International Journal of Drug Development and Research

Key words

Garlic, Stability, Tablet, Alliin. Allicin, Antioxidant, Anticancer, 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] .

Synonyms

Constituents present in different garlic preparations: -

Major organo-sulphur compounds present in different garlic preparations: - The varieties of garlic and manufacturing processes are of important considerations when choosing a garlic supplement, since products with different biologically active compounds are (fig. 1) [4] .

Formation of organo-sulphur compounds during metabolic pathways in processed garlic (fig. 2) [4] .

Reported Activities: - Garlic preparations are used as multipurpose and these are 1) Antioxidant and Antiatherosclerotic effect 2) Antihyperlipidimic effect 3) Platelet inhibitory and fibrinolytic effect 4) Antihypertensive effect 5) Anticancer effect 6) Antimicrobial effect 7) 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 peroxidase [5] . 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 3-Hydroxy-3-Methyl-Glutaryl Coenzyme A (HMG-CoA) reductase, an enzyme involved in cholesterol biosynthesis. A low concentration of garlic compounds has no direct inhibition of HMGCoA reductase. This activity is carried out by an indirect mechanism [8] .

Platelet inhibitory and fibrinolytic effect: - Some form of garlic which increases cyclic adenosine monophosphate levels and thus decreases thromboxane formation. 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 [12-14] .

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 and the limits of garlic are.

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 (relative centrifugal force) 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 and store at -20 0C until being used. 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 4ºC 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 is not used as an internal standard and no need to store at – 20 0C.

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

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] .

Determination of degradation rate constant and half life of allicin by biological assays

The biological half life of allicin is calculated according to the equation: t1/2 = 0.693/k The biological activity of allicin decreases proportionally to the incubation time as shown in Table 1[19] .

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 nhexane 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] .

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

1) Fresh garlic : 4g approx 1 clove (4–12 mg of alliin or 2– 5 mg of Allicin) 2) Dehydrated garlic powder : 600–1200 mg in divided doses 3) Aged garlic : 1–7.2 g/day 4) Fresh air-dried bulb : 2–5 g 5) Garlic oil : 2–5 mg 6) Dried bulb : 2–4 g three times daily 7) Tincture (1:5 in 45% alcohol): 2–4 ml three times daily [9] .

Marketed preparation of garlic (Table 3)

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.

Conflict of Interest

NIL

Source of Support

NONE

Tables at a glance

Table 1 Table 2 Table 3

Figures at a glance

Figure 1 Figure 2 Figure 3

References

1. Indian Pharmacopoeia. Garlic. The Indian Pharmacopoeia Commission, Ghaziabad 2007; 2047-2048.
2. United States Pharmacopoeia. Garlic. United States Pharmacopoeial Convention, Inc, Rockville 2004; 1997-2002.
3. The Ayurvedic Pharmacopoeia of India. Garlic. The Controller of Publications Civil Lines, Delhi 2001; 108-109.
4. Martinez MC, Corzo N, Villamiel M. Biological properties of onions and garlic. Trends Food Sci Technol 2007; 18: 609-625.
5. Al-Numair KS. Hypocholesteremic and Antioxidant Effects of Garlic (Allium sativum L.) Extract in Rats Fed High Cholesterol Diet. Pak J Nutr 2009; 8: 161- 166.
6. Mathew BC, Biju RS. Neuroprotective effects of garlic a review. Libyan J Med 2007; 23-33.
7. Alexander NO, Grunwald J. Effects of Garlic on Atherosclerosis. Nutrition 1996; 13: 656-663.
8. Ackermann RT, Mulrow CD, Ramirez G, Gardner CD, Morbidoni L, Lawrence VA. Garlic shows promise for improving some cardiovascular risk factors. Arch Int Med 2001; 161: 813-824.
9. Bathaei FS, Akhondzadeh S. Cardiovascular Effects of Allium Sativum (Garlic): An Evidence-Based Review. J Teh Univ Heart Ctr 2008; 1: 5-10.
10. Shadkchan Y, Shemesh E, Mirelman D. Efficacy of allicin, the reactive molecule of garlic, in inhibiting Aspergillus spp. in vitro, and in a murine model of disseminated aspergillosis. J Antimicrob Chemother 2004; 1-5.
11. Shariatzadeh SMA, Mehranjani MS, Mahmoodi M. Effect of Garlic (Allium Sativum) on Blood Sugar and Nephropathy in Diabetic Rats. J Biol Sci 2008; 1-6.
12. Thomson M, Al-Amin ZM, Al-Qattan KK, Shaban LH, Ali M. Anti-diabetic and hypolipidaemic properties of garlic (Allium Sativum) in streptozotocin-induced diabetic rats. Int J Diabetes & Metab 2007; 15: 108-115.
13. Eidia A, Eidib M, Esmaeilia E. Antidiabetic effect of garlic (Allium sativum L.) in normal and streptozotocin-induced diabetic rats. Phytomedicine 2006; 13: 624-629.
14. WHO monographs on selected medicinal plants. Bulbus Allii Sativi., vol 1, 1999.
15. Diego MD, Avello M, Mennickent S, Fernandez M, Fernandez P. Validated liquid chromatographic method for quantitative determination of allicin in garlic powder and tablets. J Sep Sci 2007; 30: 2703- 2707.
16. Baghalian K, Ziai SA, Naghavi MR, Badi HN, Khalighi A. Evaluation of allicin and botanical traits in Iranian garlic (Allium Sativum L.) Ecotypes. Sci Hort 2005; 10: 155-166.
17. Fujisawa H, Suma K, Origuchi K, Seki T, Ariga T. Thermostability of allicin determined by chemical and biological assays. Biosci Biotechnol Biochem 2008; 72: 2877-2883.
18. Fujisawa H, Suma K, Origuchi K, Kumagal H, Seki T, Ariga T. Biological and chemical stability of garlic-derived allicin. J Agric Food Chem 2008; 56: 4229-4235.
19. Blatt Y, Cohen D, Kimmelman E, Friedman O, Rotman A. US Patent No. 6270803 B1 2007.