FIELD OF THE INVENTION
The present invention relates to synergistic herbal compositions. More particularly, the present invention relates to synergistic herbal composition, comprising Guggulu oleoresin, Asafoetida oleoresin, Dill oil, Fennel oil and Ajwain oil, as active ingredients in effective amount and pharmaceutical acceptable excipients, which is effective in reducing body lipids.
BACKGROUND OF THE INVENTION
Nature has endowed us with a variety of herbs that possess numerous medicinal properties, and that are used to prepare herbal medicines. Herbs with proven beneficial effects include Commiphora wightii, Ferula asafoetida, Foeniculum vulgare, Anethum graveolens, and Trachyspermum ammi.
Guggulu consists of oleoresin obtained as an exudate from the tapping of stem and branches of Commiphora wightii [Family, Burseraceae]. The plant is commonly known as Guggul tree and is found in arid areas of India, Bangladesh, and Pakistan. In India, it is found in Rajasthan, Gujarat, Assam, Madhya Pradesh, and Karnataka. It is a small, bushy tree with thorny branches and produces a yellowish gum resin (Guggulu) in small ducts located throughout its bark. The trees are tapped by making an incision on the bark. The resin, which flows out, is allowed to harden before it is collected. The tree is tapped from November to January and the resin is collected through May to June. A Guggul tree yields between 250 to 500?g of dry resin during each collection season. (Sarup et al.,“Pharmacology and Phytochemistry of Oleo-Gum Resin of Commiphora wightii (Guggulu),” Scientifica, vol. 2015, Article ID 138039). Ancient Sanskrit medical text, Sushruta Samhita, also discloses prescription of guggul for the treatment of medoroga, a disease that closely resembles the symptoms of high cholesterol and hardening of the arteries.
Asafoetida consists of oleoresin obtained from the rhizome or tap root of several species of Ferula, a perennial herb, which is prized as a condiment in India and Iran. Asafoetida is also known as food of the gods, jowani badian, hing, hengu, ingu, kayam, and ting. Asafoetida is been used as traditional medicine for various diseases like flatulence, constipation, topical treatment for abdominal injuries, asthma, bronchitis, hysteria etc. Asafoetida also has antimicrobial, Antiepileptic and contraceptive/abortifacient properties as disclosed in literature.
Fennel (Foeniculum vulgare) is a flowering plant species in the carrot family. It is a hardy, perennial herb with yellow flowers and feathery leaves. It is indigenous to the shores of the Mediterranean but has become widely naturalized in many parts of the world, especially on dry soils near the sea-coast and on riverbanks. It is a highly aromatic and flavorful herb with culinary and medicinal uses.
Dill (Anethum graveolens) is an annual herb in the celery family Apiaceae. It is the only species in the genus Anethum. Dill is widely grown in Eurasia where its leaves and seeds are known for flavouring food and medicinal uses.
Ajwain (Trachyspermum ammi), is also known as Ajowan caraway, Bishop's weed or Carom, is an annual herb in the family Apiaceae. It originated in India and Pakistan. Both the leaves and the fruit (often mistakenly called seeds) of the plant are consumed by humans. Ajwain is traditionally used for flavouring food and treatment of disorders such as indigestion, flatulence, diarrhoea and colic. Ajwain is also used as antacid and cleanser.
Several herbal extracts contain powerful ingredients, which are bio-active chemical entities occurring in nature that can be harnessed to remedy the myriad pathologies of the human body. Elevated level of lipid in the blood is one such pathology affecting an ever increasing percentage of the population. Lipids are a class of chemical compounds that are essential for healthy cell membranes, hormone production, and vitamin storage. Even the brain depends on lipids for proper functioning. Lipids become a problem when too much of it is produced in the body or ingested through regular eating of unhealthy foods.
Excessive lipids in the blood accumulate over time, forming plaques on the walls of the arteries and blood vessels. This narrows the openings, producing turbulent blood flow through the vessels, and cause the heart to use more force to get the blood through the constricted areas.
Lifestyle modification remains the best strategy for both preventing and treating hyperlipidemia. This involves adhering to a healthy diet, regular exercise, no smoking, and maintenance of a healthy weight. However, our modern fast paced lifestyle leaves no room for such lifestyle modifications.
Other options for reducing the amount of body lipids include drugs belonging to the class of statins and fibrate. However, these therapeutic agents are not free from side effects. Statins (HMG- CoA reductase inhibitors) the most widely used drugs today which hitherto were thought to be very safe drugs, have exhibited side effects following long term therapy. The adverse effects, which have become the source of concern, are increase in hepatic transminases and myopathies.
US patent publication 20040253327A1 discloses composition of guggul extract for controlling blood cholesterol and discloses use of guggulipids for lowering blood cholesterol.
US patent publication 20070172468A1 discloses use of guggul lipid a guggulsterone source in composition with phytosterol source, soluble fiber source, policosanol source as dietary supplement for reducing blood cholesterol levels.
DE patent publication 19633446A1 patent publication discloses use of Anethum graveolens in composition with Fumara officinalis, Cichorium intybus, Cynara scolymus, and Citrus limonum risso for lowering blood lipids.
However, there is a need of to develop a synergistic herbal composition with enhanced efficacy, which is free from mentioned drawbacks and decreases the level of lipids in the blood, thereby preventing cardiovascular complications and damage to vital organs such as the liver.
The inventors of the present invention have found that a synergistic herbal composition comprising Guggulu oleoresin, Asafoetida oleoresin, Dill oil, Fennel oil and Ajwain oil is very effective in reducing the levels of lipids in the body.

OBJECTS OF THE INVENTION
It is, therefore, an important object of the present invention to provide a synergistic herbal composition, which overcomes the disadvantages of the prior art.
It is yet another object of the present invention to provide a synergistic herbal composition which reduces body lipids and prevents cardiovascular complications and damage to vital organs.
It is yet another object of the present invention to provide a synergistic herbal composition which is better alternative to synthetic drugs.
It is yet another object of the present to provide a synergistic herbal composition, which is free from side effects.
