FIELD OF THE INVENTION
The present invention relates to novel salts of Boswellic acid(s) with alkaline earth metals and transition metals of biological importance and their preparation. Salts can be prepared from pure Boswellic acids as well as partially enriched or with crude extracts of stem exude gum resin of the plants from various species of Boswellia genus (Family: Burseraceae). This includes Boswellic acids of Ursane type (P series) as well as Oleane type (a series).
BACKGROUND OF THE INVENTION
Boswellia serrata, also known as Indian frankincense belongs to the family Burseraceae. Oleo-gum-resin of Boswellia serrata, is known for various medicinal properties and has been used traditionally in Asian, African and-Middle Eastern folk medicine. A series of Boswellic Acids are the active constituents for the same, which belongs to the groups of Pentacyclic triterpenic acids of Ursane series (p-Boswellic Acids) and Oleane series (a -Boswellic Acids). In addition to Boswellia serrata, there are several other plant sources of Boswellic acids from Burseraceae family.
These acids are specific, non-redox inhibitors of leukotriene synthesis interacting directly with 5-lipoxygenase (5-LO). Inflammation is part of the complex biological response of body tissues to harmful stimuli such as irritation, injury or infection characterized by redness, heat, pain, swelling and decline function, inflammation can also arise due to age related factors.
Arthritis is one of the most common diseases and now days NSAIDs (Non-steroidal anti-inflammatory drugs) are the most commonly used remedies for the treatment of arthritis. Rheumatoid arthritis is a chronic, systemic, inflammatory autoimmune disorder that affects causes inflexibility, swelling, weakness and pain. Osteoarthritis is a chronic degenerative disorder, it occurs when the cartilage or cushion between.joihts breaks down leading to pain, stiffness and inflammation.
Rheumatoid arthritis is a chronic disease affecting over 1.3 million Americans and as much as 1% of the worldwide population. According to Arthritis India arthritis affects 15% people that mean 180 million people inlndia. The data is based on more than 6.4 million samples received for arthritis testing over the last 3.5 years since January 2014 at SRL Laboratories acrosslndia.
The resinous gum of the bark known, as guggulu in Ayurveda and reported to be a powerful anti-inflammatory drugs and the resin obtained from the plant source is recommended for rheumatoid arthritis, osteoarthritis, fibromyositis and spondylistis. Patient treated with Boswellia reported reduces in knee pin, joint inflammation and enhances in knee flexion and walking distance [Anilkumar, M., Ethnomedicine: A

source of complementary therapeutics., 2010;267-93.]. Four pentacyclic triterpene acids including the B-boswellic acids which interferes with lukotriene biosynthesis and it is specific and dose dependent 5-lipoxygenase, 5-eicosatetraenoic acid and leukotrierie B4 inhibitor [Amnion, H.P., Mack, T., Singh, G.B., . Safayhi, H., Planta medica., 1991;57(03):203-7].
In one of the clinical trials, Boswellia serrata have also showed fair to excellent anti-inflammatory results in 88% of the patients with no adverse side effects [Iram, F., Khan, S.A., Husain, A., Asian Pac J Trop Biomed., 2017;7(6):513-23]. The anti-inflammatory activity of mixture of Boswellic acid showed 25-46% inhibition of paw edema in rats and mice. In chronic test of formaldehyde arthritis it exhibited 45-67% antiarthritic activity in a similar dose range. The fraction was effective in both adjuvant arthritis (35-59%) as well as established arthritis (54-84%) [Singh, G.B. and Atal, C.K., Indian J Pharmacol., 1984; 16, 51]. A randomized double blind placebo controlled crossover study was conducted to assess the efficacy, safety and tolerability of Boswellia serrata Extract (BSE) in 30 patients of osteoarthritis of knee, 15 each receiving active drug or placebo for eight weeks. After the first intervention, washout was given and then the groups were crossed over to receive the opposite intervention for eight weeks. All patients receiving drug treatment reported decrease in knee pain, increased knee flexion and increased walking distance [Kimmatkar, N., Thawani, V., Hingorani, L., Khiyani, R., Phytomedicine., 2003;10(l):3-7].
Calcium is very essential nutrient of human body, lack of calcium in the body leads to Osteoprosis, which porous bones in the human body and low calcium level in the body is also results into arthritis specially psoriasis arthritis. Animals as well as in-vitro studies data specify a possible mechanism through which lack of magnesium induces increased inflammation resulting in a risk of chronic disease. Scientific statement of American Heart Association/Centre for Disease control and Prevention described many substances have been used to determine whether increased chronic low-grade-inflammation has occurred that could lead to pathological effects and many of these substances are used to associate magnesium deficiency with increased inflammation.
A study has provided an in-vitro model to explore anti-inflammatory effect of calcium for preventing endothelial cell activation in preeclampsia, in this study Calcium appears to exert its effect, by reducing endothelial activation, induced by the dangerous/ toxic trophoblastic debris from preeclamptic placentae. Anti-inflammatory effects appear to be involved in this protective pathway by calcium supplementation [DeSousa, J., Tong, M., Wei, J., Chamley, L., Stone, P., Chen, Q., J. Hum. Hypertens., 2016;30(5):303.]. A randomized, double-blind, placebo-controlled clinical trial on patients, Calcium and vitamin D3 prevented loss of bone mineral density in the lumbar spine and trochanter in patients with rheumatoid

