Description:FORM2
THE PATENTS ACT 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETESPECIFICATION
(Seesection10andrule13)
1. TITLEOFTHEINVENTION
Alpha Pinene enriched Anti-arthritic formulation and method of preparation thereof
2. APPLICANT
(i) NAME : GD Goenka University
(ii) NATIONALITY: INDIAN
(iii) ADDRESS : Sohna Gurugram Road, Sohna, Haryana, India,122103
(i) PREAMBLETOTHEDESCRIPTION
COMPLETE
The following specification particularly describes the invention and the manner in which it is to be performed.
FIELD OF THE INVENTION
The present therapy for RA involve nonsteroidal anti-inflammatory drugs corticosteroids, disease modifying anti-rheumatic drugs and some recently developed biologic agents, but all of these are linked with side effects. The present invention relates to a composition for the treatment of Rheumatoidarthritis comprising Alpha Pinene isolated from Boswellia serrata extract and pharmaceutical excipient. Moreover, it also relates to a method for extracting Alpha Pinene compound from Boswellia serrata.
BACK GROUND OF INVENTION
Rheumatoidarthritis(RA)isanautoimmuneallergicconditioncausesongoinginflammationandsynovial fibroblast proliferation, which leads to the permanent loss of articular cartilage and bonebroughtonbyatherosclerosis[YanagisawaSetal.,2022].TheprimarycauseofRAisinflammation-induced loss of articular cartilage, however the exact mechanism is yet unknown.Thesamemightcausesymptomssuchasjointpainandswelling,stiffnessinthejoints,insomnia,exhaustion,weightloss,andflu-likesymptoms[AnjanaGoel1andSunandaKulshrestha,2021].As aresult,challengeswithdaily tasks includingdressing,grooming,walking, and cooking are frequent. Additionally, discomfort and exhaustion can make it difficulttoworkandfrequentlyhaveabadimpactonsociallife[TuressonWadellAetal.,2021].
Worldwide, the prevalence of RA ranges from 0.5 to 1% ofthe population, and in the case of the Indian population, more than 20%of the population hasanyformofarthritis[LinYen-Juetal.,2020].Comparedtomen(2%),women(4%)encounteritmore frequently. More over 50% of RA patients in wealthy nations stopped working full-timewithin ten years after the disease's inception. Every year, 41 out of every 100,000 people receivea RA diagnosis, and 1.3 million Americans have the disease. Diagnoses for the condition can bemade anytime between three months after the commencement of the illness and even after twoyearslater,whenithasbecomemoresevere[ChandoAnitaetal., 2021].
RA patients typicallyneedanalgesic and anti-inflammatorymedications (NSAIDs)totreatillnesssymptoms [PagliaMDGetal.,2021]. The leadingfactorcontributingtoincreasedmortalityinRAiscardiovasculardisease(CVD),primarilybroughtonbyatherosclerosis[DeyabG et al., 2021]. RA is very different from osteoarthritis, a degenerative joint condition that solelyaffects joint function and also link to number of body
functions as such gastrointestinal issues,renal dysfunction, and an elevated risk of cardiovascular disease [Kumar Janakiraman et al.,2018].
Nonsteroidalanti-inflammatorymedicines(NSAIDs),steroids,disease-modifyinganti-rheumaticmedications (DMARDs) and glucocorticoids are the main pharmacological classes of drugs usedto treat RA. These medications can only slow the RA's progression, which raises the danger ofinfection.Theprolongeduseofthesetreatmentswillresultindrugdependenceandastringnegative side effect, including rashes, liver damage and hair loss [Zhihao Zeng et al., 2021]. Inthis autoimmune illness, a number of immunological processes are carried out simultaneouslyandthesynoviumfoundalsoplaysacrucialroleintheseprocesses.AcuteinflammationinRAischaracterised by synoviocyte activation and proliferation, which results in the generation ofcytokines and proteases. Pro-inflammatory cytokines like TNF-, IL-6, and IL-1 are produced bysynoviocytes that resemble macrophages,whereas fibroblast-like synoviocytes are primarilyresponsible for producing IL-6, proteolytic enzyme (MMPs), prostaglandins, and leukotrienes.[Gurleen Kour et al., 2021]. Invasion of the synovium damages bone and cartilage. Additionally,T cells, ß cells and adaptive immune cells infiltrate the synovium as a result of RA. Rheumatoidfactors and ACPAs are produced by ß cells, which also serve as cells that deliver antigen andhelpstoactivateTlymphocytes[ArzooPannuetal.,2019].Numerousimmune-mediatedsubstances canpromoteinflammationinsynovialjoints.[AdityaSetal.,2016].Themostprevalent kinds of arthritis are gout, juvenile idiopathic arthritis, reactive arthritis, psoriaticarthritis,rheumatoidarthritis,septicarthritis,andankylosingspondylitis[JainSetal.,2021].
The diagnosis of RA is made using clinical imaging methods and laboratory examinations.Testableconditionsinlaboratoriesincludeanaemia,thepresenceofrheumatoidfactor,antibodiesagainstcycliccitrullinatedpeptides,andanincreaseinerythrocytesedimentationrate.Whilethecondition'ssymptoms,suchaspersistentmorningstiffnessandsoreness,providesomedetailsabout it.EventhoughtheyhelpinRAidentification[YanagisawaS etal.,2022].
Tablets, capsules, oral liquids, topical treatments, parenterals, paediatric/geriatric medicines, andtransdermal patches are only a few of the standard dose forms used to treat RA [Aisha Mobasharet al., 2020]. Ointment, cream, gels, and paste are examples of topical dose forms forthetreatmentofRA[JurcaTetal.,2021].Transdermalpatchesaretheadvancedformattinga
topicaldrug delivery device that uses minimally invasive means to deliver medication via the skin. Poorpatient compliance, a brief half-life (t/2), low bioavailability, and poor solubility were the maindements with conventional dosage forms used to treat RA. These issues may be resolved bydevelopingnewdosageformsexamplemicroparticles,nanoparticles,nanoemulsions,nanomicelles,nanodispersions,nanocapsules,nanosuspensions[JanakiramanKet al., 2018].