SUMMARY OF THE INVENTION
In accordance with an embodiment of the invention, there is provided a novel synergistic herbal composition comprising an amount effective of (a) Guggulu oleoresin; (b) Asafoetida oleoresin; (c) Dill oil; (d) Fennel oil; and (e) Ajwain oil for reducing body lipids
In another embodiment of the present invention, there is provided a synergistic herbal composition comprising (a) Guggulu oleoresin is present in the range of 0.50% – 10.00%; (b) Asafoetida oleoresin is present in the range of 0.05% – 5.00%; (c) Dill oil is present in the range of 0.50% – 10.00%; (d) Fennel oil is present in the range of 1.00% - 20.00%; and (e) Ajwain oil is present in the range of 0.25% – 10.00%.
In an embodiment of the present invention, there is provided a synergistic herbal composition comprising one or more pharmaceutically acceptable excipients.
In an another embodiment of the present invention, there is provided a synergistic herbal composition comprising pharmaceutically acceptable excipients selected from the group comprising of diluents, antioxidants, adsorbents, glidants, lubricants, antimicrobial preservatives, emulsifiers, buffering agents, solvents, colouring agents, plasticizers, co-solvents, capsule shell and so forth but not limited to it.
In an another embodiment of the present invention, there is provided a synergistic herbal composition comprising diluents selected from group comprising of Till oil, Arachis oil, Microcrystalline cellulose, and water; wherein till oil is present in the range of 10.00% to 50.00% w/w; Arachis oil is present in range of 25.00% to 75.00% w/w; Microcrystalline cellulose is present in range of 50.00% to 80.00% w/w and water is present in range of quantity sufficient.
In an another embodiment of the present invention, there is provided a synergistic herbal composition comprising antioxidant is Butylated Hydroxy Toluene present in range of 0.01% to 0.50% w/w.
In an another embodiment of the present invention, there is provided a synergistic herbal composition wherein adsorbents is selected from group comprising of Microcrystalline cellulose and Colloidal silicon dioxide; wherein Microcrystalline cellulose is present in range of 40.00% to 80.00% w/w and Colloidal silicon dioxide is present in range of 0.50% to 5.00% w/w.
Yet in another embodiment of the present invention, there is provided a synergistic herbal composition comprising lubricants is Magnesium stearate present in range of 0.10% to 2.50% w/w.
In an another embodiment of the present invention, there is provided a synergistic herbal composition comprising antimicrobial preservatives selected from group comprising of Sodium benzoate, Potassium sorbate, Methylparaben or sodium salt, Propylparaben or sodium salt thereof; wherein, Sodium benzoate is present in range of 0.01% to 0.20% w/w, Potassium sorbate is present in range of 0.01% to 0.20% w/w, Methylparaben or sodium salt thereof is present in range of 0.01% to 0.20% w/w, and Propylparaben or sodium salt thereof is present in range of 0.005% to 0.020% w/w.
In an another embodiment of the present invention, there is provided a synergistic herbal composition comprising emulsifiers selected from group comprising of Polysorbate 80 and Polyoxyl 40 hydrogenated castor oil, wherein Polysorbate 80 is present in range of 2.00% to 20.00% w/w, Polyoxyl 40 hydrogenated castor oil is present in range of 0.10% to 7.50%.

In an another embodiment of the present invention, there is provided a synergistic herbal composition comprising buffering agents selected from group consisting of Sodium citrate and Citric acid; wherein Sodium citrate is present in range of 0.05% to 5.00% w/w and Citric acid is present in range of 0.05% to 5.00% w/w.
In an embodiment of the present invention, there is provided a synergistic herbal composition comprising solvent is water present in range of quantity sufficient.
In an another embodiment of the present invention, there is provided a synergistic herbal composition comprising colouring agents selected from group consisting of Quinoline Yellow, Brilliant Blue FCF and Tartrazine; wherein Quinoline Yellow is present in range of 0.001% to 0.100% w/w, Brilliant Blue FCF is present in range of 0.001% to 0.100% w/w and Tartrazine is present in range of 0.001% to 0.100% w/w.
In an another embodiment of the present invention, there is provided a synergistic herbal composition comprising plasticizers selected from group consisting Glycerine, Propylene glycol and Sorbitol solution 70%; wherein Glycerine is present in range of 0.50% to 12.50% w/w, Propylene glycol is present in range of 0.50% to 12.50% w/w, and Sorbitol solution 70% is present in range of 0.50% to 12.50% w/w.
In an another embodiment of the present invention, there is provided a synergistic herbal composition comprising co-solvent is Propylene glycol present in range of 2.00% to 30.00% w/w.
In an another embodiment of the present invention, there is provided a synergistic herbal composition comprising capsule shell selected from group consisting of gelatine and Hydroxypropylmethylcellulose; wherein gelatin is present in range of 10.00% to 30.00% w/w and Hydroxypropylmethylcellulose is present in range of 10.00% to 30.00% w/w.
In an another embodiment of the present invention, there is provided a synergistic herbal composition wherein composition is formulated in form selected from group consisting of soft-gelatin capsule, hard-gelatin capsule, starch capsule, Liquid-filled HPMC capsule, HPMC capsule and liquid formulations.
In an another embodiment of the present invention, there is provided a process for preparing the herbal formulation in the form of a tablet, liquid (syrup), soft-gel capsule, hard gelatin capsule and capsules made from hydroxypropyl methylcellulose (HPMC) or starch.
In an embodiment of the present invention, there is provided synergistic herbal composition effective in reducing the lipid levels in body.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1: shows the percentage inhibition of lipid synthesis by Test Items in HepG2 cells against oleic acid stimulation
Figure 2: shows percentage inhibition of cellular cholesterol levels by Test Items in HepG2 cells against oleic acid stimulation
DETAILED DESCRIPTION OF THE INVENTION
The present invention describes novel synergistic herbal composition and process for preparation of same.
The expression of various quantities in terms of “% w/w” means the percentage by weight, relative to the weight of the total solution or composition unless otherwise specified.
The term "quantity sufficient" used in reference to the amounts of ingredients is commonly understood in the art to mean the quantity required to make up volume of the composition and is generally readily determined by the persons skilled in the formulation arts. Quantity sufficient can be interchangeably used as "q.s."
The present invention describes synergistic herbal composition comprising an effective amount of (a) Guggulu oleoresin; (b) Asafoetida oleoresin; (c) Dill oil; (d) Fennel oil; and (e) Ajwain oil as an active ingredient for reducing body lipids.
