Garlic consumption has more than tripled in the United States since 1980 and now stands at about 2.5 pounds per capita. Around 24,000 to 26,000 acres of garlic are planted annually in the United States with a total production of around 400 million pounds. U.S. production is concentrated in California, with smaller acreages in Oregon, Nevada, New York, and Washington. About 30 percent of U.S. garlic production is sold in the fresh market; the rest is dehydrated. The average price of garlic has been around $60–70 per cwt., but wholesale fresh- market prices have typically been around three times higher. The United States is the world's largest importer of garlic, primarily from China, Spain, Mexico, and Argentina.

Taxonomical Classification:

• Kingdom: Plantae
• Clade: Angiosperms (flowering plants)
• Clade: Monocots (plants with a single seed leaf)
• Order: Asparagales
• Family: Amaryllidaceae
• Subfamily: Allioideae
• Genus: Allium
• Species: Allium sativum

       
           
       
History:

Morphology:

The leaves are green in colour and grow to be 12-15 cm tall (some species can grow up to 60 cm tall). Garlic leaves, also known as garlic scapes, have a different morphology compared to the bulb. The leaves are narrow, flat, and lance-shaped, growing in a tuft from the base of the plant. Garlic flowers are part of the garlic scape, which is the flower stalk that emerges from the garlic plant. The flower cluster is called an umbel. The garlic umbel consists of small, star-shaped flowers arranged in a spherical or semi-spherical shape at the top of the scape. The individual flowers typically have six petal-like tepals, which are the floral structures that resemble both petals and sepals.

       
           
       
Garlic flowers can vary in colour, ranging from white to shades of pink or purple. The flowers may have a delicate fragrance, although it's not as strong as the characteristic garlic scent associated with the bulbs. Garlic fruit has a pungent odour and is pale or slightly yellowish. Depending on the cultivar, the tall leaves may develop from small, rigid stems above the bulb or a softer pseudo stem consisting of overlapping leaf sheaths. ^[3]

       
           
       
The bulbs are covered with membrane skin and contain up to 20 edible bulblets known as cloves. The bulb is divided into individual sections called cloves. Each clove is enclosed in a thin, papery skin and is capable of growing into a new garlic plant when planted. The clove clusters create the bulb, which is a subterranean portion. It has long leaves with blades grouped in an alternate arrangement. The entire bulb is covered in multiple layers of thin, papery skin. These layers help protect the cloves and keep them intact. The base of the bulb is known as the root plate. This is the part from which roots emerge and anchor the garlic plant into the soil. The basal plate is the bottom part of each clove that is attached to the root plate. It's the point of attachment for the cloves within the bulb. Sometimes, a hard stem or remnant of the garlic scape (flower stalk) may be visible at the top of the bulb. This part is usually removed before culinary use. The stem, or pseudo-stem, is very short and forms a tray at the base where adventitious roots emerge. It is constituted of a string of leaves joined together by their sheaths. It has an adventitious root system, which is thick and sparsely branching, with an epidermis, a multicellular cortex, and an endoderm surrounding the central stele. It can be white, brown, light brown, violet, light violet, or dark violet, with rounded, elliptical, or circular shapes, as well as transverse broad and transverse narrow elliptical shapes. ^[4]

       
           
       
Phytoconstituents:

Garlic contains at least 33 Sulphur compounds, minerals germanium, many enzymes, potassium, copper, selenium, iron, calcium, magnesium, and zinc, as well as vitamins A, B1, and C, fibres, and water. Lysine, aspartic acid, histidine, arginine, threonine, swine, glycine, glutamine, alanine, cystine, proline, valine, leucine, methionine, phenylalanine, isoleucine, tryptophan are among the 17 amino acids included in it. Garlic contains more Sulphur compounds than other Allium species, which are responsible for garlic‘s pungent flavour as well as its therapeutic properties. Figure 2 represents the major constituents of garlic. When garlic is minced or crushed, the enzyme allinase is triggered, and allicin is produced from allin (present in intact garlic). Other important compounds discovered in garlic homogenate include ally methyl thiosulfate and 1-propenyl ally thiosulfate. Garlic contains minerals including germanium and selenium, as well as enzymes and vitamins like peroxidase, alliinase, and tyrosinase. ^[5]

Structure of Phytoconstituents:

Traditional Uses:

Garlic is widely used due to its seasoning and flavour qualities as well as health-enhancing properties. Moreover, it is used in folk medicine as a remedy for the treatment of bacterial and viral diseases. Garlic has been a kitchen superhero for centuries! Apart from adding flavour to dishes, it has a bunch of traditional uses. It's believed to have medicinal properties and has been used to treat various ailments like colds, infections, and even as a natural antibiotic. Leaves have been one of the important sources of medicine since dawn of human civilization. These are the gifts of nature to mankind for treating different types of diseases. Almost from prehistoric period herbal medicine are used for alleviation of suffering caused by different diseases in human. Further they are well documented in India and other countries: even today they are in great use in these countries. There are several beliefs or claims regarding the therapeutic utility of herbs and herbal formulation i.e;

• Herbal medicines exhibit fewer side effects and are safe.
• Herbs and herbal formulations are cheaper and easily available.
• For certain disease like hepatitis, herbs and herbal drugs are the only remedies.
• Certain chemical constituents from the herbs are serving as prototype molecules for the discovery of more effective drugs than existing ones.

Medicinal leaves are treated as a subject of serious study and undergoing intense research all over the World to identify the lead molecules. The immense surge of scientific interest in natural products as a potential source of drugs has contributed to the development of phytopharmacology research. In many developing countries, phyto- pharmaceuticals form the main base of National Health Care Programmed e.g. the use of herbal extracts in the treatment of various diseases in different countries and they are also known to possess a rich traditional medicine. In South Africa it is estimated that 80% of the black population consults traditional practitioners, perhaps this is not one and only reason for the developing countries to invest in leaves based pharmaceuticals but also has been a global resurgence of interest in leaves based drugs. [8]

Herbal therapy provides rational means for the treatment of many diseases which are considered to be obstinate and incurable in other system of medicine. It lays a great deal of emphasis upon the maintenance of positive health of an individual and thus aims at both the prevention and cure of diseases. Hyperacidity / Ulcers which require the treatment for chronic period of time and the usage of such drugs may cause various side effects, sometimes with permanent damage of vital organs. The ulcer is one of chronic disease which is normally treated with H2-receptor blockers (Cimetidine, Ranitidine) and proton pump inhibitors (Omeprazole, Pentaprazole). These drugs are known to effect microsomal enzyme system of the liver resulting in the pharmacokinetic type of drug- drug interaction with other drugs. But in herbal / traditional medicines, there are several preparations prescribed to treat hyperacidity and ulcers. [9]

Inflammation is a normal protective response to tissue injury caused by physical trauma, noxious chemical or microbiological agents. It is body‘s response for tissue repair and is triggered by the release of chemical mediators from injured tissue and migrating cells. The mechanism of anti-inflammatory drugs is considered to be inhibition of prostaglandins synthesis at the site of injury. The anti-inflammatory potency of different compounds roughly corresponds with their ability to inhibit cyclooxygenase enzyme (COX). [10]

The traditional medicine is largely gaining popularity over allopathic medicine because of the following reasons.

• Rising cost of medical care.
• Natural products are free from side effects.
• No reoccurrence after the treatment.
• Easy availability of the drugs from natural sources especially in Developing countries.
• Cure of disease by the changes in life style and social pathology.
• Renewable sources.

In the present study, we have chosen a leaves Allium sativum for which different pharmacological activities are reported with different parts of the leaves. From the literature Ayurvedic texts we found that leaves of Allium sativum have been traditionally indicated for the treatments of anti-ulcer and anti-inflammatory condition. However anti- ulcer and anti- inflammatory activities of this leaves are not reported. But for its medicinal properties reported in the Ayurvedic texts prompted us to select leaves extracts for the study of anti- ulcer and anti-inflammatory activities in different experimental animal models. ^[11]

MATERIAL AND METHODS:

Table No: 4.1 List of materials and equipments used during experiment

Preparation of Different Extracts:

1. Preparation of alcoholic extract:

Leaves of Allium sativum were collected in the month of August from the native place Ambale, Dist. Pune and dried in shade at room temperature then subjected to size reduction to a fine powder with the help of mixer grinder. [12] The leaves powder was packed in a soxhlet apparatus and extracted with 95% alcohol for 18 hr. Appearance of colourless solvent in the siphon tube was taken as the termination of extraction. The extract was then transferred into the previously weighed empty beaker and evaporated to a thick paste on the water bath, maintained at 50oC to get alcoholic (AELAS) extract. The extract was finally air dried thoroughly to remove all traces of the solvent and the percentage yield was calculated. [13]

2. Preparation of aqueous extract:

About 100 g of leaves powder was taken in a round bottom flask (2000 ml) and macerated with 500 ml of distilled water with 10 ml of chloroform (preservative) for 24 h with shaking for every hour in a closed vessel. Then the marc was removed by filtering the extract (AQELAS) and then it was concentrated on a water bath maintained at 50 ?.