Boswellia serrata commonly known as Indian frankincense tree, belongs to Burseraceae family,holdingbothtreesandshrubsoftropicalandsubtropicalgeographicaldistribution,withapproximately 700 species originating from 18 genera [Upaganlawar A. and Ghule B. et al.,2014]. The genus Boswellia was named in an honour of Johann Boswell (1719-80) who hasdescribed 25 species of Boswellia; some of them, however, appear now as synonyms of the 21known species. The genus Boswelliahas mostly found in Africa (Sudan, Eritrea, Ethiopia,Somalia, and Kenya), southern Arabia (Oman and Yemen), and India [Pawankumar Rai, 2019].In India, Boswellia serrata mainly established in the region of the States of Andhra Pradesh,Gujarat, Madhya Pradesh, Jharkhand and Chhattisgarh. Boswellia serrata has also shown theability to inhibit pro-inflammatorycytokines. Itsuppresses interleukin-1ß(IL-1ß), tumornecrosisfactor-a(TNF-a),interferon-?(IFN-?),and enhances productionIL-10in arthritisinduced rats. These cytokines have a crucial role in chronic inflammation and tissue damageduringtheprogression ofRA[KumarRetal.,2019].
Pinene (C10H16) is a bicyclic, double bond, terpenoid hydrocarbon. a- and ß-pinene are twoisomers found in nature, e.g., in pine (coniferous trees) essential oils (EOs). They are among thebest-known representativesofabroadfamilyofmonoterpenes.a-andß-pineneenantiomersaredifferent in their interactions with polarized light, and their mirror image does not overlap. Bothstructural isomers have two enantiomers (+) and (-). This difference yields four active isomers.a-pinene is a colorless, water-insoluble but oil- and ethanol-soluble organic liquid. Its boilingpoint is 155 °C. a-pinene has been detected in at least 40 different EOs. ß-pinene is also acolorless organic liquid which is oil-soluble but ethanol- and water-insoluble. It has a boilingpoint rangingfrom 163–166 °C. It is obtained commercially by distillation or by a-pineneconversion[SalehiB.etal.,2019].
Hu et al., 2022 said that investigation of the active ingredients and pharmacologicalmechanismsofPoranasinensisHemsl.Againstrheumatoidarthritis usingnetworkpharmacologyandexperimentalvalidationwasperformed.Thepotentialcomponentsan
dtargets of P. sinensis against RA were analyzed using network pharmacology, and fivecompounds,twenty-fivetargets,andeightpathwayswereidentified.ExperimentalvalidationsuggestedthatP.sinensisextractandfivecompounds(esculetin,umbelliferone,trans-N-feruloyltyramine, caffeic acid andscopolin)could inhibitthereleaseofinflammatorymediators(NO,TNF-a,IL-1ßandIL-6)inLPS-inducedRAW264.7cell.
Lee et al., 2021 in this Anti-inflammatory, and anti-arthritic effects by the twigsof Cinnamomumcassia onCompleteFreund'sadjuvant-inducedarthritis(CFA)inrats.C.cassia twigsmay act as aviably sufficient therapeutic or preventivecandidateforosteoarthritis andrheumatoidarthritis.
El-Ghazalyetal.,2020thestudyofameliorationofadjuvant-inducedarthritisbyexposuretolowdosegammaradiationandresveratroladministrationinrats.Administration of RSV augmented the modulatory activity of low dose radiation withminimumexposurelevelonthediseaseprogression.
Abate et al., 2010 suggested that VS (viscosupplementation) therapy with HyaluronicAcid (HA) appears to be a safe and effective treatment option for RA patients who havefailed to react to conventional medications. The FDA (Food and Drug Administration)hasapprovedthistreatmentforkneeRA,buttheevidencefortheotherjointsispromising but not conclusive. When NSAIDs (Non-steroidal anti-inflammatory drugs)arecontraindicatedorpoorlytolerated,orwhenNSAIDsorcorticosteroidsareineffective,HAisutilised.VShasasignificantpain-relievingeffectwithinthreemonths,and this effect lasts for a long time (12-18 months). Patients can resume work and socialactivitiesmorerapidlynowthattheirarticularfunctionhasimproved.
The problem is Rheumatoid arthritis. These problem is increasing rapidly in day todays life, and becoming very serious during the days. These invention work on anti-arthritic activity. The herbal constituent alpha-pinene used to treate arthritis. It is found that alpha-pinene exhibits anti-inflammatory activity through the suppression of Mitogen-activated protein kinases (MAPKs) and the nuclear factor-kappa B (NF-?B) pathway in mouse peritoneal macrophages. Many different plants and different constituents were used to treated arthritis. The main disadvantage is slow action.The constituent alpha-pinene is new to be used in invention. Alpha-pinene exhibits anti-inflammatory activity through the suppression of Mitogen-activated protein kinases (MAPKs) and the nuclear factor-kappa B (NF-?B) pathway in mouse peritoneal macrophages.
The present study aims to provide scientific evidence regarding the anti-arthritic activity of alphapinene,whichisderivedfromBoswelliaserrata.Byquantifyingandidentifyingthebioactiveconstituent and conducting pharmacological evaluations, the study aims to contribute to theunderstanding of the potential therapeutic benefits of this compound in the management ofarthritis.
SUMMARY:
Rheumatoid arthritis (RA) is a severe autoimmune condition that causes persistent joint inflammation that increases the risk of early death. It is with almost 0.3% to 1% worldwide prevalence. Compared to males, women are three times more likely to be impacted by RA in the ratio of 3:1. The present therapy for RA involve nonsteroidal anti-inflammatory drugs corticosteroids, disease modifying anti-rheumatic drugs and some recently developed biologic agents, but all of these are linked with side effects. Herbal medicines are more accepted with the opinion that they are safer and have less adverse effects than synthetic ones. Phytochemicals of herbal medicines are particularly useful in the management of arthritis additional to being highly beneficial in the treatment of inflammatory, autoimmune and infectious disorders. One of the main causes of disability for millions of individuals worldwide is arthritis. Some of the phytoconstituents of herbal drugs, of Boswellia serrata have been reported to have anti-inflammatory activity. The utilisation of biologically active molecules derived from plants is used to treat autoimmune diseases like rheumatoid arthritis (RA) and the pharmacological effects of phytochemicals have long been known. The plant Boswellia serrata commonly known as "Salai Guggal," this species is significant and useful in many different ways and mostly to be more effective for arthritis. The plant's most active component that helps treat rheumatoid arthritis is “Alpha pinene”, it is extracted from its leaf using the percolation process. These bioactive natural materials can be used to treat conditions like arthritis, an asymptomatic disorder characterised by persistent joint pain, swelling and inflammation. The model used is CFA which is principally contains 1 mg/ml of heat-killed Mycobacterium tuberculosis suspended in mineral oil. Complete Freund's adjuvant (0.1 ml) was injected into the left hind paw of the rats.To engage in arthritic-relieving activities that improve one's physical and functional capacities while reducing pain and stiffness.The present therapy for RA involve nonsteroidal anti-inflammatory drugs corticosteroids, disease modifying anti-rheumatic drugs and some recently developed
biologic agents, but all of these are linked with side effects. The present invention relates to a composition for the treatment of Rheumatoidarthritis comprising Alpha Pinene isolated from Boswellia serrata extract and pharmaceutical excipient. Moreover, it also relates to a method for extracting Alpha Pinene compound from Boswellia serrata.