In the present invention components of synergistic herbal composition are obtained or made available from chemical suppliers as follows: (a) Guggulu oleoresin is obtained from M/s Sanat Products Limited, Asafoetida oleoresin is obtained from M/s Phyto Life Sciences Private Limited, Dill oil is obtained from M/s V. MANE FILS SA, Fennel oil is obtained from M/s Kancor Ingredients Limited and Ajwain oil is obtained from M/s Menthol and Allied Products.
The present invention describes synergistic herbal composition comprising (a) Guggulu oleoresin is present in the range of 0.50% – 10.00%; (b) Asafoetida oleoresin is present in the range of 0.05% – 5.00%; (c) Dill oil is present in the range of 0.50% – 10.00%; (d) Fennel oil is present in the range of 1.00% - 20.00%; and (e) Ajwain oil is present in the range of 0.25% – 10.00%.
The present invention describes synergistic herbal composition comprising one or more pharmaceutically acceptable excipient.
The present invention describes synergistic herbal composition comprising, pharmaceutically acceptable excipient wherein pharmaceutically acceptable excipient are selected from the group consisting of diluents, antioxidants, adsorbents, glidants, lubricants, antimicrobial preservatives, emulsifiers, buffering agents, solvents, colouring agents, plasticizers, co-solvents, capsule shell and so forth but not limited to it.
The present invention describes synergistic herbal composition comprising, diluents selected from group consisting of Till oil, Arachis oil, Microcrystalline cellulose, water but not limited to it; wherein till oil is present in the range of 10.00% to 50.00% w/w; Arachis oil is present in range of 25.00% to 75.00% w/w; Microcrystalline cellulose is present in range of 50.00% to 80.00% w/w and water is present in range of quantity sufficient.
The present invention describes synergistic herbal composition comprising, antioxidants consisting of Butylated Hydroxy Toluene, but not limited to it; wherein Butylated Hydroxy Toluene present in range of 0.01% to 0.50% w/w.
The present invention describes synergistic herbal composition comprising, adsorbents are selected from group microcrystalline cellulose, colloidal silicon dioxide, but not limited to it; wherein Microcrystalline cellulose is present in range of 40.00% to 80.00% w/w and Colloidal silicon dioxide is present in range of 0.50% to 5.00% w/w.
The present invention describes synergistic herbal composition comprising, lubricants consisting of magnesium stearate, but not limited to it; wherein Magnesium stearate present in range of 0.10% to 2.50% w/w.
The present invention describes synergistic herbal composition comprising, antimicrobial preservatives are selected from group consisting of sodioum benzoate, potassium sorbate, methylparaben or sodium salt, propylparaben or sodium salt, but not limited to it; wherein Sodium benzoate is present in range of 0.01% to 0.20% w/w, Potassium sorbate is present in range of 0.01% to 0.20% w/w, Methylparaben or sodium salt thereof is present in range of 0.01% to 0.20% w/w, and Propylparaben or sodium salt thereof is present in range of 0.005% to 0.020% w/w.
The present invention describes synergistic herbal composition comprising, emulsifiers are selected from group consisting of polysoprbate 80, Polyoxyl 40 hydrogenated castor oil, but not limited to it; wherein Polysorbate 80 is present in in range of 2.00% to 20.00% w/w, Polyoxyl 40 hydrogenated castor oil is present in range of 0.10% to 7.50%.
The present invention describes synergistic herbal composition comprising, buffering agents are selected from group consisting of sodium citrate and citric acid, but not limited to it; wherein Sodium citrate is present in range of 0.05% to 5.00% w/w and Citric acid is present in range of 0.05% to 5.00% w/w.
The present invention describes synergistic herbal composition comprising, solvent consisting of water, but not limited to it; wherein water is present in quantity sufficient.
The present invention describes synergistic herbal composition comprising, colouring agents are selected from group consisting of Quinoline yellow, Brilliant Blue FCF, Tetrazine, but not limited to it; wherein Quinoline Yellow is present in range of 0.001% to 0.100% w/w, Brilliant Blue FCF is present in range of 0.001% to 0.100% w/w and Tartrazine is present in range of 0.001% to 0.100% w/w.
The present invention describes synergistic herbal composition comprising, plasticizers are selected from group consisting of glycerine, Propylene glycol, Sorbitol solution 70%, but not limited to it; wherein Glycerine is present in range of 0.50% to 12.50% w/w, Propylene glycol is present in range of 0.50% to 12.50% w/w, and Sorbitol solution 70% is present in range of 0.50% to 12.50% w/w.
The present invention describes synergistic herbal composition comprising, co-solvents consisting propylene glycol, but not limited to it; wherein co-solvent is present in range of 2.00% to 30.00% w/w.
The present invention describes synergistic herbal composition comprising, capsule shells are selected from group consisting of gelatin and Hydroxypropylmethylcellulose, but not limited to it; wherein gelatin is present in range of 10.00% to 30.00% w/w and Hydroxypropylmethylcellulose is present in range of 10.00% to 30.00% w/w.
The present invention describes synergistic herbal composition is formulated in form selected from group consisting of Soft-gelatin capsule, hard-gelatin capsule, starch capsule, Liquid-filled HPMC capsule, HPMC capsule and liquid formulations, but not limited to it.
The present invention describes synergistic herbal composition is soft gelatin capsules, wherein pharmaceutical acceptable excipients are selected from diluents, antioxidants, plasticizers, colouring agent, antimicrobial preservative and gelatin soft shell but not limited to it.
The present invention describes synergistic herbal composition is liquid-filled HPMC capsule, wherein pharmaceutical acceptable excipients are selected from diluents, antioxidants and HPMC capsule shell but not limited to it.
The present invention describes synergistic herbal composition is hard gelatin capsule or a capsule made from hydroxypropyl methylcellulose (HPMC), wherein pharmaceutical acceptable excipients are selected from adsorbents, diluents, glidants, lubricants, antimicrobial preservatives, and antioxidants and gelatin/HPMC capsule shell but not limited to it.
The present invention describes synergistic herbal composition is liquid formulation, wherein pharmaceutical acceptable excipients are selected from emulsifiers, buffering agents, solvents, antimicrobial preservatives, antioxidants and colouring agents but not limited to it.
The present invention describes synergistic herbal composition is tablet formulation, wherein pharmaceutical acceptable excipients are selected from adsorbents, diluents, glidants, lubricants, antimicrobial preservatives, antioxidants, and colouring agents but not limited to it.
