The Structural Changes Of Garlic Biology Essay

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Garlic and its preparations have been widely recognized as agents for prevention and treatment of cardiovascular and other metabolic diseases, atherosclerosis, hyperlipidemia, thrombosis, hypertension and hypoglycemia. This review discusses the possible mechanisms of therapeutic actions of garlic, different extraction procedures along with determination of its constituents, its stability and dissolutions method of garlic tablet.

Keywords: Garlic, Stability, Tablet, Alliin. Allicin, Antioxidant, Antimicrobial, Anticancer, Antihyperlipidemia, Antihypertension and Hypoglycemia.

Introduction

Garlic consists of fresh or dried compound bulbs of Allium Sativum Linn. (Family Lilliaceae).1

It contains not less than 0.2% of alliin (the main constituent present in the garlic), calculated on the dried basis. Its bulbs are made up of cloves and are wrapped in a white papery sheath with pungent taste and odour. 2

Constituents present in different garlic preparations3: -

Garlic homogenate : Allicin

Heat treated garlic : Alliin

Garlic powder : Alliin

Aged garlic extract : S-allyl cystine

Steam distilled garlic oil : Diallyl disulfide

Oil-macerated/ether-extracted garlic oil : 2-vinyl-4-H-1, 3-dithiin

Major metabolite of raw garlic on blood : Allyl mercapton

Synonyms 4

Sanskrit : Rasona, Yavanesta

English : Garlic

Gujrati : Lasan, Lassun

Hindi : Lahasun

Punjabi : Lasan

Urdu : Lahsan, Seer

Spanish : Ajo

Major organo-sulphur compounds present in different garlic preparations: - The garlic variety and manufacturing process are important considerations when choosing a garlic supplement, since products with different biologically active compounds and effects (fig. 1)3

Formation of organo-sulphur compounds during metabolic pathways in processed garlic (fig. 2)3

Reported Activities: - Garlic preparations are used as multipurpose and these are

Antioxidant and Antiatherosclerotic effect

Antihyperlipidimic effect

Platelet inhibitory and fibrinolytic effect

Antihypertensive effect

Anticancer effect

Antimicrobial effect

Hypoglycemic effect

Antioxidant: - The antioxidant effects of certain substances (garlic metabolites) of the secondary plant metabolism belong to the non-enzymatic protective mechanisms. Garlic enhances the serum levels of two antioxidant enzymes viz catalase and glutathione peroxidase5. It also decreases plasma malondialdehyde concentration and thus plays a role in the prevention of diseases of cardiovascular as well as providing protection against environmental sources of free radicals.6

Antiatherosclerotic effect: - Atherosclerosis results by accumulation of intra and extra cellular lipids. Garlic powder extract inhibits biosynthesis of cholesteryl esters and triglycerides in atherosclerotic cells and it also inhibits the activity of acyl-CoA cholesterol acyltransferase, the enzyme involved in the formation of cholesteryl esters, the main component of the excessive fat accumulated by cells. On the other hand, garlic extract stimulates cholesteryl ester hydrolase that degrades cholesteryl esters in atherosclerotic cells. 7

Antihyperlipidimic effect: - The inhibition of cholesterol synthesis in the liver is exerted at the level of HMG-CoA reductase, an enzyme involved in cholesterol biosynthesis. A low concentration of garlic compounds has no direct inhibition of HMG-CoA reductase. This activity is carried out by an indirect mechanism.8

Platelet inhibitory and fibrinolytic effect: - The inhibition of thromboxane production is caused by the inhibition of cyclooxygenase, and not lipoxygenase. There may be a direct inhibition of thromboxane. β-thromboglobulin release is decreased, which suggests that the effect may be more on the platelet activation phase. 9

Antihypertensive effect: - The hypotensive action of garlic may be due to a direct relaxant effect on smooth muscles. Aqueous garlic extract and its individual components, allicin and ajoene also open K+ channels, causing a membrane hyperpolarization and thus lead to a decrease of the Ca2+ inward current into the vascular smooth muscle cell and finally induce vasodilation resulting from intracellular Ca2+ decrease.9

Anticancer effect: - Treatment of human melanoma cells with S-allylcysteine reduces expression of cell-surface ganglisides, the tumor associated markers of differentiation and transformation. S180 tumor cells when exposed to a garlic extract displayed delayed progression to S phase. 10