OBJECTS OF THE INVENTION
The main objective of thepresentstudy toevaluate the anti-arthritic activityof alphapinene, achemical compound extracted from the leaves of Boswellia serrata. The study has the followingobjectives:
a. Another objective of the present invention to extractthedesiredchemicalcompound:The desired chemicalcompound,alphapinene,will be extractedfrom the leaves of Boswellia serrata. The extraction processmay involve techniques such as solvent extraction, percolation methods to obtain thecompoundinapureform.
b. Another objective of the present invention toidentifythequantitativeandqualitativeestimation:Thestudywillinvolvequantifyingand identifying the bioactive constituent, alpha pinene, present in the extracts obtainedfromBoswelliaserrata.TechniquessuchasUVspectroscopy,TLC(thin-layerchromatography), HPLC (high-performance liquid chromatography), analytical methodsmaybeused forthispurpose.
c. Another objective of the present invention to evaluate pharmacological activity: The anti-arthritic activity of the Boswellia serrataextracts containing alpha pinene will be assessed. Pharmacological studies may include invitro experiments using himatological analysis or in vivo studies using animal models ofarthritis. The extracts will be tested for their potential to reduce joint inflammation, painandotherarthriticsymptoms.
The study aims to provide scientific evidence regarding the anti-arthritic activity of alphapinene,whichisderivedfromBoswelliaserrata.Byquantifyingandidentifyingthebioactiveconstituent and conducting pharmacological evaluations, the study aims to contribute to theunderstanding of the potential therapeutic benefits of this compound in the management ofarthritis.
BRIEF DESCRIPTION OF FIGURES:
These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying figures in which like characters represent like parts throughout the figures, wherein:.
Figure 1a: Calibration curve for Standard by UV spectroscopy.
Figure 1b: Calibration curve for extract by UV spectroscopy.
Figure 2a: TLC plate under UV-Cabinet (254nm)
Figure 2b: TLC plate under UV-Cabinet (366nm)
Figure 3: RepresentativeHPLCchromatogramsofextractmixtureat260nm
Figure 4: RepresentativeHPLCchromatogramsofstandardmixtureat260nm
Figure 5: H-and E-stained histopathological imag?es of a typical ankle joint of each group
a. Normal(0.5ml/kg)(4X)
b. Normal(1ml/kg)(10X)
c. Negative(0.5ml/kg)(4X)
d. Negative(1ml/kg)(10X)
e. Standard(0.5ml/kg)(4X)
f. Standard(1ml/kg)(10X)
g. Drugtreated(0.5ml/kg)4X
h. Drugtreated(0.5ml/kg)10X
i. Drugtreated(1ml/kg)4X
j. Drugtreated(1ml/kg)10X
DETAILED DESCRIPTION OF THE INVENTION
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the figures and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system,
and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.
It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.
Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrase “in an embodiment”, “in another embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method. Similarly, one or more devices or systems or elements or structures or components proceeded by "comprises... a" does not, without more constraints, preclude the existence of other devices or other systems or other elements or other structures or other components or additional devices or additional systems or additional elements or additional structures or additional components.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting.
The terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items.
The terms “having”, “comprising”, “including”, and variations thereof signify the presence of a component.
Before going into details about the present invention, it should be noted that the invention is not limited to the details of construction and arrangement of parts shown in the accompanying
drawings, as the invention is capable of other embodiments and can be applied or carried out in a variety of ways. It should also be noted that the phraseology and language used below are intended to describe rather than constrain.
Examples
Example 1: ExtractionofB.serrataleaves
Boswelliaserrata(leaves)werecollectedinthemonthofJanuaryfrom Haryana,India.Theleaveswere submittedinSAIFIASCIENCECOLLEGE,BHOPALforplantauthenticationandisbeingauthenticatedon3rdJan,2023.TheAuthenticationno.is054/Bot/Saf/23.
The leaves of the selected plant B. serrata were separately macerate (200g) of the air dried, coarselypowdered, with (320:80) ml of methanol and water of the specified strength in a closed flask for fourhours, shaking often during six hours and allow to stand for 2 days. Than filter the filtrate by takingprecautions against loss of solvent by Whatman filter paper, evaporate the filtrate to dryness by usingheating mantel. The temperature maintained of heating mantle is 500C. The percentage of alcohol-solubleextractwithreferencetotheair-dried drugwascalculated.
The shade dried coarsely powdered B. serrata leaves were taken in a tared glass bottle and the initialweight was taken. The crude drug was air dried and weighed. The same was repeated till a constantweightwasobtained.TheLODcontentofthesamplewascalculatedaspercentagewithreferencetotheshadedried material.
Lossondrying=initialweightofsample –weightofsampleafterdryingX100
The Loss on Drying (LOD) is 70%. The LOD value represents thepercentage of moisture or volatile components present in the sample before drying, which wasreducedto3gramsafterthedrying process.
Thisextractionmethodisparticularlyusefulforextractingcompoundsfromthermolabile(heat-sensitive)plantmaterial,asitminimizesexposuretohightemperatures.Itisaconvenienttechniquethatallowsforcompleteextractionofbioactivecompoundsfromtheplantmaterial.Thechoiceofmenstruumandextractiontimeadjustedbasedonthespecificplantmaterialanddesiredcompounds.