The present invention describes a process for preparing the synergistic herbal composition in the form of a tablet, liquid (syrup), softgel capsule, hard gelatin capsule and capsules made from hydroxypropyl methylcellulose (HPMC) or starch.
The present invention describes synergistic herbal composition of the present invention is effective in reducing the lipid levels without the side effects associated with synthetic drugs.
The present invention is more particularly described in the following non-limiting examples that are intended as illustrations only since numerous modifications and variations within the scope of the present invention will be apparent to a skilled artisan. Unless otherwise noted, all parts, percentages, and ratios reported in the following examples are on a weight basis, and all reagents used in the examples were obtained or made available from the chemical suppliers.
Example 1:
Lists the ingredients and their amounts present in the synergistic herbal composition in Soft-Gelatin Capsule formulation of the present invention. The unit of each value is percent weight of the composition.
Table 1 – Synergistic herbal composition in soft-gelatin capsule formulation:
Sr. No. Ingredient Qty. (% w/w)
1. Fennel oil 13.0 – 17.0
2. Dill oil 3.5 – 5.5
3. Ajwain oil 3.0 – 4.5
4. Guggulu oleoresin 5.5 – 8.0
5. Hing oleoresin 0.5 – 0.8
6. Till oil 25.0 – 40.0
7. Butylated hydroxy toluene 0.01 – 0.05
8. Gelatin soft shell 15.0 – 25.0
9. Glycerine 4.0 – 8.0
10. Sorbitol solution 70% 2.0 – 5.0
11. Water q.s.
12. Quinoline yellow 0.005 – 0.020
13. Sodium benzoate 0.01 – 0.05
14. Potassium sorbate 0.01 – 0.10

Example 2:
Process for preparation of synergistic herbal composition in soft-gelatin capsule formulation
Active ingredients of herbicidal composition and Butylated hydroxy toluene are mixed with stirrer in SS vessel in step 1. Gelatin and water is added in SS jacketed gel melter and stirred, Glycerine (part) and sorbitol solution is added and mixed in step 2. Preservative is mixed in Glycerin (part) with stirrer in S.S. vessel, and mixture is added step 2 in step 3. Colours are mixed in water with stirrer in S.S vessel, and mixture is added to step 3 in step 4. Blend of step 1 is encapsulated in gelatin ribbon formed with blend of step 4 using rotary die machines. Capsules are washed to degrease and dry capsules are formed.

Example 3:
Lists the ingredients and their amounts present in the synergistic herbal composition in Liquid-filled HPMC Capsule formulation of the present invention. The unit of each value is percent weight of the composition.
Table 2 – Synergistic herbal composition in liquid-filled HPMC capsule formulation:
Sr. No. Ingredient Qty. (% w/w)
1. Fennel oil 5.0 – 9.0
2. Dill oil 1.5 – 3.0
3. Ajwain oil 1.0 – 2.0
4. Guggulu oleoresin 2.5 – 4.5
5. Hing oleoresin 0.25 – 0.45
6. Arachis oil 55.0 – 75.0
7. Butylated hydroxy toluene 0.10 – 0.25
8. HPMC capsule shell (1 No.) 15.0 – 17.5

Example 4:
Process for preparation of synergistic herbal composition in liquid-filled HPMC capsule formulation
Active ingredients of herbicidal composition and Butylated hydroxy toluene are mixed with stirrer in SS vessel in step 1. Arachis oil is added to above mixture of step 1 and mixed with stirrer in S.S vessel in step 2. Blend of above step 2 is filled in HPMC capsule and locked using suitable machine in step 3. Capsules are sealed with HPMC band using suitable machine.

Example 5:
Lists the ingredients and their amounts present in the synergistic herbal composition in hard gelatin / HPMC Capsule formulation of the present invention. The unit of each value is percent weight of the composition.
Table 3 – Synergistic herbal composition in Hard gelatin / HPMC Capsule formulation:
Sr. No. Ingredient Qty. (% w/w)
1. Fennel oil 5.0 – 9.0
2. Dill oil 1.0 – 3.0
3. Ajwain oil 1.0 – 2.0
4. Guggulu oleoresin 2.0 – 4.0
5. Hing oleoresin 0.10 – 0.40
6. Microcrystalline cellulose 60.0 – 75.0
7. Colloidal silicon dioxide 0.5 – 1.5
8. Magnesium stearate 0.5 – 1.5
9. Gelatin/HPMC capsule shell (1 No.) 15.0 – 20.0

Example 6:
Process for preparation of synergistic herbal composition in hard gelatin /HPMC capsule formulation
Active ingredients of herbicidal composition are mixed with stirrer in SS vessel in step 1. Mixture of step 1 is absorbed on required quantity of microcrystalline cellulose and sifted through #40 S.S. sieves in step 2.Colloidal silicon dioxide and magnesium stearate is sifted through #40 S.S. sieve and mixed well with bled of step 2 in step 3. Blend is filled in suitable hard gelatin or vegetarian capsule shell using manual capsule filling machine or semi-automatic capsule filling machine or automatic capsule filling machine.
Example 7:
Lists the ingredients and their amounts present in the synergistic herbal composition in liquid formulation of the present invention. The unit of each value is percent weight of the composition.
Table 4 – Synergistic herbal composition in Liquid formulation:
Sr. No. Ingredient Qty. (% w/w)
1. Fennel oil 1.0 – 4.0
2. Dill oil 0.5 – 1.0
3. Ajwain oil 0.25 – 0.75
4. Guggulu oleoresin 0.5 – 2.5
5. Hing oleoresin 0.05 – 0.20
6. Propylene glycol 20.0 – 28.0
7. Polysorbate 80 10.0 – 15.0
8. Sodium citrate 0.05 – 0.25
9. Sodium benzoate 0.05 – 0.20
10. Potassium sorbate 0.05 – 0.20
11. Quinoline yellow 0.002 – 0.020
12. Water q.s.
Example 8:
Process for preparation of synergistic herbal composition in liquid formulation
Active ingredients of herbicidal composition are mixed with stirrer in SS vessel in step 1. Propylene glycol and Polysorbate 80 is mixed well and added to above mixture of step 1 and mixed in step 2. Sodium citrate is added in water and mixed, and blend is added to mixture of step 2 and mixed in step 3. Preservative is added in water and mixed, and blend is added to mixture of step 3 and mixed in step 4. Colour is added in water and mixed, and blend is added to mixture of step 4 and mixed in step 5. Water is added to make up volume.
