Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
  • C07H15/20—Carbocyclic rings
  • C07H15/24—Condensed ring systems having three or more rings
  • C07H15/256—Polyterpene radicals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/04—Centrally acting analgesics, e.g. opioids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/06—Antimigraine agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

• B. acutangula (L.) Gaertn)
• B. acutangula has wider distribution, being found across Northern Australia from North Queensland to North Western Australia.
• Barringtonia acutangula has been further divided into two subspecies, B. acutangula ssp. acutangula and ⁇ . acutangula ssp. spicata [1]. The latter of these is found throughout the Barringtonia distribution area whereas the former is restricted to Northern Australia. Throughout their range many Barringtonia species have been used in variety of ways by local people.
• Barringtonia species are known to be edible [3, 9, 12-15].
• B. acutangula [2-4, 6-11]
• B. speciosa [5]
• B. racemosa [2, 3, 10- 12]
• ⁇ . asiatrica [2, 3, 5, 10, 11 ]
• ⁇ . calyptrate [10].
• seeds [2, 9, 10, 12] and wood [12] have also, been used.
• the leaves, fruit and seeds of several species are known to be edible [3, 9, 12-15].
• Several other properties and uses of Barringtonia species have been reported.
• ⁇ . racemosa as a tanning agent, due to the presence of tannins, [3, 12, 16] and as an insecticide reportedly to be approximately half as potent as nicotine [12, 16, 17].
• the fruit of Barringtonia has been used to poison wild pig [12].
• the seed of ⁇ . racemosa and the fruit of B. asiatica has been used for suicide and administration with "... homicidal intent " [11, 12], coconut milk being an antidote.
• HCN which has been demonstrated in high concentrations in the kernel of ⁇ . asiatica [11].
• Many of the Barringtonia species have found extensive use as traditional medicines and the fruit of B.
• acutangula has been called "Nurse fruit” [6]. All parts of the plant have been used and applications have been both internal and external. Preparation of applications may involve drying and powdering, extraction with hot or cold water, heating or juicing [3, 11, 12, 16, 18]. External applications tend to focus, as expected, on skin disease. Ailments such as general wounds, rheumatism, eczema, ulcers, scabies, tinea, ringworm, itches, inflammation and even leprosy have been treated with Barringtonia species [3, 11, 12, 16, 18]. Seeds in powered form have been used as a snuff to relieve headache whereas heated seeds are aromatic and have been used to assist in colic and parturition [3].
• Barringtonia acutangula continued to be a source of novel saponins and sapogenins. Again from the fruit of this species, a series of compounds, barringtogenol B, C, D, and E, were isolated and their structures explored [8, 26-34]. Barringtogenol C was isolated from B. acutangula fruits and the structure assigned by chemical techniques as previously described (FIG 4) (eg [8, 26, 27, 31-34]). Barringtogenol D, again isolated from B. acutangula fruit, was described by Barua et al [26] and a structure proposed by Chakraborti and Barua [29,30] (FIG 5). Barringtogenol E was isolated from the branch wood of ⁇ .
• saponins are produced as defensive agents by the plant [48].
• Increasing numbers of saponins are being isolated from lower marine organisms, but so far have been isolated from the phylum Echinodermata, in particular sea cucumbers (Holothuroidea) and starfish (Asteroidea) [46].
• Saponins consist of three main components, an aglycone (genin or sapogenin), such as a triterpene, a steroid or a steroidal alkaloid, one or more sugar chains, commonly D-glucose, D-galactose, D-glucuronicacid, D- galacturonic acid, L-rhamnose, L-arabinose, D-xylose and D-fucose and sometimes acids, such as angelic and tiglic acids [46, 47, 49]. Saponins are further classified as mono-, bi- or tri-desmosides according to the number of sugar chains which are attached to the aglycone [47].