The procedure of extraction commonly used for obtaining bioactive compounds from plant
material. Itinvolvesthefollowing steps:
a. Coarsely powdered drug material: The plant material such as leaves is ground into a coarsepowder.Thisincreasesthesurfaceareaofthematerial,facilitatingefficientextraction.
b. Placingthedrugmaterialinacontainer:Thepowderedplantmaterialisplacedinsideacontainer.The container should be large enough to accommodate the material and leave space for thesolvent.So, 200gofpowderwassuccessivelyextractedwith2L ofsolvent.
c. Adding the menstruum: The menstruum which is the solvent used for extraction, is poured ontothe powdered drug material to cover it. The choice of menstruum depends on the properties of thedesiredcompoundsandthesolubilityoftheplantconstituents.
d. Closingthecontainer:Thecontainerissealedorclosedtopreventevaporationandcontamination.Thisallowstheextractionprocesstoproceedundisturbed.
e. Allowing extraction to occur: The closed container with the drug material and menstruum is keptfor at least three days. During this time, the active constituents of the plant material dissolve intothesolvent.
f. Stirring or shaking: Periodically, the contents of the container are stirred or, if the extraction isperformed in a bottle, shaken. This ensures thorough mixing of the solvent and plant material,enhancingtheextractionefficiency.
g. Separating the micelle from the marc: After the extraction period, the mixture is separated intotwo components. The micelle refers to the solvent (containing the dissolved compounds) alongwithanysuspendedsolidsorplantdebris.Themarcreferstotheleftoverplantmaterial.
h. Filtrationordecantation:Themicelleisseparatedfromthemarcbyeitherfiltrationordecantation. Filtration involves passing the mixture through a filter to collect the liquid fractionwhile leaving behind the solid material. Decantation involves carefully pouring off the liquidportionwhileleavingthesedimentatthebottomundisturbed.
i. Separating the micelle from the menstruum: The micelle, containing the desired compounds, isthen separated from the menstruum that was used for extraction. This separation is typicallyachieved through evaporation. The micelle can be evaporated in an oven or on top of a waterbath, allowing the solvent to evaporate and leaving behind the concentrated extract. After dryingrespectiveresidueswereweighedandpercentageyieldwascalculated.
Thepercentageyieldofextractswascalculatedbyusingfollowingformula:
%yield= (Weightofextract/ Weightofpowderdrug) X 100
The percentage yield is 150%. This means that the extraction process yielded 3 gramsofextractfrom2grams ofthepowdereddrug,whichis150%oftheinitialamountofthepowder used.
Ultraviolet(UV)—visiblespectroscopyforestablishingnovelmethodsfortheanalysisandquantificationofbiosimilardrug.Determinationofmaximumabsorbanceis thefirststepfortheanalysis (quantitativelyorqualitatively) by UV. Solution (1µg/mL) was scanned using UV-Visible spectrophotometer. UVsoftwareprocessedfullscanandthe?maxwereidentifiedwiththehelpofsoftwaretobe260nm.
For quantitative analysis of five concentrations namely 2, 4, 6, 8 and 10µg/ml were used fordeveloping calibration curve byUV. The absorbance of different calibration standards wasmeasured at 260 nm using fixed wavelength mode of spectrophotometer. Absorbances of eachconcentrationwereanalyzedandforcalibrationcurvetheaverageoftheabsorbancesweretaken.Equation y = 0.009x + 0.004 for standardwas obtained for the absorbance plotted againstdifferent concentrations. Equation y = 83,337x + 45,060 for extract was obtained for the peakareasplottedagainstdifferentconcentrations(2,4,6,8and10µg/ml). The standard CalibrationcurveforStandard and extract byUVspectroscopy is provided in figure 1a and 1b.The R² value of 0.925 suggests that approximately 92.5% of the variance in the absorbanceexplainedbythelinearrelationshipwiththeconcentration.
Example 2: separation of alpha pinene compound from B.serrata leaves extract
Using Thin-LayerChromatography: Thin-LayerChromatography,isasimpleandwidelyusedanalyticaltechniquetoseparateandidentifydifferentcompoundsinamixture.Itiscommonlyusedinchemistry,biochemistry,andotherrelatedfields.Thin-layerchromatographyisatechniqueinwhichasoluteundergoesdistributionbetweentwophases,stationaryphaseactingthroughadsorptionandamobilephaseintheformofaliquid.Theadsorbentisarelativelythin,uniformlayerofdryfinelypowderedmaterialappliedtoaglass, plastic or metal sheet or plate. Precoated plates are most commonly used. Separation may also
beachievedonthebasisofpartitionoracombinationofpartitionandadsorption,dependingontheparticulartypeofsupport,itspreparation anditsusewithdifferentsolvent. Identificationcanbeeffectedbyobservation ofspotsofidenticalRfvalueandaboutequalmagnitudeobtained,respectively,withanunknownandareferencesamplechromatographedonthesameplate.Avisualcomparisonofthesizeandintensityofthespotsusuallyservesforsemi-quantitativeestimation.SamplePreparation:1000mgofBoswelliaserrataextractobtained abovewasweighedbyelectronicbalanceanddissolvedin200mlofmethanol.Spotof abovesolutionwasthenappliedontheTLCplatewiththehelpof thincapillarytube.
Solventsystem:Toluene:Ethylacetate(85:15)asmobilephase.
Detection:Afterdrying,spotofboththesamplesareidentifiedbysprayingsulphuric acidonthe TLCplate.
Stationaryphase:AluminumTLCplatecoatedwithsilicagel.
a) Flat glass plates of appropriate dimensions which allow the application at specified points ofthe necessary quantities of the solution being examined and appropriate reference solutionsand which allow accommodation of the specified migration path-length. The plates werePrepared as described below; after getting solvent selected, commercially available plates(Merck)werealso used.
b) An aligning tray or a flat surface on which the plates can be aligned and rested when thecoatingsubstancewasapplied.
c) Silica gel G was used as adsorbent. It was applied directly to the plate after preparing itsslurry.
d) A spreader was used to apply a uniform layer of adsorbent of desired thickness over the entiresurfaceoftheplate.
e) Astorageracktosupporttheplatesduringdryingandtransportation.
f) A developing chamber that can accommodate one or more plates and can be properly closedand sealed. The chamber was fitted with a plate support rack that supports the plates, back toback,withlidofthechamberin place.
g) Graduatedmicro-pipettescapableofdeliveringmicrolitrequantitiessay10µlandless.
h) UVinspection cabinetwithshort(254nm)andlong(366nm)ultra-violetwavelengths.