Example 9:
Percentages of actives in synergistic herbal composition are as follows: Considering different dosage forms viz. soft-gelatin capsule, liquid-filled capsule, hard gelatin / Hydroxypropylmethylcellulose capsule, liquid
Sr. No. Ingredient Qty. (% w/w)
1. Fennel oil 1.00 to 20.00%
2. Dill oil 0.50 to 10.00%
3. Ajwain oil 0.25 to 10.00%
4. Guggulu oleoresin 0.50 to 10.00%
5. Hing oleoresin 0.05 to 5.00%

Example 9:
EXPERIMENTAL STUDY
The above herbal composition was evaluated of anti-hyperlipidemic potential of saunfbhara and saunfbhara based formulations by determining their effect on in vitro studies of intracellular lipid accumulation and cholesterol synthesis using human hepatocyte cell line (Hepg2).
Composition used for in vitro studies are as follows:
1. DRDC/2017/006: Guggulu
2. DRDC/2017/007: Guggulu + Hing
3. DRDC/2017/008: Guggulu + Dill
4. DRDC/2017/009: Guggulu + Hing + Fennel + Dill
5. DRDC/2017/010 (Saunfbhara): Guggulu + Hing + Fennel + Dill + Ajwain
HepG2 (Human Hepatocellular carcinoma) is used as test system, where HepG2 cell line were maintained under conditions and sub-cultured by trypsinisation followed by splitting the cell suspension into fresh flasks and supplementing with fresh cell growth medium.
Hyperlipidemia is characterized by an increased flux of free fatty acids (FFAs), raised Triglycerides (TGs) and elevated serum total cholesterol. The bulk of cholesterol synthesis occurs in the liver. Since, liver plays an important role in metabolic balance of blood cholesterol and triglycerides, the human hepatic cell line HepG2 was used as test system in the present study. Also, it is reported that the morphology of HepG2 cells resembles liver parenchymal cells and can synthesize and secrete many proteins like normal human liver cells. Hence, HepG2 cells were used as test system in the present study to investigate the anti-hyperlipidemic activity of Test Items. Inhibition of lipid synthesis by Oil-O-red staining and cholesterol levels indicated the anti-hyperlipidemic potential of the Test Items.
Atrovastatin calcium is used as positive control, Atorvastatin calcium was weighed and dissolved in DMSO to obtain a stock solution of 50 mM, the stock solution was further diluted in serum free EMEM to obtain concentrations ranging from 10 µM – 100 µM (10 fold higher concentrations than the desired final concentrations in cells).
Oleic acid is used as stimulant, where oleic acid was reconstituted in ethanol to obtain a stock solution of 5M, the stock solution was further diluted in serum free EMEM to obtain a concentration of 5 mM (10 fold higher concentration than the desired final concentrations in cells).
Saunfbhara and Saunfbhara based formulations (DRDC/2017/006, DRDC/2017/007, DRDC/2017/008, DRDC/2017/009, DRDC/2017/010) were weighed and dissolved in 100% ethanol to obtain 300 mg/ml stock solution, stock solution for each formulation was further diluted in serum free EMEM to obtain concentrations ranging from 156 µg/ml – 10 mg/ml (10 fold higher concentrations than the desired final concentrations in cells; 15.6 µg/ml – 1 mg/ml
Methodology:
Determination of non-cytotoxic concentrations of Test Items
HepG2 Cells were trypsinized and a single cell suspension was prepared which were counted on a hemocytometer. Cells were seeded at a density of 10,000 cells/well/180µl in EMEM and 10% FBS in 96-well plates and were incubated in a CO2 incubator for 24 h at 37 °C, 5% CO2 and 95% humidity. Further cells were sear starved in medium containing 1% BSA for 24 h. Later medium was removed from cells after 24 h, and following treatments were provided:
Control (Untreated) group with 200 µl of EMEM and 1% BSA were added to the wells.
Test Items group with 180 µl of EMEM and 1% BSA were added to wells and 20 µl of Test item was added from the respective 10X stock solutions to achieve final concentrations ranging from 15.625 µg/ml to 1 mg/ml.
Positive Control group with 180 µl of EMEM and 1% BSA were added to wells and 20 µl of PC (Atorvastatin Calcium) was added from the respective 10X stock solutions.
Vehicle Control group with 180 µl of EMEM and 1% BSA were added to wells and 20 µl of Vehicle (ethanol for TIs & DMSO for PCs) was added to the wells from the respective 10X stock solutions.
OA stimulated group with 180 µl of 1% BSA in EMEM was added to wells and 20 µl of OA was added from the respective 10X stock solutions to achieve final concentrations ranging 10 µM to 1 mM.

The effect of Test Item on cell viability was assessed by 3-(4, 5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay after incubation for 24 h,. 20 ?L of 5 mg/ml of MTT was added to all the wells and incubated at 37?C for 3 h. Further supernatant was aspirated and 150 ?L of DMSO was added to all wells to dissolve formazan crystals. Absorbance of each well was read at 540 nm using Biotek Reader. Effect of Test Items on viability of HepG2 cells was determined as:
% Cell viability = 100-% Cytotoxicity
Where; % Cytotoxicity = {(Absorbance of Control cells (Untreated) – Absorbance of cells treated with Test Item)/Absorbance of Control cells (Untreated)}*100
Concentrations resulting in =70% cell viability were selected as safe/non-cytotoxic for further assays.
Determination of effect of Test Items on Intracellular Lipid Accumulation
HepG2 cells were counted using hemocytometer and plated in 24 well plates at the density of 0.3 X 106 cells/well in DMEM supplemented with 10 % FBS. The plates were then incubated overnight under growth conditions so as to allow cell recovery and exponential growth. After overnight incubation, the cells were subjected to serum starvation for 24 h in EMEM containing 1 % BSA. After serum starvation, following treatments were provided:
Negative Control (Untreated) group with 500 µl EMEM and 1% BSA were added.
Oleic Acid Stimulated group with 450 µl of EMEM and 1% BSA and 50 µl of Oleic Acid from 5 mM Stock Solution (Final concentration – 500 µM) was added to well
Test Items group with 400 µl EMEM and1% BSA, 50 µl of Test items (62.5 µg/ml – 1 mg/ml) and 50 µl of Oleic Acid (Stimulant; 500 µM) were added.