arthritis who were treated with low-dose corticosteroids [Buckley, L.M., Leib, E.Sis Cartularo, K.S., Vacek, P.M., Cooper, SM.,Ann. Intern. Med., 1996;125(12):961-8].
On the basis of above facts, there was a strong need.for natural non-steroidal anti-inflammatory drugs because of their conventional safety and efficacy: Boswellic acids are potential phytochemicals with various therapeutic values and will always be in demand. Above facts also suggest the utility of essential minerals like calcium, magnesium etc in our day today life and their positive role against the bones and joints related issues.
Extracts from plant sources are complex mixture of its multiple constituents. Isolation of its desirable constituent(s) from crude extract is always a big challenge for natural product chemists. It is always appreciable to have a simplest and robust method to isolate the desired constituent(s) as a single pure component or as a group of components with similar chemistry from plant originated complex mixtures. Selective crystallization, selective solubility and selective salt formation are the few methods for the same. Selective salt formation of constituents with specific chemistry is a.quick and best method to isolate a constituent-or group of constituent from complex mixture instead of other lengthy operation of extract processing.
SUMMARY OF THE INVENTION
Salt formation of active medicinal constituents is always been beneficial with respect to the ease of handling, stability, solubility, dissolution, drug delivery, bioavailability, formulation activities, as well as . for isolation and purification purposes. Importance of salt becomes more valuable, when salt forming agent is itself a nutrient and with well known biological importance.
The main objective of the present invention is to prepare and isolate the novel salts of Boswellic acid(s) with alkaline earth metals and transition metals of biological importance. Salts can be prepared by treating Boswellic acids or their derivatives with- sources of alkaline earth metals or transitions metals in appropriate conditions.
Salt formation of Boswellic acids with said metals has a lot of scope in various aspects of herbal extract processing as well as of biological importance. Followings are the scopes and possibilities with the said salt(s) of Boswellic acids.
1. Selective for Boswellic acids (Carboxylic function) during isolation, enrichment/purification
2. Good Filterable solids.
3. Non hygroscopic.

