MAOZ, Michal (16 Carcom Street, Karmiel, 21661, IL)
ASSY, Nimer (P.O. Box 428, Fassouta, 25170, IL)
GROZOVSKI, Maria (9 Hadekel Street, Karmiel, 21892, IL)
MAOZ, Michal (16 Carcom Street, Karmiel, 21661, IL)
ASSY, Nimer (P.O. Box 428, Fassouta, 25170, IL)
| Claims What is claimed is: 1. A cola beverage product which consumption does not severe syndromes of non alcoholic steatohepatitis (NASH) and other non alcoholic fatty liver disease (NAFLD) in a mammal suffering thereof comprising: (a) water, (b) a natural nutritive sweetener , (c) a colorant comprising caramel color, (d) a cola flavor substance and (e) an effective amount of an Inula viscosa extract . 2. The cola beverage product as in claim 1 wherein said syndromes are selected from the group consisting of increase of hepatic concentration of cholesterol, increase of hepatic concentration of protein C, increase of hepatic concentration of triglyceride and a combination thereof. 3. The cola beverage product as in claim 1 wherein said syndromes include one or more of the following changes: (i) increase of hepatic level of MD A (ii) decrease of the hepatic level of alpha-tocopherol and (iii) decrease of the hepatic activity of paraoxonase 4 The cola beverage product as in claim 1, wherein said water is carbonated water. 5 The cola beverage product as in claim 1 , wherein the concentration of the colorant is from about 5.0 to about 10.0 g/L. 6 The cola beverage product as in claim 1, wherein said natural nutritive sweetener is selected from the group consisting of sucrose, glucose, fructose, maltose, rhamnose, tagatose, trehalose, corn syrups, fructo-oligosaccharides, Lo Han Guo juice concentrate and combination thereof. 7 The cola beverage product as in claim 1 , wherein concentration of said cola flavor substance comprises from about 1.0% to about 2.5% by weight. 8 The cola beverage product as in claim 1, wherein said effective amount of said Inula viscosa extract comprises a weight concentration of about 0. 1% . 9 The cola beverage product as in claim 1, further comprising natural buffering salts selected from sodium and potassium salts of citric, tartaric, and lactic acids. 10 The cola beverage product as in claim 1 , further comprising natural caffeine. 11. The cola beverage product as in claim 1 , further comprising a natural preservative. 12. The cola beverage product as in claim 1 , wherein the natural preservative comprises nisin, cinnamic acid or a combination thereof. 13. The cola beverage as in claim 1 , wherein said mammal is a rat. 14. The cola beverage as in claim 1, wherein said Inula viscosa extract is obtained from leaves of Inula viscosa plant. 15. A cola beverage product which consumption diminishes an increase in hepatic concentration of cholesterol, in hepatic concentration of protein C, in hepatic concentration of triglyceride and in a combination thereof in a mammal suffering of non alcoholic steatohepatitis (NASH) and other non alcoholic fatty liver disease (NAFLD) comprising: (a) Coca-Cola® soft drink and (b) an effective amount of Inula viscosa extract . 16. The beverage product as in claim 15, wherein said effective amount of said Inula viscosa extract comprises a weight concentration of about 0. 1%. 17. A method for diminishing an increase in hepatic concentration of cholesterol, in hepatic concentration of protein C, in hepatic concentration of triglyceride and in a combination thereof, all of which occur in a mammal suffering of non alcoholic steatohepatitis (NASH) or other non alcoholic fatty liver diseases (NAFLD when consuming a naturally sweetened cola beverage, the method comprises of adding to the naturally sweetened cola beverage an effective amount of an extract derived from an Inula viscosa plant. 18. The method as in claim 17, further diminishing one or more of the following changes: (i) increase of hepatic level of MDA, (ii) reduction of the hepatic level of alpha-tocopherol and (iii) reduction of the hepatic activity of paraoxonase. The method as in claim 17, wherein said mammal is a rat. 20 The method as in claim 17, wherein said naturally sweetened cola beverage comprises substances selected from the group consisting of sucrose, glucose, fructose, maltose, rhamnose, tagatose, trehalose, corn syrups, fructo-oligosaccharides, Lo Han Guo juice concentrate and combination thereof. 21 The method as in claim 17, wherein said extract is obtained from leaves of the Inula viscosa plant. 22 The method as in claim 17, wherein said effective amount of said Inula extract comprises a weight concentration of about 0.1%. |
consumers suffering non-alcoholic fatty liver disease (NAFLD) BACKGROUND OF THE INVENTION
This invention relates to the area of exploiting therapeutically properties of compositions extracted from naturally occurring substances and more particularly, to a substance and method for decreasing the hepatic triglycerides and increasing low antioxidants levels which are caused by consuming naturally sweetened cola beverages worldwide known as "cola".