Vehicle control (TI) group 400 µl of EMEM and 1% BSA, 50 µl of ethanol vehicle from the respective 10X stock solution and 50 µl of Oleic Acid (Stimulant; 500 µM) were added.
Positive Control (PC) group with 400 µl of EMEM+1% BSA, 50 µl of Simvastatin (1 µM -25 µM) and 50 µl of Oleic Acid (Stimulant; 500 µM) were added.
Vehicle control (VC) group 400 µl of EMEM+1% BSA, 50 µl of DMSO vehicle from the respective 10X stock solution and 50 µl of Oleic Acid (Stimulant; 500 µM) were added.
Cells were incubated in CO2 incubator for 24 h after treatment and plates were taken out for assessment of lipid synthesis on Oil-O-red assay. Cell layers were washed with PBS and fixed in 10 % Formalin for 1 h at RT. After 1 h of incubation, the above cells were washed with 60 % isopropanol (v/v in PBS) and air dried. The cells were then stained with 0.5 % Oil red O solution (prepared in 60 % iso-propanol) for 1h at RT followed by washing in distilled water to remove the unbound dye. The lipid bound stain was then eluted in isopropanol. Absorbance was read at 500 nm using Biotek Synergy HT micro plate reader. Percentage inhibition in intracellular lipid accumulation was calculated as follows:
% Inhibition in intracellular lipid accumulation = {(X-R)/X}*100
Where, X = Absorbance of Control (Vehicle + Oleic acid) at 500 nm;
R = Absorbance of test group (TI/PC + Oleic acid) at 500 nm
Assessment of effect of Test Items on cellular cholesterol levels
HepG2 cells were counted using hemocytometer and plated in 24 well plates at the density of 0.3 X 106 cells/well in EMEM supplemented with 10% FBS. Cells were incubated in a CO2 incubator for 24 h at 37 °C, 5 % CO2 and 95 % humidity. After serum starvation, following treatments were provided:
Negative Control (Untreated) group with500 µl EMEM+1% BSA were added.
Oleic Acid Stimulated group with 450 µl of EMEM+1% BSA and 50 µl of Oleic Acid from 5 mM Stock Solution (Final concentration – 500 µM) was added to well
Test Items group 400 µl EMEM+1% BSA, 50 µl of Test items (62.5 µg/ml – 1 mg/ml) and 50 µl of Oleic Acid (Stimulant; 500 µM) was added.
Vehicle control (TI) group 400 µl of EMEM+1% BSA, 50 µl of ethanol vehicle from the respective 10X stock solution and 50 µl of Oleic Acid (Stimulant; 500 µM) was added.
Positive Control (PC) group 400 µl of EMEM+1% BSA, 50 µl of Atorvastatin Calcium (10µM -100µM) and 50 µl of Oleic Acid (Stimulant; 500 µM) was added.
Vehicle control (PC) group 400 µl of EMEM+1% BSA, 50 µl of DMSO vehicle from the respective 10X stock solution and 50 µl of Oleic Acid (Stimulant; 500 µM) was added.
Cells were incubated in CO2 incubator for 24 h after treatment. Further after 24 h of incubation, cells were processed for estimation of total cholesterol levels using Cholesterol Quantification kit (Sigma-MAK043) as follows:
Lysis buffer containing chloroform: Isopropanol: IGEPAL CA630 in the ratio 7:11:0.1 was prepared. 200 µl of the above buffer was added to each well, which led to detachment of cells and formation of white layer. Cells were scrapped off and transferred to labelled centrifuge tubes. The cells were homogenized in ice using a tissue homogenizer for 4-5 minutes. Solution turned turbid in appearance. After homogenizing, the cells were centrifuged at 13,000g for 10 minutes. The supernatant was collected in pre-labelled centrifuge tubes and the pellet was discarded. The tube containing the supernatant was kept at 37ºC for 24h for evaporation of buffer. After 24 h, the tube was removed from 37ºC and the dried lipids (small yellow coloured pellet) were obtained which were stored at -20 ºC until analysis. The dried lipid was then dissolved in 200 µl of cholesterol assay buffer. 50 µl of the above lipid solution or standard (provided within the kit) was added to a 96-well plate. 50 µl of reaction mix/well according to the below scheme was further added to each of the in the above plate and incubated for 60 min at 37°C:
Table 5 – Reaction mixture
Reagent Volume (µL)
Cholesterol assay buffer 44 µl
Cholesterol probe 2 µl
Cholesterol enzyme mix 2 µl
Cholesterol Esterase 2 µl
Absorbance was measured at 570 nm, after 60 min of incubation using Biotek Synergy HT micro plate reader. The amount of Cholesterol (µg) in each sample was calculated from the standard curve. The final concentrations on Cholesterol (µg/ml) were then calculated (Amount of cholesterol (µg)/Sample volume (ml) added to the wells).
Percentage inhibition in Cholesterol levels was calculated as follows:
% Inhibition in Cholesterol levels = {(X-R)/X}*100Where, X = Concentration of Cholesterol in Control (Vehicle + Oleic acid);
R = Concentration of Cholesterol in test group (TI/PC + Oleic acid)
Results:
Non cytotoxic concentrations of Test Items
Effect of Saunfbhara and Saunfbhara based formulations (DRDC/2017/006, DRDC/2017/007, DRDC/2017/008, DRDC/2017/009, DRDC/2017/010) (in the concentration range of 15.6 µg/ml – 1 mg/ml) on the viability of HepG2 cells was determined after 24 h of treatment.
Saunfbhara and Saunfbhara based formulations (DRDC/2017/006, DRDC/2017/007, DRDC/2017/008, DRDC/2017/009, DRDC/2017/010) in the concentration range of 62.5 µg/ml – 1 mg/ml demonstrated >70% cell viability as compared to control. Hence the concentration range: 62.5 µg/ml – 1mg/ml was considered non-cytotoxic and selected for further assays.
Atorvastatin Calcium (PC-1) in the concentration range of 10 µM – 100 µM demonstrated >70% cell viability as compared to control. Hence the concentration range; 10 µM – 100 µM was considered for further assays.
Effect of Test Items on intracellular lipid accumulation
Effect of Saunfbhara and Saunfbhara based formulation was determined on inhibition of lipid accumulation in oleic acid stimulated HepG2 cells. Cells were co-treated with TIs and Oleic acid (500 µM) for 24 h and levels of intercellular lipid accumulation were determined using Oil O Red Staining. The effect of Test Items on levels of lipids accumulation was determined after 24h of treatment.