TheprocedureforTLCinvolvesthefollowingsteps:
o PreparationofTLCplate:TLCplatesusedarealuminumTLCplatecoatedwithsilicagel.Themostcommon adsorbent is silica gel. Before starting the experiment, maked sure that TLC plates arecleanand freefromanycontaminants.
o Preparing the sample: Dissolve the extractwith solvent methanol and 10µl concentration wasprepared andmixture containing the compounds (alpha-pinene)want toseparate in a suitablesolventand the drug alpha-pinene dissolve with solvent methanol 10µl concentration was preparedfor comparative study. Use a small amount of the sample (usually in the microliter range) to spot itontheTLCplate.Spotscanbeapplied usingamicrosyringe.
o Developing the TLC plate: Pour asmall amount of the mobile phase (Toluene: Ethyl acetate(85:15)) into a developing chamber, which is a closed container. Place the TLC plate inside thechamber, ensuring that the solvent level is below the spot line. The mobile phase will ascendthrough the plate by capillary action, carrying the sample components with it. Allowing the solventtotravelacertaindistanceuptheplate,usuallyabouttwo-thirdsofthetotal platelength.
o Visualization of spots: Removed the TLC plate from the developing chamber once the solventreached to the highest point on the plate to the top of the plate. Marking the solvent front with apencil or a line. Quickly visualize the separated spots by using ultraviolet (UV) light by staining theplatewithdilutedsulphuricacid(dil.H2SO4),5%chemical.
o Calculation of Rf values: The phrases ultra-violet light (254 nm) and ultra-violet light (366 nm)indicate that the plate should be examined under an ultra-violet light having a maximum output atabout 254 or at about 366 nm. After the spots are visualized, measure the distance traveled by eachextractcompoundspotfromthestartinglineandthedistancetraveledbythedrugalpha-pinene.front from the starting line. The Rf value (retention factor) for each compound is calculated as theratio of the distance traveled by the extract spot to the distance traveled by the drug alpha-pinenefront.
o Rf=Distancetraveledbythecompoundspot/Distancetraveledbythesolventfront
o Interpretation of results: Compare the Rf values obtained for the spots on the TLC plate with thoseofknowncompounds(alpha-pinene)underthesameexperimentalconditions.Rfvaluesarecharacteristic for different compounds under specific TLC conditions and can be used to identifycompoundalpha-pinenein theextract.
It's essential to handle TLC plates with care and use appropriate safety measures, especially whenworking with potentially hazardous substances or solvents. It is important to choice the adsorbent,mobile phase, and visualization method which significantly impact the separation and identification ofcompoundsinTLC. In TLC, the resolution factor (Rf) value is a quantitative measure used to evaluate the separationofdifferentcompoundsonaTLCplate.Itiscalculatedastheratioofthedistancetraveledbythecompoundspot(center)tothedistancetraveledbythesolventfront(eluent)fromtheorigin (figure 2 and 3).TheRfvalueis0.45,itmeansthatthecompoundinquestiontraveled0.45timesthedistancecoveredbythesolventfrontin theTLCexperiment. TheRfvalueof 0.45suggests thatthecompoundhadmoderateretentionontheTLCplate,meaningitmovedslowerthanthesolventbutnotsignificantly slow.
Using High performance liquid chromatography (HPLC): HPLC is a versatile separation technique with wide range of application, the process is sometimescritical due to its large number of variables, which need to be properly adjusted before every single run.HPLC separation of each chemical entity from the sample mixture is based on its distinct affinitytowards the adsorbent material in the column or the mobile phase, causing various constituents to travelatdifferentvelocitiesandseparate.Itwasformerlycalledashigh-pressureliquidchromatographysince;it relies on high pressure pumps to allow quicker separation.Separation by HPLC predominantlydepends on some intrinsic tunable parameters of mobile phase like polarity, flow rate, pH, compositionand some inherent properties of sample matrix; type and nature of stationary phase; environmentalfactors like temperature and detectortype andsettings.New HPLCmethodwas developed andvalidatedaccordingtoICHguidelinestoanalyzesevenmajorchemicalconstituents.ThismethodisoneofthealternativemethodsforLC-MEmethod.
HPLC Method was performed on a PEAK chromatographic system equipped with LC-P7000 isocraticpump; injector with 20µl fixed volume loop, separation was achieved on C18 column (4.6 mm × 250mm i.d., 5 µm) and at a column temperature of 25°C; with UV-Visible detector and the output signalwas monitored and integrated by PEAK Chromatographic Software used is double beam UV-Visiblespectrophotometerwasusedtocarryoutspectralanalysisandthedatawasrecordedbysoftware. Sonicator was used to sonicating the mobile phase and samples up to 20 min., pH of the mobile phasewasadjustedwithglacialaceticacid. Themobilephasewasusedasdiluents.
High-Performance Liquid Chromatography (HPLC) is a powerful analytical technique used to separate,identify,andquantifycomponentsinamixture.Here’stheprocedureofanHPLC:
Sample preparation: Prepare the sample to be analyzed. This may involve dissolving the sample(extract of B. serrata and standard solution of alpha-pinene) in an appropriate solvent (methanol)and10µgofextractandstandardsolutionwerepreparedandfilteringittoremoveanyparticulatesthatcouldclog theHPLCcolumn.
HPLC system setup: Turn on the HPLC instrument and ensure that it is properly connected andallcomponents(pump, injector, column, detector)arefunctioningcorrectly.
Prime the HPLC system with the mobile phase to remove any air bubbles and ensure a steadyflow.
Columnselection:ChooseanappropriateHPLCcolumn(C18column)basedonthenatureoftheanalytes and the desired separation. Columns with different stationary phases and dimensions((4.6mm×250mmi.d.,5µm))areavailableforspecificapplications.