Carbonated beverages with cola flavor have been very popular in the U.S. and worldwide for decades. In 2004, it was estimated that a total of 4.8 billion cases (24 bottles of 8 oz per case) of cola beverages were consumed in the U.S. alone.
In general, cola contains water, carbon dioxide, high fructose corn syrup (HFCS) or other sugar, phosphoric acid, caramel color, and cola flavoring agents. The terms "cola" and "cola beverage" are likewise used herein consistent with their ordinary meanings in the art and nothing herein is intended to limit their meanings.
Extensive fructose consumption can cause progressive liver disease in humans as demonstrated by the inborn error of metabolism known as hereditary fructose intolerance, a rare disease which results from a deficiency of the fructose metabolizing enzyme aldolase B, see e.g. Santer et al. in "T/ze spectrum of aldolase B mutations and the prevalence of hereditary fructose intolerance in central Europe", Hum. Mutat. 2005; 25:594., hence diet beverages were introduced.
Diet cola for example, has ingredients similar to full calorie cola, except ihat HFCS or sugar is replaced by at least one artificial sweetener, for example aspartame. Artificial
preservatives such as sodium benzoate may be included to ensure microbial safety. However, use of artificial sweeteners results in beverages which have a different quality of sweetness than full- calorie beverages. So there is perceived market demand for the original and unmodified cola beverages in spite the fact that high fructose diets have induced fatty liver in rats and ducks, see e.g. Davail et al. in "Effects of dietary fructose on liver steatosis in overfed mule ducks", Horm. Metab. Res., 2005; 37:32-35.
Normally, less than 5% of the liver mass is fat by weight, but in patients with non alcoholic steatohepatitis (NASH), as much as 50%-80% of liver weight may be made up of fat, mostly in the form of triglycerides, see e.g.: Angulo, P. in "Nonalcoholic fatty liver disease", N. Engl. J. Med. 2002; 346:1221-1231.
The clinical implications of NASH are derived mostly from its common occurrence in the general population (10-24%) and its potential to progress to fibrosis (30-40%), cirrhosis (20- 30%) and hepatocellular carcinoma see e.g.: Assy et al. in "Fatty infiltration of liver in hyperlipidemic patients". Dig. Dis. Sci. 2000; 45:1929-1934, and Burke al. in "Non-alcoholic fatty liver disease, non-alcoholic steatohepatitis and orthotropic liver transplantation", Am. J. Transplant. 2004; 4:686-93.
NASH is the most common cause of cryptogenic cirrhosis and is an increasingly common indication for liver transplantation, see Burke above.
Non alcoholic fatty liver disease (NAFLD) is one of the most important emerging health issues and obesity, type 2 diabetes mellitus and hyperlipidemia are conditions frequently associated with NAFLD, see e.g.: Angulo, P. in "Nonalcoholic fatty liver disease", N. Engl. J. Med. 2002; 346:1221-1231, Assy et al. in "Fatty infiltration of liver in hyperlipidemic patients". Dig. Dis. Sci. 2000; 45 : 1929- 1934 and Burke al. in "Non-alcoholic fatty liver disease, nonalcoholic steatohepatitis and orthotropic liver transplantation", Am. J. Transplant. 2004; 4:686- 93.
Although the extent to which excessive fructose or other sugars might contribute to the high prevalence of NAFLD in western societies has not been systematically investigated, it has been shown that soft drink consumption is linked to obesity and results in an increased risk of metabolic syndrome. Individuals consuming more than 1 soft drink per day had a higher prevalence of metabolic syndrome than those consuming less than 1 drink per day.
This is so because soft drink is the leading cause of added sugar in the world. Recent evidence suggests an association between the intake of sugar sweetened soft drink and the risk of obesity and diabetes because they contain large amount of high-fructose corn syrup, which raises blood glucose similarly to sucrose see e.g.: Gaby, A.R. in "Adverse effects of dietary fructose", Alternative Medicine Review 2005; 10:294.