Figure 1 describes the percentage inhibition of lipid synthesis by Test Items in HepG2 cells against oleic acid stimulation
Figure 1 further describes Saunfbhara based formulation DRDC/2017/006 in the concentration range 62.5 µg/ml – 1mg/ml demonstrated 2.5% - 25% inhibition of intercellular lipid accumulation as compared to control cells (Veh+Oleic acid).
Saunfbhara based formulation DRDC/2017/007 demonstrated 7.4%- 52% inhibition of intercellular lipid accumulation in the concentration range 62.5 µg/ml – 1mg/ml to control cells (Veh+Oleic acid).
Saunfbhara based formulation DRDC/2017/008 demonstrated 2.9% - 20.1% inhibition of intercellular lipid accumulation in the concentration range 62.5 µg/ml – 1mg/ml as compared to control cells (Veh+Oleic acid ).
Saunfbhara based formulation DRDC/2017/009 demonstrated 11.3% - 35.8% inhibition of intercellular lipid accumulation in the concentration range 62.5 µg/ml – 1mg/ml as compared to control cells (Veh+Oleic acid).
Saunfbhara DRDC/2017/010 demonstrated 22.1% - 48.5 % inhibition of intercellular lipid accumulation in the concentration range 62.5 µg/ml – 1mg/ml as compared to control cells (Veh+Oleic acid).
Simvastatin (Positive Control) in the concentration range 1 µM -25 µM showed inhibition of intercellular lipid accumulation by 13.7% - 37.3% as compared to control cells (Veh+Oleic acid).
Effect of test item on cellular cholesterol levels
Effect of Saunfbhara and Saunfbhara based formulation on Cholesterol levels in HepG2 cells was determined against Oleic acid stimulated cells. Cells were co-treated with TIs and Oleic acid (500µM) for 24 h and levels of cholesterol in cell lysates were determined using colorimetric kit.
Saunfbhara Based Formulation (DRDC/2017/006) in the concentration range of 62.5µg/ml – 500µg/ml resulted in inhibition of cellular cholesterol levels (2.1% - 14.9%).
Saunfbhara Based Formulation (DRDC/2017/007) in the concentration range of 125µg/ml – 1mg/ml resulted in inhibition of cellular cholesterol levels 3.8% - 20.4%.
Saunfbhara Based Formulation (DRDC/2017/008) in the concentration range of 62.5µg/ml – 1mg/ml resulted in inhibition of cellular cholesterol levels 4.1% - 20.6%.
Saunfbhara Based Formulation (DRDC/2017/009) in the concentration range of 62.5µg/ml – 1mg/ml resulted in inhibition of cellular cholesterol levels 10.8% - 26.1%.
Saunfbhara (DRDC/2017/010) in the concentration range of 62.5µg/ml – 1mg/ml resulted in inhibition of cellular cholesterol levels 3.8% - 69.4%.
Atorvastatin Calcium in the concentration range 10 µM – 100 µM resulted in 16.9% - 32.4 % inhibition in the levels of cholesterol as compared to the control cells ( oleic acid alone treated cells).
On the basis of the results obtained, it can be inferred that Saunfbhara and Saunfbhara based formulations (DRDC/2017/006, DRDC/2017/007, DRDC/2017/008, DRDC/2017/009, and DRDC/2017/010) led to a reduction in Oleic acid stimulated intracellular lipid accumulation and cellular cholesterol levels in vitro. Hence, Saunfbhara and Saunfbhara based formulations (DRDC/2017/006, DRDC/2017/007, DRDC/2017/008, DRDC/2017/009, and DRDC/2017/010) may possess anti-hyperlipidemic potential. Based on the maximum extent of inhibition of lipid accumulation attained in this concentration range tested, the TIs can be graded as: DRDC/2017/007 > DRDC/2017/010 > DRDC/2017/009, DRDC/2017/008, and DRDC/2017/006.
Similarly, based on the maximum extent of inhibition of Cholesterol synthesis attained in this concentration range tested, the TIs can be graded as: DRDC/2017/010 > DRDC/2017/009 > DRDC/2017/008, DRDC/2017/007, and DRDC/2017/006.

We claim:
1. A synergistic herbal composition comprising: (a) Guggulu oleoresin; (b) Asafoetida oleoresin; (c) Dill oil; (d) Fennel oil; and (e) Ajwain oil; as active ingredients present in an effective amount for reducing body lipids.
2. The synergistic herbal composition as claimed in claim 1, comprises: (a) Guggulu oleoresin present in range of 0.50% – 10.00%; (b) Asafoetida oleoresin is present in the range of 0.05% – 5.00%; (c) Dill oil present in range of 0.50% – 10.00%; (d) Fennel oil present in of range of 1.00% - 20.00%; (e) Ajwain oil present in range of 0.25% – 10.00%.
3. The synergistic herbal composition as claimed in claim 1 comprises one or more pharmaceutically acceptable excipients.
4. The synergistic herbal composition as claimed in claim 1-3, wherein one or more pharmaceutical acceptable excipients are selected from group consisting of diluents, antioxidants, adsorbents, glidants, lubricants, antimicrobial preservatives, emulsifiers, buffering agents, solvents, colouring agents, plasticizers, co-solvents, capsule shell.
5. The synergistic herbal composition as claimed in claim 3 comprises, diluents selected from group consisting of Till oil, Arachis oil, Microcrystalline cellulose, and water; wherein till oil is present in the range of 10.00% to 50.00% w/w; Arachis oil is present in range of 25.00% to 75.00% w/w; Microcrystalline cellulose is present in range of 50.00% to 80.00% w/w and water is present in range of quantity sufficient.
6. The synergistic herbal composition as claimed in claim 3 comprises, antioxidant Butylated Hydroxy Toluene present in range of 0.01% to 0.50% w/w.
7. The synergistic herbal composition as claimed in claim 3 comprises, adsorbents selected from group consisting of Microcrystalline cellulose and Colloidal silicon dioxide; wherein Microcrystalline cellulose is present in range of 40.00% to 80.00% w/w and Colloidal silicon dioxide is present in range of 0.50% to 5.00% w/w.