Antimicrobial effect: - The antimicrobial activity of allicin is due to the inhibition of thiol-containing enzymes in the microorganisms. 11

Hypoglycemic effect: - The hypoglycemic action of garlic could possibly be due to potentiation of the insulin effect of plasma by increasing the pancreatic secretion of insulin from existing β-cells or its release from bound insulin and enhancement of insulin sensitivity. It has been previously suggested that garlic (allicin) can enhance serum insulin by effectively combining with compounds like cysteine, due to spare insulin from -SH group. 12, 13, 14

Purity Tests: - To ensure that all the analytical procedures performed allow an accurate statement of the content of impurities of an analyte, i.e. related substances test, heavy metals, residual solvents content, etc.

Total ash - Not more than 5.0%.

Acid-insoluble ash - Not more than 1.0%.

Water-soluble extractive - Not less than 5.0%.

Alcohol-soluble extractive - Not less than 4.0%.

Moisture - Not more than 7% 15

Extraction of allicin by different methods: - There are different methods to extract the allicin from the garlic. These are:-

I) 50 g of the peel garlic is cut into small pieces and homogenize in 70 ml of cold, sterile 0.9% NaCl in the presence of some crushed ice. The homogenization is carried out in a blender at high speed using 30 second bursts for a total of 10 minutes. The homogenized mixture is filtered 3 times through cheesecloth, the filtrate is centrifuge at 2000 RCF for 10 minutes and the clear supernatant is dilute to 100 ml with normal saline. The concentration of this garlic preparation is considered to be 500 mg/ml on the basis of the weight of the starting material (50 g/100 ml). The aqueous extract of garlic is store in small aliquots at -20°C until used. Determination is carried out by HPLC by taking allicin as standard. 13

II) Dried and ground bulbs (about 100 g) are submitting to extraction with 300 ml ethanol (80%) in a Soxhlet apparatus for 72 h. After extraction, the solvent is filter and then evaporated by Rotavapor. The obtained garlic alcoholic extract is store at -20 0C until being used. Determination is done by HPLC and allicin is taken as standard. 14

III) Garlic powder (125 mg) is transfer to a 25 ml volumetric flask, diluted with water to volume, vortex for 15 sec, sonicated at 8.0oC for 5 min, allow to stand at room temperature for 10 min (conversion of alliin into allicin) and centrifuged for 5 min. Then 5 ml of the supernatant is transfer to a 10 ml volumetric flask, 0.3 ml portion of internal standard 500 µg/ml (Ethyl paraben) is add (final concentration 15 µg/ml) and dilute with cold methanol to volume. Determination is carried out by HPLC and Ethyl paraben is used as internal standard. 16

IV) To 0.800 g of garlic powder, add 20.0 ml of water R and homogenise the mixture in an ultrasonic bath at 4°C for 5 min. Allow to stand at room temperature for 30 min. Then centrifuge for 30 min. Dilute 10.0 ml of the supernatant to 25.0 ml with a mixture of 40 volumes of a 1 % V/V solution of anhydrous formic acid R and 60 volumes of methanol R (stock solution). Shake and centrifuge for 5 min. Place 0.50 ml of the internal standard solution into a volumetric flask and dilute to 10.0 ml with the stock solution. 17

This is the only method in which Allicin used as an internal standard

Determination by HPLC

Column dimension : 0.25m long & 4mm internal diameter

Stationary phase : Silanised octadecylsilyl silica gel

Mobile phase : Anhydrous formic acid: Methanol (4: 6)

Flow rate : 0.8 ml/min

Internal standard : Butyl para hydroxybenzoate

Detector : Spectrophotometer at 254nm

Garlic powder

Homogenization 0.8gm powder with↓20ml of water at 4oC for 5 minutes in Ultrasonic bath

Homogenize powder

Incubate at room temperature for 30inutes ↓

Allicin containing Homogenize powder

Centrifugation for 30 minutes 6000 Xg↓

Supernatant

Dilute 10ml of the supernatant to 25ml stock solution↓

Centrifugation for 5 minutes

↓

0.50ml of the internal standard solution in a volumetric flask and dilute

to 10ml with the stock solution

↓

Determination by HPLC and Butyl parahydroxy benzoate as standard

Calculate the percentage of allicin from the expression:

22.75 X S1 M2/ S2 M1

S1 = area of the peak corresponding to allicin (most prominent peak)

S2 = area of the peak corresponding to butyl para hydroxybenzoate in the chromatogram obtained with the test solution

M1 = mass of the drug in grams

M2 = mass of butyl para hydroxybenzoate in grams in 100.0 ml of the internal standard solution

1 mg of butyl para hydroxybenzoate corresponds to 8.65 mg of allicin.18

Stability: - Stability is defined as the capacity of a drug substance or drug product to remain within established specifications to maintain its identity, strength, quality, and purity throughout the retest or expiration dating periods. Physical, chemical, and microbiological data are generated as a function of time and storage conditions (e.g., temperature and relative humidity (RH).

Thermo stability of allicin: -

Allicin is separated from garlic by using HPLC in a C18 column of MG-II (5μm, 4.6mm x 250mm)

Solvent used : 0.02M phosphate buffer (pH6.5): Acetonitrile: 1, 4 dioxane (7:1:2)

Flow rate : 0.5 ml/min

λmax : 220nm

Then compare the peak area produced by authentic allicin with that of the garlic extract. Authentic allicin is a reagent-grade preparation with 99.39% purity and is kept at -70oC until being use (fig. 3). 19

Rate constant for degradation and half life of allicin in the Garlic extract as determine by Biological assays using S.aureus and E.coli

The biological half life of allicin was calculated according to the equation:

t1/2 = 0.693/k

The biological activity of allicin was decreased in proportion to the incubation time as shown in Table 119

Stability of Allicin in different solvent: -

Ethanolic extract of allicin is more efficient than aqueous extract but the levels decreased gradually at room temperature, and allicin disappeared within a half-month, especially in 100 % ethanol or water. The 20 % and 50 % aqueous ethanol solutions are the most suitable solvents to maintain allicin for a couple of weeks at room temperature. In 20 % alcohol, allicin keeps its biological activity for longer time than in other ethanolic or aqueous solutions. . In the 50 % and 70 % ethanolic solutions, the antibacterial activity against E. coli was kept longer than in water or similar to that in water against S. aureus. In n-hexane and the vegetable oil, the amount of allicin and its biological activity decreased more rapidly than those in ethanolic aqueous solutions. Allicin is more unstable in vegetable oil than in n-hexane (Table 2). 20

Dissolution method: - Under the simulated gastrointestinal condition, the release of allicin and other allyl thiosulfinates is to be observed from the delayed-release garlic tablets. Using USP apparatus-II (paddle) dissolution method equilibrated at 37oC, one tablet was placed into round bottom glass vessels containing 750ml of 0.1N HCl and paddle stirred at 100 rpm for 2 h, after which 250ml of 0.2M Na3PO4 was added and the pH slightly adjusted if necessary, giving 1000ml at pH 6.80 ± 0.05. 1ml of the solution transfer to a test tube containing 0.05 ml of 0.21M carboxymethoxylamine hemihydrochloride immediately upon removal from the dissolution vessel in order to inhibit the alliinase enzyme and followed by HPLC analysis. The time to achieve complete disintegration was determined by observation during the dissolution test.2, 21 Contraindications: -

Garlic is contraindicated in patients with a known allergy to the drug. During pregnancy and lactation the use of garlic is not recommended and rare cases gastro-intestinal irritation or allergic reactions. 15

Precautions: -

Patients on warfarin therapy should be warned that garlic supplements may increase bleeding times. Blood clotting times have been reported to be double in patients taking warfarin and garlic supplements. 15

Recommended daily doses in humans: -

Fresh garlic : 4g approx 1 clove (4-12 mg of alliin or 2-

5 mg of Allicin)

Dehydrated garlic powder : 600-1200 mg in divided doses

Aged garlic : 1-7.2 g/day

Fresh air-dried bulb : 2-5 g

Garlic oil : 2-5 mg

Dried bulb : 2-4 g three times daily

Tincture (1:5 in 45% alcohol) : 2-4 ml three times daily 9

Conclusion:

Garlic has many health benefits and has been traditionally used worldwide. The wealth of scientific literature supports the proposal that garlic consumption have significant cardioprotective effect. Garlic is a magical medicinal herb and if consumed at regular basis, it has got the prophylactic but also curing effect.

Marketed preparation of Garlic (Table 3)

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