These two extracts were stored in airtight containers in a refrigerator below 10?. The two extracts were examined for their colour and consistency. Their percentage yield was calculated with reference to air-dried powder sample used for the extraction.

Table No: 4.2 Nature and Percentage yield of the extracts

1.         Preliminary phytochemical screening.

2.         Pharmacological activities

a.         Toxicity studies (LD50).

b.         Anti-inflammatory activity.

Preliminary phytochemical investigations:

The preliminary phytochemical investigations were carried out with AELAS and AQELAS for qualitative identification of phytochemical constituents present with each extract and test were carried out by following standard methods74, 75, and 76. All the chemicals and reagents used were of analytical grade. [14]

Table No: 4.3 Details of qualitative phytochemical tests

Pharmacological activities:

Experimental animals:

Albino rats (Wistar strain) of either sex weighing between 120-200 g and Albino mice 18-22 g were procured from National Centre for Laboratory Animal sciences, C/0 Shri. Venkateswara Enterprises, Bengaluru for experimental purpose and the animals were acclimatized for 7 days under standard husbandry condition as:

Room temperature - 26 ± 2⁰ C

Relative humidity - 45-55%

Light/ dark cycle - 12:12 h

The procedures were followed as per rules and regulations.

Determination of acute toxicity (LD50)77 Method:

The acute toxicity of AELAS and AQELAS was determined in albino rats weighing between 18- 22 g those maintained under standard husbandry conditions. The animals were fasted 3 h prior to the experiment and ?up and  down?  (OECD  Guideline  No.  425)   method   of   CPCSEA   was adopted for toxicity studies. Animals were administered with single dose of extracts and observed for its mortality during 48 h study period (short term) toxicity. Based on the short-term toxicity profile of the extracts the doses of the next animals were determined as per as OECD Guidelines No: 425. All the animals were observed for long term toxicity (7 days) and then 1/5th, 1/10th and 1/20th of the maximum dose tested for LD50 of the individual extract was taken as effective dose ED50 and were used throughout the experimental studies. [16]

Determination of anti-inflammatory activity:

1. Carrageenan induced paw edema:

Group A: Toxicant control (0.1 ml of 1% w/v Carrageenan, hind paw)

Group B:  Standard (Ibuprofen 40 mg/ kg, p.o)

Group C: AELAS (100 mg/kg p.o)

Group D: AELAS (200 mg/kg p.o)

Group E: AELAS (400 mg/kg p.o)

Group F: AQELAS (100 mg/kg p.o)

Group G: AQELAS (200 mg/kg p.o)

Group H: AQELAS (400 mg/kg p.o)

Experimental Procedure:

Male albino rats (125-150 g) of 8 groups were housed as groups of six, fasted overnight prior to and during the experiment but have free access to water. Group A was served as normal toxicant control treated with toxicant carrageenan, group B with Ibuprofen (40 mg/kg p.o.) served as standard, groups C, D and E administered with AELAS and groups F, G and H with AQELAS (low, medium and high doses p.o) respectively. The rats in Groups B, C, D, E, F, G and H were administered with 0.1 ml of 1% w/v of carrageenan into of right hind paw of rats 1 h after the administration of Ibuprofen/extracts. Immediately thereafter the oedema volumes of the injected paws were measured plethysmographically at prefixed time intervals. [27-30]

2. Histamine induced paw edema:

Group A: Toxicant control (0.1 ml of 1% w/v histamine, hind paw)

Group B: Standard (Ibuprofen 40 mg/ kg)

Group C: AELAS (100 mg/kg p.o)

Group D: AELAS (200 mg/kg p.o)

Group E: AELAS (400 mg/kg p.o)

Group F: AQELAS (100 mg/kg p.o)

Group G: AQELAS (200 mg/kg p.o)

Group H: AQELAS (400 mg/kg p.o)

Experimental Procedure:

Male albino rats (125-150 g) of 8 groups were housed as groups of 6, fasted overnight prior to and during the experiment but have free access to water. Group A was served as toxicant control treated with inflammogen histamine, group B with Ibuprofen (40 mg/kg p.o.) that served as standard. Groups C, D, E and F, G, H will administered with AELAS and AQELAS (low, medium and high dose p.o) respectively. The rats of groups B, C, D, E, F, G and H were administered with 1% w/v of histamine into sub plantar region of right hind paw of rats 1 h after administration of Ibuprofen/extracts. Immediately thereafter the oedema volumes of the injected paws were measured plethysmographically at prefixed time intervals. [31]

