to plus progesterone 0.5 mg in olive oil injected subcutaneously 8 hours prior to pairing) and placed in the cage before pairing.

The following parameters of sexual behavior will be monitored 20 min after pairing, under dim light and video recording will be done using Sony handy cam. Latency of first mount, number of mounts, latency of first intromission, inter intromission interval, number of intromissions, latency of first ejaculation, latency of second ejaculation, average ejaculation latency and number of ejaculations will be recorded.

2. Effect on Stress modulated sexual behavior in male rats.12, 13, 14, 15

Male Albino rats weighing between (150-200 g) will be divided into following six groups of six rats each and given following treatment for 28 days.

Group 1: Normal control (receives vehicle) p.o.

Group 2: Standard drug (testosterone 15mg/kg) p.o.

Group 3: Standard drug (sildenafil citrate 0.7mg/kg i.p.) p.o.

Group 4: Low dose of hydro alcoholic extract of Moringa oleifera (Lam.) p.o.

Group 5: Medium dose of hydro alcoholic extract of Moringa oleifera (Lam.) p.o.

Group 6: High dose of hydro alcoholic extract of Moringa oleifera (Lam.) p.o.

All the animals will be subjected to immobilization (IMB) stress by placing individually in Plexiglas cylinder (5 cm diameter and 16 cm large) for 6 h a day during light period starting from 8 am each day for 28 consecutive days. Water and food will be withdrawn during stress period.2 The following parameters will be studied on 28th day.

a. Sexual accessory Organ to body weight ratio:

Body weight of each animal will be measured before the IMB stress and drug treatment. The percentage change in body weight will be calculated after 28 days of treatment. Animals will be weighed just before sacrifice and body weight is noted. After sacrifying each animal by euthanasia with ether, accessory sexual organs viz, Testis, Vas

deferens, Prostate glands, Seminal vesicles, Epididymis and Adrenal glands will be isolated and weighed them in a wet condition to measure organ to body weight ratio.

b. Sperm density

Spermatozoa will be collected by flushing the vas deferens and epididymis in 2.0ml of normal saline, draw the semen in the WBC pipette up to 0.5 mark, and diluted to 11 mark by using 4 % sodium bicarbonate in 1 % phenol solution. That makes a dilution of 1 in 20. Thoroughly shake the mixture, discard the first few drops and then add 2 to 3 drops on a neubauer’s counting chamber and observe under light microscope. Sperm count/ml will be calculated as given below.

Total number of sperms / ml plasma = N x 50000

Where N is the total sperm count observed in outer four square of WBC chamber.

c. Sperm motility

The relative proportions of the normal and abnormal sperms will be calculated by smear preparation according to the method of Bauer et al., (1974). Equal volume of Cauda epididymal plasma and 5 % sodium bicarbonate will be taken in a centrifuge tube, mixed well and centrifuged for 5 minutes at 4000 g. The supernatant will be discarded and to the precipitate 5 ml of normal saline was added, mixed well and centrifuged again. The procedure will be repeated 2 to 3 times and a clear precipitate will be obtained. To the final precipitate few drops of normal saline will be added, mixed thoroughly and a smear will be prepared on a clean slide. The smear will be dried at room temperature, fixed by heating it over the flame for two to three seconds. Then the smear will be flushed with 95 % alcohol, drained and dried. It is stained in Ziehl Neelson's Carbol Fuchsin diluted with equal volume of 95% alcohol for 3 minutes and counter stained with 1:3 (v/v) aqueous solution of Loeffer's methylene blue for 2 minutes (Gurr, 1956). After staining, the smear is rinsed in water and dried in air.