Mobile phase selection: Select an appropriate mobile phase based on the solubility and chemicalproperties of the sample components. The mobile phase may consist of a single solvent (isocraticelution) oramixtureofsolvents(gradientelution) dependingontheseparationneeds.
o Calibrationandstandardsolutions(ifquantitativeanalysisisrequired):Preparestandardsolutions of 10µl of known concentrations to calibrate the HPLC system and create a calibrationcurveforquantification.
o Injection and separation: Inject the prepared sample or standard solutions of 20µl into the HPLCsystemusinganautosamplerormanualinjection.
o The sample is pumped through the HPLC column, where the components are separated based ontheirinteractionswiththestationaryphaseandmobilephase.
o Detectionanddata acquisition:Analytesare detectedasthey elutefromthecolumn byadetector(e.g.,UV-Vis).Thedetectorgeneratessignalsproportionaltotheanalyteconcentrations.
o ThedataisacquiredandrecordedbytheHPLCsoftware.
o Dataanalysisandinterpretation:Analyze the chromatogramobtained fromthedetector toidentifyandquantifythecomponentspresentinthesample.
o System maintenance: After the analysis is complete, flush the system with an appropriate solventtocleanthecolumnand preventcarryoverbetweenruns.
o PerformroutinemaintenanceandchecksontheHPLCsystemtoensureitsoptimalperformance.
The specific details of an HPLC procedure can vary based on the nature ofthe analytes, the HPLC instrument, and the objectives of the analysis. Proper training and knowledge ofHPLC principlesareessential forsuccessful operationandinterpretationofresults.
ThechromatogramoftheBoswelliaserrataextractsolutionshowstwomajorpeaksat3.305min and4.195minretention times (table 1 and figure 3).
Table1:HPLCchromatogramforExtract Peak Retentiontime Areaunderthe curve Identify 1 1.674 4141 Unknown
2
2.486
15673
Unknown 3 2.901 18487 Unknown
4
2.950
5731
Unknown 5 3.305 129762 Alpha-pinene
6
4.195
100985
Unknown 7 5.691 2599 Unknown
Afterinjection20µlofthestandardusingtheconditionsshowedpeakat3.312minretentiontime (table 2 and figure 4).
Table2: HPLCchromatogramforStandard Peak Retentiontime Areaunderthe curve Identify 1 0.191 2018 Unknown
2
0.567
6476
Unknown 3 0.970 10601 Unknown
4
1.665
23431
Unknown 5 2.500 16579 Unknown
6
2.920
355988
Unknown 7 3.312 1872308 Alpha-pinene
The major peak observed at 3.305 minutes in the Boswellia serrata extract chromatogram mightcorrespond to alpha-pinene, considering it has a very close retention time to the standard (3.312minutes). The second major peak at 4.195 minutes could be another component present in theBoswelliaserrataextract.
Confirmation of alpha-pinene: The presence of alpha-pinene in the Boswellia serrataextract is supported by the fact that the retention time (3.305 minutes) of one of the major peaksin the chromatogram closely matches the retention time (3.312 minutes) of the alpha-pinenestandard..TheHPLCpeakobservedatthespecificretentiontimeinboththeHPLCchromatogramofthe extract and the standard corresponds to alpha-pinene, providing evidence of its presence in thesample.
Example 3: anti-arthriticactivity on animal
Theanimalgroupsareasfollows (table 3):
NormalGroup: Group-I:Treatedwith(1ml/1kgbodyweight)normalsaline.
StandardGroup: Group-II:TreatedwithDiclofenacsodium(10mg/kg)bodyweight
NegativeGroup: Group-III:TreatedwithCFA(0.1ml)
DrugtreatedGroup: Group-IV:Drugextractatdoseof0.5mg/kgintra-peritoneal+CFAconsidered as(Diethylether)
DrugtreatedGroup: Group-V:Drugextractatdoseof1mg/kg intra-peritoneal +CFAconsidered as(Diethylether)
Table 3: Grouping of the animals
Sr.No.
Groupsname
Dose
Noofanimals 1 Control (Normalsaline) 1ml/kg 6
2
Positive control
(Diclofenacsodium)
10mg/kg
6 3 Negative control (CFA) 0.1ml 6
4
apinene
0.5mg
6 5Drug treated Group: apinene 1mg 6
Thevolumeofthehindpawwasmeasuredduringtreatment period at different time intervals ofconsecutive days such as (0, 7, 14, 21and28days)usingtheverniercaliper.
The experiments carried out using Albino rats weighing between 150 g and 250 g (CentralAnimal House, GD Goenka, Registration no. 2127/PO/ReRcBi/S/21/CPCSEA). The albino ratswill divide into five groups, i.e. control, standard, negative control and drug treated (two groupsof drug treated low and high dose treated group). Rats will inject with 0.1 ml of Freund'scomplete adjuvant (CFA) into the planter region of the left hind paw. The paw volumes of boththe hind paws will measure using a vernier caliper and body weight will record on the day ofadjuvant injection. The solvent-drug extract of the aerial parts of the plant (0.5 and 1 mg/kg)and diclofenac sodium (10 mg/kg) doses will administer intra-peritoneal for 14 days from theday of CFA injection. The changes in the paw volume will be measure on various days up to
21days following CFA injection. The change in the inflammatory reaction will measure by usingvernier caliper on 0th, 7th, 14th and 21st day from the day of adjuvant injection. The animalswill weigh, using digital weighing balance on 0th, 7th, 14th and 21st day from the day of CFAinjection. At the end of experiment, on the 21st day all animals will be anaesthetize (Diethylether)andbloodwilldrawnbyretro-orbitalplexusesbypunctureandcollectinEDTAcontainingtubes,respectively.Bloodanalysis,biochemicalparameteri.e.,RBC,WBC,Platelet,Hb,andhistopathologicalparameterswillbedetermined.
CompleteFreund’sadjuvant(CFA)inducedpawedemainrat: Thestudywascarried out after21days. On Day 0, all thegroups except Group1wereinoculated with CFA. About 0.1mL of the suspension was injected in sub-plantar region of lefthind paw of all groups. CFA principally contains 1mg/mL of heat-killed Mycobacteriumtuberculosissuspendedinmineraloil.Itproducesinflammatoryresponseswithin24 h.Thepawvolume and haematological parameters were taken as parameters to assess the anti-arthriticactivity[Abidin Letal.,2014].