• an aglycone such as a triterpene, a steroid or a steroidal alkaloid
• one or more sugar chains commonly D-glucose, D-galactose, D-glucuronicacid, D- galacturonic acid, L-rhamnose, L-arabinose
• haemolytic, molluscidal and piscicidal activities of saponins are well characterised and have even been used as assay techniques in bio- guided fractionations of plant and animal extracts (eg [47, 48, 50, 51]).
• haemolytic activity varies greatly or may be absent altogether and molluscicidal activity is somewhat dependent on the structure of the saponin [46, 47].
• Analgesic activity has been demonstrated in a small number of saponins. The following is an example of some of the saponins found to have analgesic effects.
• One of the active ingredients is stated as being platicodin and the dose received by the mice was equivalent to 160 mg/kg.
• the effects were comparable to 100-200mg/kg aspirin.
• Injection (ip, 100-250mg/kg) of the total saponin preparation from Panax notoginseng was found to act faster but for shorter durations than morphine and /-tetrahydropalmatine and was comparable to aminopyrine (150mg/kg) [55]. It was also noted that the saponin preparation induced a sedative effect, decreased the ED 5 o of pentobarbital in sleep induction, prolonged thiopental induced sleep and showed synergistic effects with chlorpromazine in CNS inhibition [55].
• dianosides were isolated and characterised from Dianthus superbus ver. longicalycinus [56-58]. Dianosides A and B were found to significantly inhibit acetic acid induced writhing at 10 and 30mg/kg (sc) with dianoside B the more potent [56]. In a detailed examination of the pharmacological effects of glycosidal fraction obtained from Maesa chisa var. augustifolia, Gomes etal [59] demonstrated, among other things, analgesia in the writhing test in mice. A 33% inhibition was observed in p- phenylquinonone induced writhing in contrast to a 52% inhibition observed in acetic acid induced writhing.
• R 2 is selected from hydrogen, hydroxyl, O-alkyl, O-alkenyl, O-benzoyl, O- dibenzoyl, O-alkanoyl, O-alkenoyl, O-aryl, O-heterocyclic, O-heteroaryl or
• R 5 and R 7 are independently be selected from hydrogen, alkanoyl, alkenoyl, dibenzoyl, benzoyl or benzoyl alkyl substituted alkanoyl;
• R 3 is selected from hydroxyl, O-alkanoyl, O-alkenoyl, O-benzoyl, O- dibenzoyl, O-alkyl, O-alkenyl, O-aryl, O-heterocyclic or O-heteroaryl;
• R 4 is selected from -CH 2 OH, COOH, CH 2 OCOCH 3 , COO alkyl, COO aryl,
• R 6 is selected from hydrogen or
• Ri is selected from hydrogen or alkyl; or pharmaceutically acceptable salts thereof with the proviso that when R 2 is OH, R 3 is OH, R 4 is CH 2 OH, R 6 is xylopyranosyl, Ri cannot be 4.
• alkyl refers to linear, branched, cyclic and bicyclic structures and combinations thereof, having 1 to 18 carbon atoms.
• Non- limiting examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, s- and t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.
• alkyl is selected from methyl, ethyl, propyl, isopropyl, butyl, s- and t- butyl, pentyl, and hexyl.
• alkenyl refers to unsaturated linear or branched structures and combinations thereof, having 1 to 7 carbon atoms. Non-limiting examples of alkenyl groups include, ethenyl, propenyl, isopropenyl, butenyl, s- and t-butenyl, pentenyl, hexenyl.
• Alkanoyl means alkanoyl groups of a straight or branched configuration having 1-8 carbon atoms.
• alkanoyl is selected from acetyl, propionoyl, butyryl, isobutyryl, pentanoyl and hexanoyl. More preferable alkanoyl is selected from acetyl, propionoyl, butyryl, and isobutyryl.
• alkenoyl means alkenylcarbonyl in which alkenyl is as defined above.
• alkenoyl is selected from pentenoyl, hexenoyl or heptenoyl. More preferably alkenoyl is selected from petnenoyl (tigloyl) or hexenoyl (angeloyl).