4. Potential to replace the Boswellic acids with said salt for their therapeutic applications.
5. A natural and safe medium of mineral supplement.
Salts of Boswellic acids with alkaline earth metals and transition metals of biological importance have further versatile possibilities. In addition to the known medicinal importance of Boswellic acids and healthy and safe carrier of vitals metals with it, its non-hygroscopic stability makes them more promising candidate for formulation activities, drug delivery and better bioavailability. These salts have a potential candidature to be used in place Boswellic.acids for their all originally known therapeutic applications.
DETAILED DESCRIPTION OF THE INVENTION:
Current invention discloses the novel salts of Boswellic acid(s) with alkaline earth metals and transition metals of biological importance and their formation. Boswellilc acids, a series of molecules with Ursane skeleton (P-Boswellic Acids) and Oleane skeleton (a -Boswellic Acids) were found to have tendency of forming salt with said metals in appropriate conditions. These metal salts can be prepared from pure individual Boswellic acid or from the mixture of multiple Boswellic acids. Salts of Boswellic acids can also be formed when they are partially enriched extracts or even with crude extracts from its plant source(s).
Novel metal salts of Boswellic acids of this invention with alkaline earth metals and transition metals of biological importance can be in general prepared by first converting the Boswellic acid(s) into their corresponding alkali metal salts by using alkali metal hydroxides in aqueous/alcoholic media. Resultant alkali metal salt, which is soluble in aqueous/alcoholic media, is then treated with the solution of desired said metal(s) among the. alkaline earth metals or transition metals of biological importance in form of their water soluble salt(s) to afford the corresponding desired salt of Boswellic acid(s) as filterable solids. Salt so formed can be filtered as stable solid product, with the removal of unwanted soluble substances in filtrate exhaust. Quantity of metal of interest can be used in stoichiometric ratio against the total molar equivalence of Boswellic aeid(s) considering the valancy of the metal ion.
In one of the conditions of preparing the said salts of Boswellic acids of this invention is preparing it from pure Boswellic acid or mixture of multiple Boswellic acids as found in enriched extract, which involves homogenizing the Boswellic acid(s) in an suitable alcoholic solvent, quantity sufficient to make the mass homogenous. To this, adding the solution of aqueous alkali and homogenizing it to afford corresponding alkali metal salt(s), which is soluble in existing aqueous alcoholic media. Resultant basified mass is now treated with aqueous solution of water soluble salt of desired said metal(s) resulting to form the filterable desired metal salt of Boswellic acid(s) as suspension. Solid so formed is isolated by filtration, which on drying yields the corresponding metal salts of Boswellic acids.

In one of the conditions of preparing the said salts of Boswellic acids of this invention is preparing it from crude extract of the said plant sources in a water immiscible solvent. Process involves extracting the oleo-gum-resin in a suitable water immiscible solvent. To this extract in an appropriate dilution ah optimum quantity of water is added and basified it with aqueous alkali to convert the Boswellic acids into their corresponding water soluble alkali metals salts. After stirring the biphasic mass for a while, mass is settled for layer separation. Aqueous layer, having Boswellic acids in form of their alkali metals salts is finally treated with aqueous solution of water soluble salt of desired said metal(s) resulting to form the filterable desired metal salt of Boswellic acid(s) as suspension. Solid so formed is isolated by filtration, which on drying yields the corresponding metal salts of Boswellic acids.
In one of the conditions of preparing the said salts of Boswellic acids of this invention is preparing it from crude alcoholic extract of said plant sources. Process involves first extracting the oleo-gum-resin in a suitable water miscible alcoholic solvent. This extract in an appropriate dilution is now treated with alcoholic or aqueous solution of alkali to convert the Boswellic acids into their corresponding alkali metals salts. Basified reaction mass so obtained is finally treated with aqueous solution of water soluble salt of desired said metal(s) resulting to form the filterable desired metal salt of Boswellic acid(s) as suspension. Solid so formed is isolated by filtration, which on drying yields the corresponding metal salts
One of the embodiments of current invention is to introduce a process for isolation of Boswellic acids as filterable solids from crude mixture which is selective for the constituents with carboxylic function and removing the non acidic constituents as soluble exhaust during filtration.
One of the embodiments of current invention is to introduce non hygroscopic salts of Boswellic acids with said metals, which make it stable to handle during isolation, enrichment, formulation and storage. Stable and non-hygroscopic nature of these salts have wide scope in terms of ease of handling, formulation activities, long term stability, drug delivery and better bioavailability.
One of the embodiments of current invention is to introduce a novel association of essential mineral(s) with the well known therapeutic values of Bowellic acids for their respective uses. Salts of Boswellic acids with alkaline earth metals and various transition metals of biological importance can play an additional role of supplementing the essential minerals along with the all known health benefits of Boswellc acids.
One of the embodiments of current invention is to make the said salts of Boswellic acids as a safe and healthy replacement of Boswellic acids for all of their originally known medicinal applications.