Whether or not soft drink consumption is linked with NAFLD has not been assessed yet, see e.g. Dhingra et al. in "Soft drink consumption and risk of developing cardio metabolic risk factors and the metabolic syndrome in middle-aged adults in the community", Circulation 2007; 116:480-488. Authors of the present invention disclosed that drinking Coca-Cola® per-se by healthy rats had no impact on liver cholesterol concentration but it increased hepatic triglyceride (TG) and malonic dialdehyde (MDA) levels and lowered hepatic antioxidants concentration.
Hereinafter, the term Coca-Cola® designates the classical regular (i.e. not diet, not caffeinated free, not cola zero, etc.) soft drink sold internationally in stores, restaurant and vending machines, which concentrate is produced by The Coca Cola company in Atlanta Georgia USA.
Coca-Cola® is the leading drink among cola beverages which comprises of Carbonated water, natural nutritive sweeteners, a colorant comprising caramel color and a cola flavor substance .
The nutritive sweeteners may be one of the following substances: sucrose, glucose, fructose, maltose, rhamnose, tagatose, trehalose, corn syrups, fructo-oligosaccharides, Lo Han Guo juice concentrate and combination thereof.
The concentration of the colorant may be from about 5.0 to about 10.0 g/L wherein concentration of said cola flavor substance may be from about 1.0% to about 2.5% by weight.
Other ingredients in Coca-Cola® are natural buffering salts such as sodium and potassium salts of citric or tartaric or lactic acids, natural caffeine and a natural preservative such as e.g. nisin, cinnamic acid or a combination thereof.
Drinking Coca- Cola® by rats with NAFLD syndrome severed the mentioned above symptoms. Coca-Cola® also supported liver inflammation as was seen by the enlarged rank of hepatic protein C.
Accordingly, drinking Coca-Cola® as well as other sugar or fructose containing cola beverages by NAFLD patient should be considered as an unfavorable action.
The present invention eliminates this limitation associated with drinking cola and carries additional advantages which will become apparent upon reading the following text.
SUMMARY OF THE INVENTION
The present invention is a cola product which does not increase hepatic cholesterol concentrations, hepatic triglyceride concentration, hepatic protein C concentrations and hepatic levels of MDA all of which are caused in mammals suffering of non alcoholic steatohepatitis (NASH) or other non alcoholic fatty liver diseases (NAFLD when consuming naturally sweetened cola. In accordance to the present invention there is provided a cola beverage product which does not severe syndromes of non alcoholic steatohepatitis (NASH) and other non alcoholic fatty liver disease (NAFLD) in a mammal in need thereof comprising:
(a) water; (b) a natural nutritive sweetener ; (c) a colorant comprising caramel color; (d) a cola flavor substance and (e) an effective amount of an aqueous Inula viscosa extract .
In accordance to the the present invention there is provided a cola beverage product which consumption diminishes an increase in hepatic concentration of cholesterol, in hepatic
concentration of protein C, in hepatic concentration of triglyceride and in a combination thereof in a mammal suffering of non alcoholic steatohepatitis (NASH) and other non alcoholic fatty liver disease (NAFLD) comprising: (a) Coca-Cola® soft drink and (b) an effective amount of Inula viscosa extract.
In accordance to the present invention there is provided a method for diminishing an increase in hepatic concentration of cholesterol, in hepatic concentration of protein C, in hepatic concentration of triglyceride and in a combination thereof, all of which occur in a mammal suffering of non alcoholic steatohepatitis (NASH) or other non alcoholic fatty liver diseases (NAFLD when consuming a naturally sweetened cola beverage, the method comprises of adding to the naturally sweetened cola beverage an effective amount of an extract derived from an Inula viscosa plant.
Authors of the present invention disclosed that drinking Coca-Cola® itself had no impact on cholesterol concentration in healthy rats, however it increased TG and MDA levels and lowered antioxidants cone. Coca-Cola® also support liver inflammation as can be seen by the enlarged rank of protein C.
Adding Inula viscosa extract to Coca Cola® lowered cholesterol in the healthy rats below the normal concentration. It also lowered TG and MDA levels. As for antioxidants, it decreased alpha tocopherol concentration to normal level and increased Paraoxonase activity. The protein C level was reduced to the normal control.