• benzoyl alkyl substituted alkanoyl is used to refer to straight or branched C1-C6 alkanoyl substituted with at least one benzoyl and at least one alkyl, wherein the benzoyl is attached to an straight or branched C1-6 alkyl.
• a benzoyl alkyl substituted alkanoyl is benzoyl methyl isobutanoyl.
• Heterocyclic refers to a non-aromatic ring having 1 to 4 heteroatoms said ring being isolated or fused to a second ring selected from 3- to 7- membered alicyclic ring containing 0 to 4 heteroatoms, aryl and heteroaryl, wherein said heteroatoms are independently selected from O, N and S.
• Non- limiting examples of heterocyclic include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, tetrahydrofuranyl, imidazolinyl, thiomorpholinyl, and the like.
• Aryl means a 6-14 membered carbocyclic aromatic ring system comprising 1-3 benzene rings.
• heteroaryl represents a 5-10 membered aromatic ring system containing a single ring having 1-4 heteroatoms, selected from O, S and N. Heteroaryl includes, but is not limited to, furanyl, diazinyl, imidazolyl, isooxazolyl, isothiazolyl, pyridyl, pyrrolyl, thiazolyl, triazinyl and the like.
• R 2 is hydrogen, benzoyl, dibenzoyl, tigloyl, or
• R 5 and R 7 are selected from hydrogen, tigloyl, benzoyl, or benzoyl alkyl substituted alkanoyl.
• R 3 is selected from hydroxyl, acetyl, benzoyl, dibenzoyl, isobutyryl or tigloyl.
• R is selected from -CH 2 OH, O-acetyl or hydroxy.
• the compound of formula (I) is selected from; a.
• the pharmaceutically acceptable salts may be selected from the group including alkali and alkali earth, ammonium, aluminium, iron, amine, glucosamine, choline, sulphate, bisulphate, nitrate, citrate, tartrate, bitarate, phosphate, carbonate, bicarbonate, malate, maleate, napsylate, fumarate, succinate, acetate, terephthalate, pamoate, pectinate and s-methyl methionine salts piperazine and the like.
• Another aspect of the invention resides in a composition containing one or more compounds of formula (I) when extracted from plants or parts of plants of the genus Barringtonia, preferably the species Barringtonia acutangula.
• the parts of plants include fruit, seed, bark, leaf, flower, and wood.
• the part of plants is selected from bark, flower and leaf. More preferably the parts of plants is bark.
• a pharmaceutical composition for treatment and/or control of pain comprising an effective amount of one or more compounds of formula (I) and a pharmaceutically acceptable carrier.
• Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting devices designed specifically for, or modified to, controlled release of the pharmaceutical composition.
• Controlled release of the therapeutic agent may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polyactic and polyglycolic acids and certain cellulose derivates such as hydroxypropylmethyl cellulose.
• the controlled release may be affected by using other polymer matrices, liposomes and/or microspheres.
• Pharmaceutically acceptable carriers for systemic administration may also be incorporated into the compositions of this invention.
• the pharmaceutical composition comprises a pharmaceutically-acceptable excipient.
• pharmaceutically-acceptable excipient is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration.
• carriers well known in the art may be used. These carriers or excipients may be selected from a group including sugars, starches, cellulose and its derivates, malt, gelatine, talc, calcium sulphate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water. Any suitable route of administration may be employed for providing a patient with the pharmaceutical composition of the invention.
• compositions of the present invention suitable for administration may be presented in discrete units such as vials, capsules, sachets or tablets each containing a predetermined amount of one or more pharmaceutically active compounds of the invention, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non- aqueous liquid, an oil-in water emulsion or a water in oil emulsion.
• compositions may be prepared by any of the method of pharmacy but all methods include the step of bringing into association one or more pharmaceutically active compounds of the invention with the carrier which constitutes one ore more necessary ingredients.
• the compositions are prepared by uniformly and intimately admixing the agents of the invention with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product in to the desired presentation.
