Enclosure-i brief resume of the intended work 1: Need for the study




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ENCLOSURE-I

6. Brief resume of the intended work

6.1: Need for the study:

An aphrodisiac is a food, drink, drug, scent or device that promotes, arouse or increase sexual desire or libido. A broader definition includes products that improve sexual performance named after Aphrodite, the Greek goddess of sexual love and beauty1. According to researchers at the University of Chicago, around 40 percent of women and 30 percent of men experience sexual dysfunction i.e. unable to feel satisfaction during the five stages of the sexual response cycle: desire, excitement, plateau, orgasm and resolution, hence lose interest in sex altogether. Aphrodisiacs are supposed to enhance the libido by offering a sensory experience of sight, touch, sound or smell2.

Aphrodisiac agents are used to modify the impaired sexual functions of human beings. These agents have been used since long time and the availability of a large number of sexual function improving drugs in the traditional Unani system of medicine is a unique and distinctive feature of this system. Besides having many specific drugs for enhancing sexual functions, there are certain most commonly used spices like Myristica fragrans Houtt, (Nutmeg) Syzygium aromatium (L) Merr. & Perry.(Clove) and Piper nigrum. Linn. (Black pepper) etc. which are empirically used as promising aphrodisiacs in traditional medicine practice in cases of sexual debility or depressed desire3.

In India, although population growth is a major concern, till there are a substantial number of infertile couples. Thus infertility considered as an important national problem concerning reproductive health. Infertility is defined as the state in which a couple wanting a child is unable to conceive after 12 month of regular intercourse in the absence of contraceptives and global incidence of infertility is about 13 to18%. Male infertility is found to have a role in approximately 50% of infertile couples. Approximately 15 to 20% of all cohabiting couples are infertile. Nearly 7.5 to 10% of all men in the reproductive age group are infertile i.e., incapable of fathering children4.

Sexual dysfunction may affect men during reproductive age and no limit to the race and background. The problem of infertility is closely related to stress, failing to achieve the expected goal of reproduction, so experiences the feelings of frustration and disappointment. The emotions, uncertainties and lifestyle disturbances like stressful working area, nutrient deficient food coincide with this condition and often have a significant effect on man’s self-esteem, as well as, his relationship with his partner5.

In males, stress induces suppression of testosterone secretion, spermatogenesis and libido. These altered male reproductive functions also involve reduced sperm counts, concentration, morphology, quality and motility6.

Sexual dysfunction is considered as one of the most important public health problem, since it affects larger percentage of men. 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 dysfunction7.Treatment of erectile dysfunction usually involves the psychotherapeutic approach or pharmacotherapy involves locally acting vasoactive drugs such as papaverin and alprostadil. The first-line oral therapy for erectile dysfunction 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. Central stimulants like Apomorphin and herbal drugs with aphrodisiac activity are also involved in treatment of erectile dysfunction. 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 and too are sometimes contraindicated in certain disease conditions7.

Despite the increasing availability of effective conventional medical treatments, plant-derived and herbal remedies continue to provide a popular alternative for men seeking improved sexual life8.

Hence there is an increasing demand for the alternative therapies, particularly herbal therapies that are believed to be effective, safe and economical. In Ayurvedic texts the plant F. carica fruit has been mentioned for its aphrodisiac activity. So in the present study

fruit extracts of F.carica is selected for evaluating its aphrodisiac activity in experimental animals like mice and rats.



ENCLOSURE -II

6.2: Review of Literature:

The review of various published journals and books have revealed that plant based drugs are showing promising aphrodisiac activity.



Plant Description:

The F.carica is a picturesque tropical looking tree or shrub with a dramatic spreading habit and grows up to a height of 15 to 30 ft. It is a deciduous tree with cordate or palmate leaves which are arranged alternatively with long petioles and having 3 to 5 lobes. The bark is smooth silvery and grey. This tree usually begin bearing fruit within two years9,10.



Synonyms: F.sycomorous11.

Common Names: Fig (English); Anjira, Manjula, Kakodumbar (Sanskrit); Anjir (Hindi), Anjuru, Manjimedi, Teneyatti(Telugu); Simayatti (Tamil); Simayatti (Malayalam) 12.

Location: Originally from the Eastern Mediterranean region, figs have been cultivated by humans for over 5000 years9.

Parts used: Fruit, stem, latex, and leaves10.

Family: Moraceae (Mulberry family) or Urticaceae9.

Chemical constituents:

Proteose, aminoacid, tyrosine, enzyme cravin, lipase, protease. The fleshy receptacle contains grape sugar, fat and salts. Dried fruit contain sugar, fat, pectose, gum, albumen and salts. Milky juice contains a peptonising ferment13.