Drinking Coca-Cola® by rats with metabolic syndrome severed the symptoms, i.e. it further increased the hepatic cholesterol, TG, MDA and protein C concentration levels. As for antioxidants it increased reduced alpha tocopherol concentration and Paraoxonase activity.
Adding / viscosa extract to the Coca-Cola® canceled these severities. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The present embodiments herein are not intended to be exhaustive and to limit in any way the scope of the invention; rather they are used as examples for the clarification of the invention and for enabling of other skilled in the art to utilize its teaching.
The present invention is a cola based drink containing an additive which does not contributes to an increase in hepatic cholesterol concentrations, hepatic triglyceride concentration, hepatic protein C concentrations and hepatic levels of MDA all of which were found to occur in mammals suffering of non alcoholic steatohepatitis (NASH) or non alcoholic fatty liver diseases (NAFLD) when drinking regular cola.
The new drink comprises of a therapeutically effective amount of an extract derived of the Inula viscosa plant which is added to naturally sweetened cola.
Inula viscosa (helenium), which is a member of the Compositae family, is a plant that grows to about 1 to 1.5 meter in height. The leaves and stems of the plant are coated with a sticky resin. Its flowers which blossom mainly during August to November are widely rounded and are yellowish in color. Roots are deep brown in color, whitish on the inside and have a characteristic smell. The plant is also known in Arabic as Rasen. This plant grows in the Mediterranean basin.
An aqueous extract of Inula viscosa is prepared according to the method developed by Michal Maoz: see e.g.: Maoz M. et al., in "Isolation and identification of a new antifungal Sesquiterpene Lactone from Inula viscose", Planta Medica 65, 1999, which is incorporated here by reference for all purposes as if fully set forth herein.
Preferably, leaves of Inula viscosa are collected at the end of summer in the Galilee, Israel and dried immediately after collection at an oven at 60°C for 14 hours and then the dried leaves are grinded to powder.
The powdered leaves are extracted with a borate buffer pH 9.0 (0.1M boric acid with the addition of NaOH). The concentration of the content of the dried powdered leaves in the buffer is about 10% in weight (w%). The extraction was carried out in an autoclave at 121°C for 15 minutes to get an aqueous extract.
After cooling, the aqueous extract was filtered through few layers of gauze. This filtrate was used as the source of the Inula viscosa extract in the experiments which are described below and will be referred to hereinafter as the Inula viscosa extract.
Inula viscosa extract is known for its biological activity. It is active against microorganism, especially fungi, see e.g.: Maoz, M. and Neeman I., in "Effect of Inula viscosa extract on chitin synthesis in dermatophytes and Candida albicans ", Journal of Ethnopharmacology 71; 479-482, 2000 and U.S. Patent No. 4,254,112 to Debat et al.
Other kinds of extract of Inula viscosa plant possess antiviral properties see e.g.: U.S. Patent No. 6,841,174 to Shalaby et al. and are effective in reduction blood glucose see e.g.: Zeggwaghaet al., "Study of hypoglycaemic and and have cytotoxic effect on several cancer cell cultures, hypolipidemic effects of Inula viscosa L. aqueous extract in normal and diabetic rats", J. of Ethnopharmacology, Vol. 108, issue 2, 223-227, 2006, and has cytotoxic effect on several cancer cell cultures.
The activity of Inula viscosa extract is attributed to the compound- tayunin which is a sesquiterpen lactone, see e.g.: "Tayunin - A new compound from Inula viscosa leaves and its antifungal activity against dermatophytes and the yeast Candida albicans ", A dissertation of the Faculty of Food Engineering and Biotechnology, Technion-IIT, Haifa, Israel 1997, and Berdicevski et al., in "Antimycotic Activity ofTayunin-Inula Viscosa Extract-SEM Observations", 41st Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago 111., 2001.
Recently, inventors of the present invention disclosed an effective reduction of hepatic cholesterol concentration, hepatic triglyceride concentration, hepatic protein C concentration and hepatic levels of MDA together with an increase of hepatic antioxidant activity in mammals suffering non alcoholic steatohepatitis (NASH) and other non alcoholic fatty liver disease (NAFLD) which is achieved by administrating to the sick mammals the Inula viscosa extract, see e.g. IL patent application No.199847 which is incorporated here by reference for all purposes as if fully set forth herein.