• a method of treating and/or controlling pain comprising administering to a subject in need of such treatment an analgesically effective amount of one or more compounds according to formula (I).
• an analgesically effective amount of one or more compounds according to formula (I) there is provided the use of one or more of the compounds according to formula (I) in the manufacture of a medicament for the treatment and/or control of pain.
• FIG 1 shows the structure of A1 - barringenol
• FIG 2 shows the structure of barringtogenic acid and barringtogenol
• FIG 3 shows (a) initial and (b) revised structures of barringtogenol ⁇
• FIG 4 shows structure of barringtogenol C
• FIG 5 shows structure of barringtogenol D
• FIG 6 shows structure of barringtogenol E
• FIG 7 shows compounds from B.
• FIG 8 shows structure of barrinic acid
• FIG 9 shows compounds from B acutangula including acutangulic acid, tangulic acid and barringenic acid
• FIG 10 shows structure of 2a, 3 ⁇ , 19a tri hydroxy-olean-12-ene dioc acid 28 - O - ⁇ - D glucopyranoside
• FIG 11 shows structure of barringtoside A, barringtoside B and barringtoside C
• FIG 12 shows normal grooming response as control in Formalin assay
• FIG 15 shows schematic for preparation of crude saponin mixtures
• FIG 16 shows acid and base hydrolysis scheme for insoluble active portion of water extract
• FIG 21 shows preparative gel permeation column;
• FIG 23 shows C18 separation of TSK - 4a;
• FIG 24 shows C18 preparative separation of TSK - 4a;
• FIG 25 shows preparative C18 chromatogram of H 2 0 extract;
• FIG 26 shows outline of numbering system in regard to various fractions for compound F70.
• FIG 27 shows separation of fraction elating at 70% MeOH (F70);
• FIG 28 shows separation of fraction 70.2 (40% MeCN in 1% TFA);
• FIG 29 shows chromatogram of F.70.2.6;
• FIG 30 shows separation of fraction F.70.2.2 at 254nm (left) and 233nm (right);
• FIG 31 shows separation of fraction F70.2.5 at 220nm (left) and 233nm (right);
• FIG 32 shows separation of fraction F70.3;
• FIG 33 shows chromatograms of F.70.3.5 and F70.3.7;
• FIG 34 shows analytical separation of fraction F.70.3.4 (predominantly single compound);
• FIG 35 shows separation of F70.4;
• FIG 36 shows separation of F70.4.2;
• FIG 37 shows separation of F70.4.3;
• FIG 38 shows preparative chromatograms showing loss of peaks F.80.2 and F.80.3;
• FIG 39 shows preparative chromatograms of F.80.4;
• FIG 40 shows separation of fraction F.
• FIG 65 is a graph of the mean ( ⁇ SEM) paw withdrawal threshold versus time curves for (A) ipsilateral (inflamed) and (B) contralateral (non- inflamed) hindpaws of FCA-rats;
• FIG 66 is a graph of the mean ( ⁇ SEM) paw withdrawal threshold versus time curves for the (A) ipsilateral (inflamed) and the (B) contralateral (non-inflamed) hindpaws of FCA-rats;
• FIG 68 is the mean ( ⁇ SEM) dose-response curves for the antinociceptive effects of i.v. bolus doses of F70.3.2 and F70.3.6 in the ipsilateral hindpaws of FCA-rats; and
• FIG 69 is a graph of paw volume pre and post FCA treatment.
• EXPERIMENTAL SECTION SECTION A - PAIN ASSAYS The Formalin Assay.
• the formalin assay involves the subcutaneous injection of a small amount of formalin into the fore or hind paw of a rat or mouse and the behavioural response to this injection is recorded as a measure of pain response.
• a modification of this method was described by Dubuisson and Dennis [60] and the behavioural response detailed for both rats and cats. Independently the pain produced was described as being initially intense, sharp, stinging and burning and was given a 3/5 on a standard pain questionnaire. Some 4 to 5 minutes later this intense pain gave way to a steady throbbing ache which gradually disappeared over 30 to 60 minutes leaving a mild tenderness at the injection site [60].