Medicinal uses:

The fruit is sweet, cooling used as an antipyretic, tonic, purgative, alexiteric, aphrodisiac, lithiontriptic; useful in inflammations, weakness, paralysis, thirst, diseases of the liver and spleen, pain in the chest, cures piles; stimulates growth of hair. It is also having emollient, demulcent and nutritive properties. The fresh and dried fruit are used in constipation. Roasted fruit are used as a poultice for boils and carbuncles. A poultice of dried fruit in milk removes unpleasant odors from ulcers and cancers. The pulp is mucilaginous and has long been estimated as a pectoral emollient for coughs. In its fresh green state the fruit secretes a milky acrid juice, which will destroy warts. The root is useful in leucoderma and ringworm12.



ENCLOSURE -III

6.3. Main objectives of study:

There are no published reports available regarding aphrodisiac effect of F.carica fruit extracts. The main objective of the proposed work is to evaluate the beneficial effect of F.carica fruit extracts for their aphrodisiac activity in different experimental animal models as the fruit are reported for its aphrodisiac activity in ethnic medicine. In the light of the available literature on the F.carica, the present work is planned with the following objectives.



In Phase-I:

  • Preparation of various extracts alcoholic (AEFFC) and aqueous (AQEFFC) with fruit of F.carica.

  • To investigate preliminary phytochemical constituents by following standard methods.

  • Determination of LD50 and dose selection as low, medium and high doses with respect to the LD50 values of AEFFC and AQEFFC.

In Phase-II

To study the effect of AEFFC and AQEFFC on sexual behavior in albino mice and rats in the following models:

A. Mounting behavior in mice14

B. Assessment of mating in mice15

C. Effect on fertility in mice15

D. Histopathological study of testes in mice16

E. Sexual behavior on prolonged immobilization-induced stress in rats1

It is also planned to evaluate the following parameters in the above models

1. Mounting latency

2. Number of mounts

3. Thrusting

4. Examination of vaginal smears

5. Number of sperm positive females

6. Litter size

7. Number of Pups

8. Histopathological study of testis



ENCLOSURE-IV

7. Materials and Methods:

7.1: Source of the data

The whole work is planned to generate data from laboratory based experimental animal studies as described in various National/International Journals and books available with our college and other reputed Institutions of India and through e-publishing and Helinet of RGUHS, Bengaluru.



ENCLOSURE-V

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

The whole study is divided into the following phases:



Phase-I

  • Preparation of AEFFC and AQEFFC using maceration:

The fruit powder will be macerated with (95%) alcohol for 24 h to obtain the alcoholic extract, and the extract will be concentrated by distilling off the solvent and then evaporated to dryness on a water bath below 500 C. Similarly in the above manner fruit powder will be macerated with chloroform water (chloroform acts as a preservative) to obtain the aqueous extract.

  • Preliminary Phytochemical Screening:

The preliminary phytochemical investigations of AEFFC and AQEFFC will be carried for qualitative identification of phytoconstituents using standard procedures. All the chemicals and reagents proposed to be used will be of analytical grade.

  • Determination of LD50 of AEFFC and AQEFFC:

The acute toxicity of the AEFFC and AQEFFC will be determined by using albino mice of either sex (16-20 g), maintained under standard husbandry conditions. The animals will be fasted for 3 h prior to the experiment and were be administered with single dose of AEFFC/ AQEFFC and observed for its mortality up to 48 h study period (Short term toxicity). Based on the short-term toxicity profile, the next dose will be determined as per OECD guidelines No: 425. From the LD50 dose of the individual extracts 1/20th, 1/10th and 1/5th doses are to be selected and considered as low, medium and high doses respectively for the selected study.

Phase- II

Models for aphrodisiac activity

1. Mounting Behavior in mice 3,14,20,21:

Experimental Procedure

Adult Swiss albino male mice of (25-35g) each consisting of 6 animals will be divided into 8 groups and the treatment protocol is given below.



Group I: Normal control (Gum acacia 10 ml/kg, p.o)

Group II: Standard Drug (Tentex Forte 171 mg/kg, p.o)

Group III: Low Dose of AEFFC (p.o)

Group IV: Medium Dose of AEFFC (p.o)

Group V: High Dose of AEFFC (p.o)

Group VI: Low Dose of AQEFFC (p.o)

Group VII: Medium Dose of AQEFFC (p.o)

Group VIII: High Dose of AQEFFC (p.o)

A ‘mount’ is operationally defined as a male assuming the copulatory position but failing to achieve intromission. To quantify mounting behavior, non estrous female mice were paired with males following treatment with the investigational fruit extract for a period of 30 days. At the end of treatment period animals were observed individually for 4 h and their behavior was scored. For this they were placed individually in a clear glass chamber and allowed to acclimatize for 15 min, then non estrous female was introduced into the arena. Animals were paired during observation. The number of mounts was recorded during a 15 min observation period at the start of 1st, 2nd, 3rd and 4th h using digital recorder. Females will be separated following observation at each hour. All experiments were performed from 09.00 to 12.00 h on sunny days (room temp 27 -280 C) because the hormone levels are increased in the morning than in evening. In similar manner aphrodisiac activity of vehicle/standard drug/plant extracts treated groups are to be evaluated.