Inductionofarthritis:Pre-induction baseline was taken prior to the injection of Complete Freund’s Adjuvant (CFA)measured by left hind paw volume of each animal at 0th day for the induction of arthritis inalbinoWistarrats.Normalsaline wasgivenorallytothenormalgroup.CFAwasinjectedtothesub- planter region of the left hind paw to the other groups such as negative, standard and drugtreated groups.A 24G needle was introduced into the sub-planter region of the left hind pawpercutaneously. With the injection, a firm resistance to injection was characteristically felt aftertheinjection of0.1mlofCFAwasintroduced.
Measurementofratpawsedema: The severity of adjuvant arthritis was quantified by measuring the volume of the left hind pawby Vernier caliper. Paw volume was measured at 0th day and there after 7th, 14th, 21st days ofCFA post-inoculation. Data were expressed as the increase in paw volume with respect to day0thpawvolume (table 4).
The volume of the hind paw was measured during treatment period at different time intervals ofconsecutivedayssuchas(0,7,14,21and28days)usingtheverniercaliper.
Table4: Day-wiseInflammationofratspaw
Groups Treatement Pawreadingin(cm) Percentageinhibition 0thday 7thday 14thday 21stday 28thday
1
Normal
0.45
±0.04*
0.514±0.0
507*
0.488±0.06
05*
0.51±0.033
9*
0.512±0.07
59*
2 Negative 0.538±0.0 334* 0.754±0.0 658 0.816±0.04 03 0.842±0.03 114 0.87±0.040 6**
3
Standard
0.536±0.0
384*
0.772±0.0
228*
0.834±0.03
43*
0.864±0.02
70*
0.548±0.02
38**
37.01 4 Drugtreated(0.5ml /kg) 0.552±0.0 502 0.77±0.06 20 0.832±0.05 40* 0.87±0.072 8* 0.678±0.03 03** 22.06
5
Drugtreated(1ml/kg)
0.57±0.04
47
0.858±0.0
729
0.912±0.06
09*
0.95±0.057
0*
0.594±0.05
22**
31.72
The differences in the mean values among the treatment groups are greater than would beexpectedbychance;thereisa statisticallysignificantdifference(P=<0.001)**,(p<0.050)*.
TheresultsindicatethatRAinductioncausedasignificantincreaseinpawvolume,representinginflammationandswelling.However,treatment with diclofenac sodium and alpha-pinene resulted in a significant reduction in paw volume,indicatingpotentialanti-inflammatoryeffects of these treatments. The"P< 0.01"notationsuggests that the differences between the treatment groups and the RA-induced group werestatisticallysignificant,meaningthat the results are unlikelyduetochance andare likelyattributedtothetreatments'effects.Themeanpawvolumeinnormalcontrolgroupwas(0.642±0.0795)cm.TherewasasignificantincreaseinpawvolumeinRAinducedrats(0.840±0.046)cm. Paw volume was reduced significantly in the groups treated with diclofenacsodiumandthealpha-pinene(P<0.01).
PerformedCBCcount: Body weights were observed at 0th, 7th, 14th, 21st days by using weighing balance. All animalswereanaesthetizedwithdiethyl-etherandbloodwascollectedfromtheretro-orbitalpunctureoftheentire arthritis andnon-arthritic animals inplain EDTA containingtubes respectively.Samples were subjected to hematology analyser and the results were found. The red blood cell(RBC),whitebloodcell(WBC),hemoglobin(HGB)andplatelet(PLT) weremeasured.
Body weights were observed at 0th, 7th, 14th, 21st days by using weighing balance. All animalswereanaesthetizedwithdiethyl-etherandbloodwascollectedfromtheretro-orbitalpunctureoftheentirearthritisandnon-arthriticanimalsinplainEDTAcontainingtubesrespectively.
Samplesweresubjectedtohematologyanalyserandtheresultswere found.Theredbloodcell(RBC),whitebloodcell(WBC),hemoglobin(HGB)andplatelet(PLT)weremeasured in table 5.
Table5: Changesinhematologicalparametersadministeredtreatedratgroups
Groups WBC(103/µL) RBC(106/µL) HGB (g/dL) PLT(103/µL) Normal 6.52±1.52** 6.16±1.46** 13.64±1.70** 973.6±272.51**
Negative
15.18±2.15**
3.85±0.32**
9.88±0.52**
4882±712.49** Standard 5.88±0.65** 4.89±1.62** 14.44±2.45** 1171±411**
Drugtreated(0.5ml/kg)
10.86±1.04**
4±0.60**
9.87±0.54**
3135.2±697.26** Drugtreated(1ml/kg) 7.50±2.05** 4.59±0.81** 12.80±0.67** 1619±352.58**
Dataareexpressedasmean±SD(n=5)
Meanvalueswithdifferentsuperscriptsaresignificantlydifferentfromeach otherasrevealedbysigmastate(P=<0.001)**.
Induction of arthritis resulted in a significant reduction in RBC count, Hblevel and an increase in
total WBC count and platelets. Treatment with diclofenac sodium andalpha-pinene significantly increase in Hb and RBC count and decrease in the total WBC countandplatelets.
Performedhistopathology: The histopathology studies were performed the knee joints and hind paws of rats were removedfor histological analysis. The joints were fixed in 10% formaldehyde solution, decalcified in10%EDAnti-arthritic activity of Alpha Pinene enriched Boswellia serrata extract and method of preparation thereofTAfor30 days,embeddedin paraffin,andthencutintoabout5 mmthicknesssections,stained with hematoxylin-eosin (H&E) and observed by light microscopy. Sections were scoredas described previously by two independent examiners who were blinded to the experimentalprotocol.