• the formalin assay was chosen for the current work for several reasons. Firstly, and most importantly, this assay is often reported in journals such as Pain, which would indicate that ethical considerations have been overcome. It has also been demonstrated that the two distinct phases observed in the assay reflect two distinct phases of nociception. The first phase, early or acute, begins immediately after injection of formalin and lasts some 3 - 5 minutes. This phase is considered to be the direct chemical stimulation of nociceptors. A period of minimal activity lasting for 10 - 15 minutes follows this initial phase. Subsequently a second, late or tonic phase, begins and lasts for 20 - 40 minutes.
• the response shown in both early and late phases can be reduced using known analgesics, such as morphine, codeine, nefopam and orphendarine [61].
• the late phase was affected both by non-steroidal anti-inflammatory compounds, such as indomethacin and naproxen and steroids such as dexamethasone and hydrocortisone (e.g. [61]).
• non-steroidal anti-inflammatory compounds such as indomethacin and naproxen and steroids
• dexamethasone and hydrocortisone e.g. [61]
• aspirin and paracetamol were shown to be analgesic in a dose dependent manner in both phases of the formalin test [61].
• mice Male Quackenbush mice weighing 25 - 35g were used. They were housed in colony cages (400 x 300 x 130mm; Wiretainers) with ad libitum access to food (standard rat/mouse pellets; Norco Feeds) and water. For short term storage a maximum of 15 - 16 mice were housed per cage while for longer periods this number was reduced to less than 10 mice per cage. Bedding material was wood shavings or more commonly recycled paper pellets (Breeders Choice).
• a 12 hour light/dark cycle was maintained in the animal holding facility with lights on at 06:00hrs. All testing was performed during the light phase to minimise any diurnal variation in behaviour.
• the temperature of the facility was maintained at 21°C with humidity ranging between 45 and 65%. Newly acquired animals were housed in the holding facility for a minimum of two days prior to being used for testing.
• Test compounds Formalin (Ajax) was supplied as solution of approximately 37.5% formaldehyde containing 10% methanol as a preservative. This stock solution was diluted 1 :20 with water to give a 2% formaldehyde solution (5% 5- formalin) which was used as the nociceptive agent.
• Morphine hydrochloride was kindly donated by Extal, a division of Kenyan Alkaloids Pty. Ltd. and appropriate concentrations were made by dilution with sterile isotonic (0.9%) saline. Testing method.o In the absence of a dedicated room for testing, all tests were performed in the laboratory on weekends during times consistent with the first hours of the light phase in the holding facility. These times were chosen to minimise disturbances during testing due to other workers in the laboratory and to minimise any variation in response due to time of the day.s The mice were brought into the laboratory at least one hour prior to testing. Subsequently the mice were individually placed into empty colony cages, which also served as observation chambers. They were allowed a further 30 minutes to explore their environment.
• FIG 12 indicates that the amount of time that the mice spend grooming their hind paws is approximately 4 seconds every 5 minutes. Although there was some small variation in this time, it was decided that these values were not significant and were therefore not included in further calculations.
• the second control experiment involved an ip injection of isotonic saline followed, 30 minutes later, by injection of formalin.
• the results forthe controls are shown in FIG 13.
• the characteristic biphasic nature of the response reported by previous workers is shown in FIG 13.
• the final control experiment conducted to ensure the validity of the technique was to construct a dose response curve for morphine.
• a calculation of analgesic activity over the total experimental time period was required to evaluate the extracts as analgesics. Any differences observed between the acute and tonic phases of the nociceptive response were noted.
• a comparison between the duration of experimental and control pain for the entire experimental period (45min) was needed.
• This last step may involve several techniques, including Sephadex LH20, silica, Diol or reverse phase (C8 and C18) chromatography, alone or in combination.
• the technique of counter current chromatography and its variations (DCCC, RLCC, CPC) has also found application in the separation of saponins.