2. Assessment of Mating 3,15:

Experimental Procedure

Adult Swiss albino male mice of (25-35 g) each consisting of 6 animals will be divided into 8 groups and the treatment protocol follow as below.



Group I: Normal control (Gum acacia 10 ml/kg, p.o)

Group II: Standard Drug (Tentex Forte 171 mg/kg, p.o)

Group III: Low Dose of AEFFC (p.o)

Group IV: Medium Dose of AEFFC (p.o)

Group V: High Dose of AEFFC (p.o)

Group VI: Low Dose of AQEFFC (p.o)

Group VII: Medium Dose of AQEFFC (p.o)

Group VIII: High Dose of AQEFFC (p.o)

Different groups of animals (mice) were treated as mentioned above in the evening (17.00 to 18.00 h) and each male mouse was placed in separate cage. After one hour, eight estrous females will be placed into each cage and cohabitated overnight. The vaginal smear of each female mouse was examined next day under the microscope to confirm mating by the presence of sperms. The number of sperm positive females was recorded in each group. In similar manner aphrodisiac activity of vehicle/standard drug/ plant extracts treated groups are to be evaluated.



3. Effect on fertility in mice 15,20:

Experimental Procedure

Adult Swiss albino male mice of (25-35 g) each consisting of 6 animals

will be divided into 8 groups and the treatment protocol follows as given below.

Group I: Normal control (Gum acacia 10 ml/kg, p.o)

Group II: Standard Drug (Tentex Forte 171 mg/kg, p.o)

Group III: Low Dose of AEFFC (p.o)

Group IV: Medium Dose of AEFFC (p.o)

Group V: High Dose of AEFFC (p.o)

Group VI: Low Dose of AQEFFC (p.o)

Group VII: Medium Dose of AQEFFC (p.o)

Group VIII: High Dose of AQEFFC (p.o)

Different groups of animals (mice) were treated as mentioned above in the evening (17.00 to 18.00 h) and each male mouse was placed in separate cage. After one hour, one estrous female with proven fertility will be placed into each cage and cohabitated overnight. These females will be watched for pregnancy and birth of offspring’s. The litter size and number of male and female pups were recorded in each group. In similar manner aphrodisiac activity of vehicle/ standard drug/plant extracts treated groups are to be evaluated.



4. Histological analysis of testes16,21-23:

Experimental Procedure

Adult Swiss albino male mice of (25-35g) each consisting of 6 animals will be divided into 8 groups and the treatment protocol follows as given below.



Group I: Normal control (Gum acacia 10 ml/kg, p.o)

Group II: Standard Drug (Tentex Forte 171 mg/kg, p.o)

Group III: Low Dose of AEFFC (p.o)

Group IV: Medium Dose of AEFFC (p.o)

Group V: High Dose of AEFFC (p.o)

Group VI: Low Dose of AQEFFC (p.o)

Group VII: Medium Dose of AQEFFC (p.o)

Group VIII: High Dose of AQEFFC (p.o)

Animals will be administered with vehicle/standard drug/ and fruit extracts for a period of 30 days and at the end of treatment period animals were sacrificed by overdose of Ether anesthesia and histopathological studies of testis were done by fixing the testes in Bouin's fluid and passed through ascending series of ethanol and then through xylene, and embedded in paraffin wax. Tissues were sectioned at 5 mm and stained with haematoxylene and eosin.



5. Sexual behavior on prolonged immobilization-induced stress in rats17,24:

Experimental Procedure

Adult albino rats of (150-200g) each consisting of 6 animals will be divided into 8 groups with the treatment protocol mentioned below.



Group I: Normal control (Gum acacia 10 ml/kg, p.o)

Group II: Standard Drug (Tentex Forte 171 mg/kg, p.o)

Group III: Low Dose of AEFFC (p.o)

Group IV: Medium Dose of AEFFC (p.o)

Group V: High Dose of AEFFC (p.o)

Group VI: Low Dose of AQEFFC (p.o)

Group VII: Medium Dose of AQEFFC (p.o)

Group VIII: High Dose of AQEFFC (p.o)

Different groups of prepubertal (40 days of age) male albino rats were housed under controlled environmental conditions and had free access to Laboratory Chow diet and tap water. Animals were treated as mentioned above in the morning time. Stress immobilization was attained by wrapping the animals in wire mesh for 3 h a day during the light period, starting at 8:00 A.M., for 15 days. Control animals were left undisturbed in their cages. The males were placed in the observation 2 h after the beginning of the dark phase and 10 min before the females for adaptation. The latency, number of mounts and thrusting were recorded simultaneously by two investigators with light provided by a 40-watt red lamp. In the mount behavior the male places his forepaws on the female without pelvic movements, while in the thrusting behavior the rat executes repeated deep pelvic thrusts.



STATISTICAL ANALYSIS

All results 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 Post hoc test (Dunnett’s ‘t’ test). P< 0.05* 0.01** and 0.001*** will be considered as statistically significant.



ENCLOSURE-VI

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