3. Formalin Induced Paw Oedema:

Group A: Toxicant control (Formalin 1%, hind paw)

Group B: Standard (Ibuprofen 40 mg/ kg)

Group C: AELAS (100 mg/kg p.o)

Group D: AELAS (200 mg/kg p.o)

Group E: AELAS (400 mg/kg p.o)

Group F: AQELAS (100 mg/kg p.o)

Group G: AQELAS (200 mg/kg p.o)

Group H: AQELAS (400 mg/kg p.o)

Experimental Procedure:

Male albino rats (125-150 g) of 8 groups were housed as groups of 6, fasted overnight prior to and during the experiment but have free access to water. Group A was served as toxicant control treated with toxicant Formalin, group B with Ibuprofen (40 mg/kg p.o.) that served as standard. Groups C, D, E and F, G, H were administered with AELAS and AQELAS (low, medium and high dose p.o) respectively. The rats of groups B, C, D, E, F, G and H were administered with 1% of Formalin into sub plantar region of right hind paw of rats 1 h after administration of Ibuprofen/extracts. Immediately thereafter the oedema volumes of the injected paws were measured plethysmographically at prefixed time intervals. [32] For comparison purpose, the volume of oedema was measured at prefixed time intervals. The difference between paw volumes of the treated animals was measured and the mean oedema volume was calculated. Percentage reduction in oedema volume was calculated by using the formula,

                                    Vo - Vt

Percentage reduction =              x 100

                                       Vo

Where,

Vo = Volume of the paw of control at time?t‘.

Vt = Volume of the paw of drug treated at time?t‘.

Statistical analysis:

All results will be expressed as mean ± SEM from 6 animals. Statistical difference in mean will be analysed using one-way ANOVA (analysis of variance) followed by Post hoc test (Dunnett?s?t‘ test). P< 0>

RESULTS AND DISCUSSION:

Preliminary Phytochemical studies Preliminary phytochemical screening:

AELAS and AQELAS were subjected for phytochemical screening and found to contain

tannins, carbohydrate, sterols, flavonoids, glycosides, alkaloid and triterpenes both in alcoholic and aqueous extracts. [34]

 Pharmacological activities

Acute oral toxicity study:

The mice treated with AELAS and AQELAS at a dose of 2000 mg/kg,  p.o. exhibited normal behaviour, without any signs of passivity, stereotypy and vocalization. Their motor activity and secretory signs were also normal and no sign of depression. AELAS and AQELAS even up to the dose level of 2000 mg/kg body weight did not produce any behavioural symptoms or mortality. So 1/5th, 1/10th and 1/20th doses of LD50 (maximum dose tested for each extract) was selected as low, medium and high doses and were used in the present study to explore anti-ulcer and anti-inflammatory activities.

Anti-inflammatory activity:

1. Anti-inflammatory activity of AELAS and AQELAS in Carrageenan induced paw oedema model in rats:

The AELAS and AQELAS with three selected doses i.e. 100, 200 and 400 mg/kg have exhibited a significant reduction in paw oedema volume in carrageenan induced paw oedema in rats at different time intervals. Results are tabulated in Table No. 5.7. Ibuprofen (40 mg/kg) was used as standard reference and it has significantly reduced paw oedema volume by 43.35% at 1st h, 61.93% at 2nd h, 79.79% at 3rd h and 85.83% at 4th h, thus standard drug has exhibited time dependent reduction in oedema volume. During 1st h of study AELAS and AQELAS with low, medium and high doses have significantly reduced oedema volume by 5.35%, 14.78%, 24.50% and 10.21%, 19.64%, 29.07% respectively, which was found to be a time dependent effect. During 2nd h of study AELAS and AQELAS with low, medium and high doses have significantly reduced oedema volume by 8.12%, 23.59%, 31.21% and 11.94%, 27.28%, 38.83% respectively a time dependent effect.

During 3rd h of study AELAS and AQELAS with low, medium and high doses have significantly reduced oedema volume by 14.16%, 28.03%, 38.38% and 17.64%, 31.55%, 45.22% respectively a time dependent effect was noted. During 4th h of study AELAS and AQELAS with low, medium and high doses have significantly reduced oedema volume by 22.72%, 33.83%, 59.75% and 30.05%, 41.16%, 67.16% respectively a time dependent effect was noted and result are graphically represented in Fig No.5.7. ANOVA indicates a significant difference among the AELAS and AQELAS treated groups. Dunnett‘s?t‘ test confirms a significant anti-inflammatory activity with both the extracts, but more with AQELAS than AELAS.