• the major problem associated with the isolation of saponins using chromatographic techniques is the lack of a suitable chromophore for UV detection. Although these problems can be overcome by using RI, mass detection and by derivatisation and UV detection, each of these techniques 5 has its own inherent advantages and difficulties which have been discussed elsewhere [67].
• the use of bioassay to guide the purification may, however, dictate the isolation methods used.
• Spectra were collected in both negative and positive ion modes, at a range of cone voltages ( ⁇ 50V, ⁇ 100V, ⁇ 150V and ⁇ 200V), and were analysed using MassLynx software.
• High resolution ESMS HR-ESMS
• HR-ESMS High resolution ESMS
• the instrument was a Bruker (Billerica MA, USA) BioApex 47e FTMS equipped with an Analytica of Branford (Branford CT, USA) external electrospray source. The instrument was calibrated in either positive or negative mode prior to sample infusion and molecular masses were reported to within 5ppm. Plant material.
• the plant sample was identified as Barringtonia acutangula (L.) Gaetrn. ssp.
• Bark was removed from the trees by the aboriginal people who lived in the area in such a manner as to inflict minimal damage and ensure the continued growth of the tree.
• the bark samples were air or oven dried at a maximum temperature of 50°C.
• the dried samples were mill ground to a coarse powder and the powdered sample stored in an airtight container at room temperature until ready for extraction.
• the small sample flowers and leaves were dried, powdered and stored in a similar manner the bark. Extraction of plant material.
• the dried and powdered bark was soaked for several hours in approximately ten volumes (w/v) of demineralised water (dH 2 0).
• the resulting mixture was filtered through several layers of muslin, centrifuged (Damon IEC Division DPR6000, 4500g for 45min) and the supernatant freeze dried (Virtis Freezemobile 12).
• the dried extract was stored in airtight containers at 4°C.
• the flowers (0.25g) and leaves (1.35g) were extracted with dH 2 0 in a similar fashion to the aqueous extraction of the bark.
• Method 1 The water extract was redissolved in ddH 2 0 for assay or further purification.
• Extracts were assayed as described previously.
• the active fraction from the gel permeation (TSK-4a) column was dissolved in MeOH, filtered (0.45:m nylon, Activon) and applied to a preparative C18 column (vide infra).
• the column was eluted using a step gradient of MeOH in ddH 2 0 (0, 35, 70 and 100% MeOH) and the fractions collected according to the resultant chromatogram.
• Method 3 The method utilised in this section was similar in all respects to method 2, the only difference was in the step gradient used for preparative C18 chromatography. In this instance a step gradient was employed which consisted of 10% increments of MeOH in ddH 2 0 (0-100% MeOH).
• the water extract was applied directly to a C18 preparative column omitting the need for a gel permeation step.
• the water extract was dissolved in dH 2 0, centrifuged (Sorval RC5B Plus, 12000g for 20min) and the supernatant applied a C18 preparative column (vide infra) and the column eluted as in method 3.
• Separation of active fractions The methods used for separation of crude, active fractions into pure compounds were developed on an individual basis.
• the columns used to facilitate separations included C18, C8, diol and phenyl bonded silica.
• Suitable mobile phases included MeOH, MeCN, isopropanol (/-PrOH), hexane, EtOAc, H 2 0, 0.01 M HAc and 1 % TFA.
• the approach used was to begin with a C8 or C18 column and a gradient of MeOH or MeCN with H 2 0 or 1% TFA (early separations used 0.01 M HAc). Modifications to the method were made until it became necessary to use a different column (e.g. phenyl or diol) and the process repeated in order to provide maximal separation and resolution. The methods adopted for each fraction are discussed in the relevant areas of the results and discussion sections. Chromatography.