8. The synergistic herbal composition as claimed in claim 3 comprises lubricants Magnesium stearate present in range of 0.10% to 2.50% w/w.
9. The synergistic herbal composition as claimed in claim 3 comprises, antimicrobial preservatives selected from group consisting of Sodium benzoate, Potassium sorbate, Methylparaben or sodium salt, Propylparaben or sodium salt thereof; wherein Sodium benzoate is present in range of 0.01% to 0.20% w/w, Potassium sorbate is present in range of 0.01% to 0.20% w/w, Methylparaben or sodium salt thereof is present in range of 0.01% to 0.20% w/w, and Propylparaben or sodium salt thereof is present in range of 0.005% to 0.020% w/w.
10. The synergistic herbal composition as claimed in claim 3 comprises, Emulsifiers selected from group consisting of Polysorbate 80 and Polyoxyl 40 hydrogenated castor oil, wherein Polysorbate 80 is present in range of 2.00% to 20.00% w/w, Polyoxyl 40 hydrogenated castor oil is present in range of 0.10% to 7.50%.
11. The synergistic herbal composition as claimed in claim 3 comprises, buffering agents selected from group consisting of Sodium citrate and Citric acid; wherein Sodium citrate is present in range of 0.05% to 5.00% w/w and Citric acid is present in range of 0.05% to 5.00% w/w.
12. The synergistic herbal composition as claimed in claim 3 comprises, solvent is water present in range of quantity sufficient.
13. The synergistic herbal composition as claimed in claim 3 comprises, colouring agents selected from group consisting of Quinoline Yellow, Brilliant Blue FCF and Tartrazine; wherein Quinoline Yellow is present in range of 0.001% to 0.100% w/w, Brilliant Blue FCF is present in range of 0.001% to 0.100% w/w and Tartrazine is present in range of 0.001% to 0.100% w/w.
14. The synergistic herbal composition as claimed in claim 3 comprises, plasticizers selected from group consisting Glycerine, Propylene glycol and Sorbitol solution 70%; wherein Glycerine is present in range of 0.50% to 12.50% w/w, Propylene glycol is present in range of 0.50% to 12.50% w/w, and Sorbitol solution 70% is present in range of 0.50% to 12.50% w/w.
15. The synergistic herbal composition as claimed in claim 3 comprises co-solvent Propylene glycol present in range of 2.00% to 30.00% w/w.
16. The synergistic herbal composition as claimed in claim 3 comprises, Capsule shell selected from group consisting of gelatine and Hydroxypropylmethylcellulose; wherein gelatin is present in range of 10.00% to 30.00% w/w and Hydroxypropylmethylcellulose is present in range of 10.00% to 30.00% w/w.
17. The synergistic herbal composition as claimed in claim 1, wherein composition is formulated in form selected from group consisting of Soft-gelatin capsule, hard-gelatin capsule, starch capsule, Liquid-filled HPMC capsule, HPMC capsule and liquid formulations.
18. The synergistic herbal composition as claimed in claim 1, wherein synergistic herbal composition is effective in reducing the lipid levels in body.
19. The synergistic herbal composition as claimed in 1, wherein said synergistic composition is soft-gelatin capsule comprising (a) Gugglulu oleoresin present in range of 5.5%-8.0%; (b) Asafoetida Oleoresin present in range of 0.5%-0.8%; (c) Dill Oil present in range of 3.5%-5.5%; (d) Fennel oil present in range of 13.00%-17.00%; (e) Ajwain oil present in range of 3.0%-4.5%;
Wherein pharmaceutical acceptable excipients consists of till oil present in range of 25.0% – 40.0%, butylated hydroxyl toluene present in range of 0.01 – 0.05, gelatine soft shell present in range of 15.0% – 25.0%, glycerine present in range of 4.0% – 8.0%, sorbitol solution 70% present in range of 2.0% – 5.0%, water present in quantity sufficient, quinoline present in range of 0.005% – 0.020%, sodium benzoate present in range of 0.01%– 0.05%, and potassium sorbate present in range of 0.01% – 0.10%.
20. The synergistic herbal composition as claimed in claim 1, wherein said synergistic composition is liquid-filled HPMC capsule comprising (a) Gugglulu oleoresin present in range of 2.5%-4.5%; (b) Asafoetida Oleoresin present in range of 0.25%-0.45%; (c) Dill Oil present in range of 1.5%-3.0%; (d) Fennel oil present in range of 5.00%-9.00%; (e) Ajwain oil present in range of 1.0%-2.0%;
Wherein pharmaceutical acceptable excipients consists of arachis oil present in range of 55.0% – 75.0%, butylated hydroxyl toluene present in range of 0.10% – 0.25%, and HPMC capsule shell present in range of 15.0% – 17.5%.
21. The synergistic herbal composition as claimed in claim 1, wherein said composition is hard gelatin/ HPMC capsule comprising, wherein hard gelatin/ HPMC capsule comprising (a) Gugglulu oleoresin present in range of 2.0%-4.0%; (b) Asafoetida Oleoresin present in range of 0.10%-0.40%; (c) Dill Oil present in range of 1.0%-3.0%; (d) Fennel oil present in range of 5.00%-9.00%; (e) Ajwain oil present in range of 1.0%-2.0%;
Wherein pharmaceutical acceptable excipients consists of Microcrystalline cellulose present in range of 60.0%– 75.0%, Colloidal silicon dioxide present in range of 0.5% – 1.5% , Magnesium stearate present in range of 0.55 – 1.5%, and hard gelatin/HPMC capsule shell present in range of 15.0%– 20.0%.
22. The synergistic herbal composition as claimed in claim 1, wherein said composition is liquid composition comprising (a) Gugglulu oleoresin present in range of 0.5% – 2.5%; (b) Asafoetida Oleoresin present in range of 0.05% – 0.20%; (c) Dill Oil present in range of 0.5% – 1.0%; (d) Fennel oil present in range of 1.0% – 4.0%; (e) Ajwain oil present in range of 0.255 – 0.75%;
Wherein pharmaceutical acceptable excipients consists of propylene glycol present in range of 20.05–28.0%, Polysorbate 80 present in range of 10.0%–15.0%, Sodium citrate present in range of 0.055 – 0.25%, sodium benzoate present in range of 0.05% – 0.20%, potassium sorbate present in range of 0.05%– 0.20%, quinoline yellow present in range of 0.002% – 0.020% and water present in quantity sufficient.