AELAS- Alcoholic Extract of Leaf of Allium Sativum, AQELAS- Aqueous Extract of Leaf of Allium Sativum.

2. Anti-inflammatory activity of AELAS and AQELAS in Histamine induced paw oedema model in rats:

The AELAS and AQELAS with three selected doses i.e. 100, 200 and 400 mg/kg have exhibited a significant reduction in paw oedema volume in histamine induced paw oedema in rats at different time intervals. Results are tabulated in Table No. 5.9. Ibuprofen (40 mg/kg) was used as standard reference and it has significantly reduced paw oedema volume by 56.89% at 1st h, 70.94% at 2nd h, 87.30% at 3rd h and 93.43% at 4th h, thus exhibited a time dependent reduction in oedema volume. During 1st h of study AELAS and AQELAS with low, medium and high doses have significantly reduced oedema volume by 4.67%, 17.73%, 39.66% and 9.11%, 26.60%, 48.28% respectively, which was found to be a time dependent effect. During 2nd h of study AELAS and AQELAS with low, medium and high doses have significantly reduced oedema volume by 11.61%, 26.50%, 44.94% and 18.94%, 30.05%, 52.27% respectively noted a time dependent effect. During 3rd h of study AELAS and AQELAS with low, medium and high doses have significantly reduced oedema volume by 17.10%, 37.16%, 57.16% and 27.10%, 43.90%, 63.90% respectively a time dependent effect was noted.

During 4th h of study AELAS and AQELAS with low, medium and high doses have significantly reduced oedema volume by 25.55%, 46.96%, 68.38% and 36.26%, 54.03%, 75.44% respectively a time dependent effect was noted and result are graphically represented in Fig No.5.8.

ANOVA indicates a significant difference among the AELAS and AQELAS treated groups. Dunnett‘s?t‘ test confirms a significant anti-inflammatory activity with both the extracts, but more with AQELAS than AELAS.

AELAS- Alcoholic Extract of Leaf of Allium Sativum, AQELAS- Aqueous Extract of Leaf of

Allium Sativum.

Table No: 5.9 Anti-inflammatory effects of AELAS and AQELAS in Histamine induced paw oedema model in rats at different time intervals

3. Anti-inflammatory activity of AELAS and AQELAS in Formalin induced paw oedema model in rats:

The AELAS and AQELAS with three selected doses i.e. 100, 200 and 400 mg/kg have exhibited a significant reduction in paw oedema volume in Formalin induced paw oedema in rats at different time intervals. Results are tabulated in Table No. 5.11. Ibuprofen (40 mg/kg) was used as standard reference and it has significantly reduced paw oedema volume by 54.75% at 1st h, 64.73% at 2nd h, 84.40% at 3rd h and 90.14% at 4th h, which was found to be a time dependent effect. During 1st h of study AELAS and AQELAS with low, medium and high doses have significantly reduced oedema volume by 4.75%, 17.25%, 38.00% and 8.75%, 21.25%, 42.25% respectively noted as time dependent effect. During 2nd h of study AELAS and AQELAS with low, medium and high doses have significantly reduced oedema volume by 7.78%, 21.91%, 43.32% and 11.20%, 29.19%, 50.60% respectively noted as time dependent effect. During 3rd h of study AELAS and AQELAS with low, medium and high doses have significantly reduced oedema volume by 19.45%, 32.80%, 55.42% and 19.84%, 39.18%, 61.81% respectively recorded as time dependent effect.

During 4th h of study AELAS and AQELAS with low, medium and high doses have significantly reduced oedema volume by 24.51%, 45.22%, 65.92% and 34.86%, 52.25%, 72.96% respectively which was recorded as time dependent effect and result are graphically represented in Fig No.5.9.

ANOVA indicates a significant difference among the AELAS and AQELAS treated groups. Dunnett‘s?t‘ test confirms a significant anti-inflammatory activity with both the extracts, but more with AQELAS than AELAS.

AELAS- Alcoholic Extract of Leaf of Allium Sativum, AQELAS- Aqueous Extract of Leaf of Allium Sativum.

AELAS- Alcoholic Extract of Leaf of Allium Sativum, AQELAS- Aqueous Extract of Leaf of Allium Sativum.