The present invention is based on carefully designed laboratory tests in a study which was conducted using rats and which is described below; the study had two aims:
Firstly, to find out what are the effect of drinking cola on lipid metabolism and peroxidation, liver protein C and antioxidants levels (alpha tocopherol and paraoxonase) in healthy and fatty liver rats, and secondly to inquire the effect of the addition of an Inula viscosa extract to cola on the above parameters.
Procedures Used in carrying out the study
Materials and Methods
Forty eight male Sprague-Dawley rats (Harlan Laboratories Ltd., Jerusalem, Israel) weighing 200±20 grams were studied.
Rats were housed in regular cages situated in an animal room at 22°C, with a 14/10-hour light/dark cycle. Rats were maintained on standard rat chow diet (SRCD) (pellets #19520; Koffolk, Tel Aviv, Israel) and were given tap water to dnnkad libitum. All animal studies were conducted according to the regulations for the use and care of experimental animals.
At the beginning of the study rats were randomly divided in two diet groups. First diet group (24 rats) served as liver control rats and was maintained on standard chow diet (control liver) for 12 weeks, whereas the other diet group (24 rats) was given fructose enriched diet (FED) only, (TD 89247; Harlan Teklad, Madison, WI, USA) for 12 weeks.
The FED contained (as supplied by Harlan Teklad) 20.7% (per weight basis) protein (as casein), 5% fat (as lard), 60% carbohydrates (as fructose), 8% cellulose, 5% mineral mix
(#170760; R-H) and 1% vitamin mix (#40060; Teklad). The SRCD (Koffolk) contains 21.9% protein, 4.5% fat, 41% starch, 5% sugar and 3.7% crude fiber.
Both diets were in pellet form. 12 weeks after the initiation of FED each diet group was randomized for establishing four treatment groups having six repetitions each as shown in table 1.
The first group remained untreated, the second group was given 25 ml of water containing 5.6 mg of Inula viscosa extract per day per day . The third group was given 4ml regular Coca-Cola® per day wherein the Coca-Cola® was diluted in 25 ml of water and the fourth group was given 25 ml of drinking water containing 5.6 mg of Inula viscosa extract together with 4ml of regular Coca-Cola® (which corresponds to a weight ratio of 0.14 % betwen the extract and the Cola respectively) per day.
Treatments were given to both diet groups: control rats and FED rats
Table 1 - Treatments groups
Hepatic lipid extraction: Total hepatic lipids were extracted from freeze-dried liver samples by chloroform: methanol (2:1) according to Folch et al. (Folch et al. in "A simple method for isolation and purification of total lipids from animal tissues. ' ' ' ' J. Biol. Chem.; 226,497-509 1957.
Hepatic enzymes extraction: Hepatic antioxidant enzymes were measured in the rat liver cytosolic fraction. Approximately 0.5 g of liver was homogenized in 5 ml of ice-cold 50 mmol phosphate buffer (pH 7.4). The liver homogenate was centriiuged for 10 minutes at 5000 rpm at 4°C and the pellet was discarded. The supernatant was centrifuged for a further 30 minutes at 8000 rpm at 4°C.
Results
A. The effect of the treatments on liver control rats
Results are summarized in Tables Nos. 2-4.
1. Inula viscosa extract reduced hepatic cholesterol and protein C by -8% and -3%, respectively and increased hepatic level of MDA (+ 5%) as compared to untreated group. Inula viscosa extract had also increased concentrations of hepatic triglyceride, alpha tocopherol and paraoxonase (PON) (+6.5%, +54% and+9%, respectively), compared to untreated group.
2. Coca-Cola® had increased hepatic triglyceride, protein C and MDA by +270%, +30% and +99%, respectively as compared to untreated group. The hepatic levels of alpha- tocopherol and paraoxonase (PON) activity were decreased (-25% and -44%, respectively) as compared to untreated group.
3. Combination of Inula viscosa extract and Coca-Cola® reduced hepatic cholesterol, hepatic triglyceride, hepatic MDA, protein C and alpha-tocopherol concentrations by -20%, - 34%, -50%, -10% and -28%, respectively, and increased paraoxonase (PON) activity by +130% as compared to control group with Coca Cola® (group No. 3).