One of the embodiments of current invention is to replace the use of Boswellic acids with the said salts of Boswellic acids, which are safe and healthy for all of their originally known therapeutic applications for Boswellic acids.
EXAMPLE: 1
Boswellia serrata extract (250 gm) with its.chemical assay (by titration): 62.46 % as beta Boswellic acids and total known p-Boswellic acids: 22.63 % by HPLC was homogenized in ethanol (500 mL) at room temperature. To the homogenized mass, solution of sodium hydroxide (26.4 gm) dissolved in de-mineralized water (500 mL) was added in about 30 min duration at room temperature. Basified mass was stirred for about 60 min at room temperature and filtered the mass to remove insoluble mass in it. Filtrate solution was diluted with water (1250 mL) and stirred for a while at room temperature. To this, solution of Calcium chloride dihydrate (49.5 gm) dissolved in water (2000 mL) was added at room temperature in 90 min duration. Suspension so formed was stirred for about 5 h at roc)m temperature. Filtered the suspension and washed the wet solid with water (2x250 mL) under vacuum. Drying of wet solid in hot air oven for 12 h afforded the calcium salt of Boswellic acids (250 gm). Salt so formed was found with total known (3-Boswellic acids: 25.62 % by HPLC and Calcium content: 2.69% w/w (by complexometric titration)
EXAMPLES
Boswellia serrata extract (250 gm) was homogenized in ethanol (500 mL) at room temperature. To the homogenized mass, solution of sodium hydroxide (26.5 gm) dissolved in de-mineralized water (250 mL) was added in about 30 min duration at room temperature. Basified mass was stirred for about 60 min at room temperature and charged into the stirred aqueous solution of Calcium chloride dihydrate (49.5 gm) dissolved in water (2000 mL) at room temperature in about 45 min duration. Suspension so formed was stirred for about 60 min at room temperature, diluted with water (750 mL) and stirred for about 3 h at room temperature. Filtered the mass in buchner funnel under vacuum and washed the wet cake with water (2x250 mL) under vacuum. Drying of wet solid in hot air oven afforded the calcium salt of Boswellic acids (251 gm).
EXAMPLE: 3
Boswellia serrata extract (250 gm) was homogenized in ethanol (800 mL) at room temperature. To the homogenized mass, solution of sodium hydroxide (26.5 gm) dissolved in de-mineralized water (500 mL) was added under stirring in about 30 min duration at room temperature. Basified mass was stirred for about 60 min at room temperature and charged into the stirred aqueous solution of Zinc chloride (44.8

gm) dissolved in water (1125 mL) at room temperature in about 45 min duration. Suspension so formed was stirred for about 60 min at room temperature. Raised the temperature of stirred mass up to 50 °C and stirred at this temperature for lh. Diluted the stirred mass with water (1625 mL) and stirred for about 3 h at about 50 °C. Filtered the mass in buchner funnel under vacuum and washed the wet cake with water (2x250 mL) under vacuum. Drying of wet solid in hot air oven afforded the calcium salt of Boswellic acids (272.3 gm)
EXAMPLE: 4
Boswellia serrata extract (250 gm) was-homogenized in ethanol (500 mL) at room temperature. To the homogenized mass, solution of sodium hydroxide (26.5 gm) dissolved in de-mineralized water (500 mL) was added under stirring in about 30 min duration at room temperature. Basified mass was stirred for about 60 min at room temperature and charged into the stirred aqueous solution of Magnesium chloride hexahydrate (67.0 gm) dissolved in water (2000 mL) at room temperature in about 45 min duration. Suspension so formed was stirred for about 60 min at room temperature. Diluted the stirred mass with water (750 mL) and stirred for about 3 h at room temperature. Filtered the-mass in buchner funnel under vacuum and washed the wet cake with water (2x250 mL) under vacuum. Drying of wet solid in hot air oven afforded the calcium salt of Boswellic acids (251 gm).
EXAMPLE: 5
Boswellia serrata extract (250 gm) was homogenized in ethanol (500 mL) at room temperature. To the homogenized mass, solution of sodium hydroxide (26.5 gm) dissolved in de-mineralized water (500 mL) was added under stirring in about 30 min duration at room temperature. Basified mass was stirred for about 60 min at room temperature and charged into the stirred aqueous solution of Ferous sulphate heptahydrate (91.3 gm) dissolved in water (2000 mL) at room temperature in about 45 min duration. Suspension so formed was stirred for about 60 min at room temperature. Diluted the stirred mass with water (750 mL) and stirred for about 3 h at room temperature. Filtered the mass in buchner funnel under vacuum and washed the wet cake with water (2x250 mL) under vacuum. Drying of wet solid in hot air oven afforded the calcium salt of Boswellic acids (271.1 gm).
EXAMPLE: 6
Boswellia serrata oleo-gum-resin (250 gm) was hydro-distilled with water (165 mL) to recover the essential oils. Remaining herb was made water free, extracted with Ethyl acetate (2x500 mL) and distilled under vacuum to remove the solvent for analysis purpose. Extract residue so obtained (113 gm, with total p-Boswellic acids: 19.37 % by HPLC) was dissolved in Ethyl acetate (500 mL), charged water (1000 mL)