The abnormal sperms included categories like double tailed, detached head, detached tail, mid piece bending and irregular head. The relative proportions of the normal and abnormal sperms obtained from the smear and are expressed in terms of percentage.

d. Histology of testis

Two-left testis of each group will be excised and rinsed in 0.9% saline blotted dry of saline and excess blood. They will be fixed in 12 % formalin for 24 hr. The tissues, after fixation, will be washed in water to remove excess fixative. Washed tissues will be then dehydrated through a graded series of ethyl alcohol, cleared with xylene and embedded in paraffin wax. Sections will be cut at 3 μm with microtone blade, and mounted on clean glass slide. The sections will be routinely stained with haemotoxyllin and eosin. The stained slides will be observed (400 X) in research microscope and photographed.

STATISTICAL ANALYSIS

All values will be expressed as mean ± SEM from 6 animals. Statistical difference in mean will be analyzed using one-way ANOVA (analysis of variance) followed by Dunnet‘t’ test. p< 0.05 will be considered as statistically significant.

7.3: Does the study require any investigations or interventions to be conducted on patients other human or animals? If so, please describe briefly:

Study requires investigation on rats.

7.4: Has ethical clearance been obtained from your institute in case of 7.3?

YES: IAEC NO.: 576/2002/bc/IAEC/CPCSEA

ENCLOSURE – VI

8. List of references:

1. Wight R, Arnott GA. Species wikipedia free species directory GRIN 2006;07:47-48, http:// wki/L-reticulata, accessed on 27th October 2008.

2. Retana MS, Salazar ED, Velazquez MJ, Javier V. Effect of acute and chronic stress on masculine sexual behavior in the rat. J Psychoneuroendocrinol 1996; 231(21): 39-50.

3. Neychev VK, Mitev VI. The aphrodisiac herb Tribulus terrestris does not influence the androgen production in young men. Dep Chem Biochem Med

16 Sept 2004; 12:35-37. 

4. Hosseinzadeh H, Ziaee T, and Sadeghi A. The effect of saffron, Crocus sativus stigma, extract and its constituents, safranal and crocin on sexual behaviors in normal male rats, Mashhad University of Medical sciences. Phytomedicine 25th October 2007.

5. Yoon H, Chung W S, Park Y and Cho I H. Effect of stress on female rat sexual function. Int J Impo Res 2005; 17:33-38.

6. Kirtikar and Basu. Indian Medicinal Plants. Periodical Experts Book Agency, 2nd ed, 1935; volume-I: 677-81.

7. Fuglie LJ (1999) The Miracle Tree: Moringa oleifera: Natural Nutrition for the

Tropics. Church world service, 68 pp ; revised in 2001 and published as The

Miracle Tree, Multiple Attributes of Moringa, 172 pp.

http// bookstores/advanceds accessed on 27th October 2008.

.

.

8. OECD 2001 guidelines on acute oral toxicity. Environmental health and safety monograph series on testing and adjustment no.425.

9. Boolell M, Gepi-Attee S, Gingell JC, Allen MJ. Sildenafil, a novel effective oral therapy for male erectile dysfunction. Br J Urol 1996; 78:257–61.

10. Hooi H, Hung SC, Ahmad PY. Effects of Eurycoma longifolia Jack (Tongkat Ali) on the Initiation of Sexual Performance of Inexperienced Castrated Male Rats. Exp Anim 2000; 49(1): 35-38.

11. Ratnasooria, Dharmasiri MG. Effect of Terminalia Catappa seeds on sexual behavior & fertility of male rats. Asian J Androl 2000; 2:213-19.

12. University of Plymouth Dept. of psychology, Salmon study materials on-line, PSY128 Lecture Support Material, Hormones and Sexual Behavior.environment, accessed on 27th October 2008.

13. Inoko M, Kiharay Y, Morii I, Fujiwara H, dasayana S. Transition from compensatory hypertrophy to dilated failing heart ventricles in Dahi-salt sensitive rats. Am J Physiol 1994; 267(36):H 2471-482.

14. Jain AK. Manual practical physiology for MBBS, Arya publications Sirmour, Semen analysis sperm count and motility 2003; 1:176-77.

15. Bauer JD, Ackerman PG, Toro G. Clinical Laboratory Methods. St. Louis Missouri, USA: Mosby Co, 1974.

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