Histopathological examination of H&Estained joint tissuesections obtainedfromnormalcontrol rats showed normal looking synovial membrane, no edema and no inflammatory cells.On the other hand, cases of the untreated arthritis group showed severe acute inflammatoryreaction with marked edema of the subcutaneous tissue, dense infiltrate of acute inflammatory.Cells mainly polymorphs, lymphocytes, scattered giant cells. The diffuse inflammatory reactioninfiltratesthesubcutaneoustissue,muscles,jointspaces,synovialmembrane,eventhebonetrabeculae. Inflammation was more remarkable on the left hind paw than that observed insectionsoftherightortheforepaw.Alltheexaminedsectionsobtainedfromthetreatedarthritisrats showed remarkable regression of inflammation and moderate infiltrates of inflammatorycells with more predominance of chronic inflammatory cells; lymphocyte, histiocytes scatteredgranulomata with epithelioid cells, and giant cells. It is to be noted that inflammation is limitedtothesubcutaneous tissue.Noinflammationisseeninmusclesorsynovialmembranes.Similarly, relative regression of inflammatory reaction with moderate arthritis and residual fewpolymorphswasobservedintissuesectionsofthetreatedarthritisgroup (figure 5)
The4Xand10Xresolution.S,synoviocytesandsynovialhyperplasiaindiseasedcontrol.Adoublearrowindicatesspacebetweencartilageboneofjoints,andincreasedspacebetweenbonecartilagewasfoundindiseasedcontrol;showinflammationinthebonemarrow.
In present study anti-arthritic activity of leaves of Boswellia serrata wasinvestigatedplantswasselectedonthebasisoftheirtraditionaluseintreatmentofarthritis.
Plantmaterialswereextractedbymacerationprocessandextractionwith80%methanolic solution. Further phytochemical investigation carried out on constituent includingalpha-pinene. ExtractswerealsocharacterizedbyUV,TLCandHPLC.
Further on dosing to animals of groups IV and V used to performed accordingly as 0.5mg/kgand1mg/kgwereselectedasdosefordrugtreatedgroupforinvivoinvestigation.
Arthritiswasinducedinexperimentalanimals(AlbinoWistarrats)usingCFAadministered byinjectedinthesub-planterregionoftheratshindpaw.Potentialwasassessed onthebasisofUV,TLCandHPLCandhematologyandhistopathologywereperformed.
In another embodiment, thealpha-pinene enriched Boswellia serrata extract is prepared with the pharmaceutical acceptable excipient for the the preparation of pharmaceutical composition. Furthermore, the pharmaceutical acceptable excipient is selected from the the group consisting of diluents, carriers, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers and lubricants.
It was observed that all drug treated animals provided significant effect on all assessedparameters. Level of inflammation significantly decreased in drug treated group in arthritis. Indrug treated group level of WBC, Platelets were found to be significantly high and RBC andHemoglobin were significantly less. Histological examination also revealed that increased spacebetween bone cartilage was found in diseased control; show inflammation in the bone marrowwhichwastreatedin drugtreated group.
Thusfrompresentinvestigationitcanbeconcludedthatthedrugalpha-pinenecomparable to the extract of Boswellia serratapossess significant anti-arthritic potential. Thiseffect is mediated through decreasing inflammation ofpaw of rats and decreasing anti-arthriticactivity.
I/We claim:
1. A method for extracting Alpha Pinene from Boswellia serrata, comprising the steps:
a. coarsely powdering the leaves of boswellia serrata;
b. pouring a solvent onto the powdered material to cover it;
c. sealing the container to prevent evaporation and contamination for 4 hours;
d. allowing the extraction process to occur for 2-3 days with periodically shaking;
e. separating the resulting mixture into a solvent-containing fraction (micelle) and leftover plant material (marc);
f. employing filtration or decantation to separate the micelle from the marc;
g. separating the micelle from the solvent through evaporation ;
h. drying the concentrated extract;
i. isolating of Alpha Pinene through the use of Thin-Layer Chromatography (TLC) or High-Performance Liquid Chromatography (HPLC).
2. The method as claimed in claim 1, wherein the solvent is methanol.
3. The method as claimed in claim 1, wherein the evaporation is done in oven or water bathat 500C. 4. A pharmaceutical composition for treating rheumatoid arthritis comprising Alpha Pinene enriched Boswellia serrata extract and pharmaceutical acceptable excipient. 5. The composition of claim 1, wherein the dose of Alpha Pinene is in the range of 0.5 to 1 mg/kg. 6. The composition as claimed in claim 4, wherein the pharmaceutical acceptable excipient is selected from the group consisting of diluents, carriers, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers and lubricants. 7. The composition as claimed in claim 4, wherein the use of said composition results in an inhibition of inflammation in the range of approximately 20-35%. Date-30/11/2023 G D Goenka University, Sohna Gurugram Road, Sohna, Haryana, India, 122103
Abstract
Alpha Pinene enriched Anti-arthritic formulation and method of preparation thereof
Rheumatoid arthritis (RA) is a severe autoimmune condition that causes persistent joint inflammation that increases the risk of early death.The present therapy for RA involve nonsteroidal anti-inflammatory drugs corticosteroids, disease modifying anti-rheumatic drugs and some recently developed biologic agents, but all of these are linked with side effects. The present invention relates to a composition for the treatment of Rheumatoidarthritis comprising Alpha Pinene isolated from Boswellia serrata extract and pharmaceutical excipient. Moreover, it also relates to a method for extracting Alpha Pinene compound from Boswellia serrata. , Claims:method for extracting Alpha Pinene from Boswellia serrata, comprising the steps:
a. coarsely powdering the leaves of boswellia serrata;
b. pouring a solvent onto the powdered material to cover it;
c. sealing the container to prevent evaporation and contamination for 4 hours;
d. allowing the extraction process to occur for 2-3 days with periodically shaking;
e. separating the resulting mixture into a solvent-containing fraction (micelle) and leftover plant material (marc);
f. employing filtration or decantation to separate the micelle from the marc;
g. separating the micelle from the solvent through evaporation ;
h. drying the concentrated extract;
i. isolating of Alpha Pinene through the use of Thin-Layer Chromatography (TLC) or High-Performance Liquid Chromatography (HPLC).
2. The method as claimed in claim 1, wherein the solvent is methanol.
3. The method as claimed in claim 1, wherein the evaporation is done in oven or water bathat 500C. 4. A pharmaceutical composition for treating rheumatoid arthritis comprising Alpha Pinene enriched Boswellia serrata extract and pharmaceutical acceptable excipient. 5. The composition of claim 1, wherein the dose of Alpha Pinene is in the range of 0.5 to 1 mg/kg. 6. The composition as claimed in claim 4, wherein the pharmaceutical acceptable excipient is selected from the group consisting of diluents, carriers, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers and lubricants. 7. The composition as claimed in claim 4, wherein the use of said composition results in an inhibition of inflammation in the range of approximately 20-35%.