into it and stirred biphasic mass was basified to pH 9.8 using 10% aqueous sodium hydroxide solution. After stirring the mass for 15 min, it was settled for 2h, separated the layers. Collected the aqueous layer and was stripped under vacuum to make it free of ethyl acetate. To this added the solution of Calcium chloride di-hydrate (5.0 gm) dissolved in water (500 mL) under stirring in 30 min at room temperature. Suspension so formed was stirred for 4h at. room temperature, filtered under vacuum and washed with water (2x50 mL). and dried affording the calcium salts of Boswellic acids (12.7 gm) with total known [3-Boswellic acids: 47.24 % by HPLC.

We claim:
1. Salts of Boswellic Acid(s) of both, Ursane series (P-Boswellic Acids) and Oleane series (a-Boswellic Acids) with alkaline earth metals and transition metals of process and biological importance.
2. Alkaline earth metals, as described in claim-1 may be among Calcium, Magnesium or other relevant metals of process or biological importance.
3. Transition metals as described in claim-1 may be among zinc, iron, copper or other relevant metals of process or biological importance.
4. Process for preparation of salts of Boswellic Acid(s) of Ursane series ((3-Boswellic Acids) or Oleane series (a -Boswellic Acids) with alkaline earth metals or transition metals of process or biological importance, which involves following steps
a. Dissolving the pure Boswellic acid, mixture of Boswellic acid(s) or extract having
Boswellic acid(s) in suitable water miscible solvent or in combination of water and water
immiscible solvent.
b. Treating the above (4a) with aqueous alkali to convert the Boswellic acids into their
corresponding alkali metals salts. If the combination of water and water immiscible
organic solvent was used (4a), remove the layer of water immiscible solvent before its
next operation.
c. Treating the resultant aqueous or aqueous alcoholic phase with aqueous solution of salts
of desired metal among alkaline earth metals or transition metals of process or biological
importance to form the corresponding salt of Boswellic acids as filterable solid
suspension.
d. Filtering the suspension to isolate the solid, washing it with water and its drying to afford
the desired salt of Boswellic acid(s)
5. Boswellic acid(s), as described in. claim-1 and claim-4, used to convert into their said salts can either in pure form, as mixture of multiple Boswellic acids or even as crude or partially enriched extracts originated from stem exude oleo-gum-resin of the plants from various species of Boswellia genus from Burseraceae family.
6. Water miscible solvent as described in claim 4a may be among methanol, ethanol, n-propanol or isopropanol etc.
7. Water immiscible solvents, as described in claim 4b may be among Hydrocarbons, Esters, Ethers, Ketones or Chlorinated solvents.

8. Aqueous alkali, as described in claim 4b may be among Lithium hydroxide, sodium hydroxide or potassium hydroxide, most preferably sodium hydroxide.
9. Use of salts of alkali earth metals or transition metals as described in claim4-c are among halides, sulphate, carboxylates etc, which are soluble.in water.