Table 2. Average values of hepatic cholesterol and hepatic triglycerides
in control rats as a result of the treatments
* Compared to untreated group
** Compared to group No.3 Table 3. Average values of hepatic Protein C and hepatic malon dialdehyde in control rats as a result of tiie treatments
* Compared to untreated group
** Compared to group No. 3
Table 4. Average values of hepatic alpha tocopherol and Paraoxonase activity in control rats as a result of the treatments
* Compared to untreated group
** Compared to group No.3 Conclusions for the various treatments on the healthy liver control rats are the following:
Drinking Coca-Cola® itself had no impact on cholesterol concentration. However it increased TG and MDA levels and lowered antioxidants cone. Coca-Cola® also support liver inflammation as can be seen by the enlarged rank of protein C.
The effect of the I.viscosa extract alone is mainly to increase the alpha tocopherol concentration and Paraoxonase activity.
Adding I.viscosa extract to Coca-Cola® lowered cholesterol below the normal concentration. It also lowered TG and MDA levels. As for antioxidants it decreased alpha tocopherol concentration to normal level and increased Paraoxonase activity. The protein C level was reduced to the normal control.
B. Effect of fructose diet on rats
Fatty liver induced by the fructose enriched diet (FED) had +10% increases in the hepatic cholesterol concentrations as compared to liver control rats. Hepatic triglyceride, hepatic protein C concentrations and hepatic levels of MDA were significantly higher (+240%, +58.7% and +109%, respectively). Fatty liver rats had significantly lower concentrations of alpha- tocopherol and paroxonase activity when compared with control liver (-30% and -47%, respectively).
The observed redox imbalance in NAFLD as a consequence of decreased levels of antioxidants, along with an increased MDA levels in circulation may be important factor in the development of NASH. C. Effect of the treatments on FED rats
Results are shown in Tables Nos. 5-7.
1. Inula viscosa extract reduced hepatic triglyceride, hepatic cholesterol and protein C by -34%, -31% and -43%, respectively and the hepatic levels of MDA were significantly lower (- 49%) as compared to untreated group. Inula viscosa extract had significantly increased
concentrations of alpha-tocopherol and paroxonase activity (+94% and +97%, respectively).
2. Coca-Cola® had increased hepatic triglyceride and hepatic cholesterol by +78% and +5%, respectively as compared to untreated rats. The hepatic levels of MDA, alpha-tocopherol and paraoxonase (PON) activity were decreased (-13%, -30% and -32%, respectively) compared with untreated rats. 3. Combination of Inula viscosa extract and Coca-Cola® reduced hepatic
triglyceride, hepatic cholesterol, hepatic MDA, hepatic protein C and alpha-tocopherol by -59%, - 39%, -51% , -20% and -17%, respectively as compared to FED rats fed with Coca Cola®.
Paraoxonase activity in the same group was increased by +1 14%. Table 5. Average values of hepatic cholesterol and hepatic triglycerides in FED rats as a result of the treatments
* Compared to untreated group.
** Compared to group No. 3.
Table 6. Average values of hepatic Protein C and hepatic malon dialdehyde in FED rats as a result of the treatments
* Compared to untreated group
** Compared to group No.3
Table 7. Average values of hepatic alpha tocopherol and Paraoxonase activity in FED rats as a result of the treatments
* Compared to untreated group
** Compared to group No.3 Conclusions for the treatments on the FED rats are the following:
Treating with I.viscosa extract diminishes the increases in the hepatic cholesterol, hepatic triglyceride, hepatic protein C concentrations and hepatic levels of MDA concentrations of FED rats as compared to liver untreated rats. Inula viscosa extract had significantly increased concentrations of alpha-tocopherol and paraoxonase activity as compared to liver untreated rats.
Drinking Coca-Cola® by FED rats possessing the metabolic syndrome severed the mentioned above symptoms.
Adding a small amount of about 0.03% in weight of I.viscosa extract to regular Coca- Cola® drink canceled the severity which is caused by drinking regular Coca-Cola® and enables it consumption by both healthy as well as sick rats without harming their liver.
While the invention has been described with respect to a limited number of embodiments, it will be appreciated that many variations, modifications and other applications of the invention may be made without departing from the spirit and scope of the invention.