• Analytical and semi-preparative chromatography Semi-preparative and analytical chromatography were performed using a Waters 600 HPLC system fitted with a photodiode array detector (PDA 996), an autosampler (717 plus) and a fraction collector. Chromatographic information was collected and stored using Millennium 2010 chromatography manager software (version 2.10). Analytical chromatography was performed using Dynamax columns in one of two formats. Reverse phase (C18 or phenyl) columns were either 4.6 x 250mm (8:m irregular silica, 60 A pore size, 1 mL/min) or 4.6 x 50mm ("Short Ones", 3:m spherical silica, 60 A pore size, 1 mL/min).
• a slurry was made using dry toluene (400mL) and dry silica gel (200g, 40-63:m, Merck) to which octadecyl trichlorosilane (40mL, Fluka) was added and the mixture stirred for24hr.
• the slurry was subsequently filtered via a Buchner funnel and the product washed sequentially with 400mL each of dry toluene, dry MeOH and dry DCM. Finally the non-end capped C18-silica product was dried at 37°C for 24hr.
• End capping was achieved by suspending the non-end capped C18- silica in 400mL of dry toluene, adding 40mL of trimethylchlorosilane (Fluka) and allowing the mixture to stand for 24hr. The final product was washed successively with 400mL dry toluene and 400mL dry DCM before being dried at 37°C for a further 24hr. Acetylation of the insoluble component of the water extract.
• the pellet (100mg) was added to 10mL of 2M NaOH, refluxed for 2hrs and allowed to cool overnight.
• the mixture was partitioned successively into DCM (20mL)o and n-butanol (20mL) and the solvent evaporated in vacuo.
• the pH of the aqueous layer adjusted to 7 with 1 M HCI.
• the aqueous phase was successively partitioned into DCM and n-butanol and the solvent removed in vacuo.
• the aqueous phase was acidified (pH 1) with 1M HCI, partitioned into DCM and /7-butanol and the solvent removed as previously.s
• the solvent remaining in the aqueous phase was also removed in vacuo.
• Method 1 The powdered bark was extracted in dH 2 0 to give a crude water extract (9.8%). The crude water extract was dissolved in ddH 2 0 at a concentration of 10 mg/mL/kg and assayed for analgesic activity as previously described. These results can be seen in FIG 18.
• the water extract was at least as active in the mouse formalin assay as the extracts from other solvents. As no additional extraction could be achieved by the other solvents it was decided to characterise analgesic activity in the water extract. At this time it was decided to construct a dose response curve for both the water soluble and the water insoluble extract and compare the results (FIG 20). From these results it can be seen that the amount of extract required to reduce the pain response by 50% (ED 50 ) is approximately 36 mg/kg (soluble) and 5 mg/kg (insoluble). These results, combined with the ease of extraction, further supported the decision to concentrate further purification effort on the water soluble material.
• Method 2 This method involved a preparative gel permeation separation of the water soluble extract. Initially five fractions were collected and, with respect to the starting H 2 0 extract, the following average yields were obtained, TSK- 1 0.4%, TSK-2 15%, TSK-323%, TSK-40.7%, TSK-520% and an insoluble portion (22%). Fractions TSK 1 to 5 were assayed at a crude water soluble equivalent dose of 100 mg/kg (i.e. approximately 2.5 times the ED 5 o) and the results can be seen in Table 2. Although the greatest apparent reduction in the pain response was seen in fraction 2 (74%), this required 15.8 mg/kg of material.
• the resulting chromatogram can be seen in FIG 24 and the yields obtained, with respect to the TSK-4a starting material, were 0%-MeOH (F0) 61.5%, 10%- MeOH (F10) 3.6%, 20%-MeOH (F20) 3.2%, 30%-MeOH (F30) 2.9%, 40%- MeOH (F40) 2.6%, 50%-MeOH (F50) 1.6%, 60%-MeOH (F60) 0.9%, 70%- MeOH (F70) 2.4%, 80%-MeOH (F80) 0.2%, 90%-MeOH (F90) 0.1 %, 100%- MeOH (F100) 0.2% and an insoluble portion 3.1%. Again each fraction was assayed and the results can be seen in Table 4. The results of these assays indicated that the most potent fractions, on a
• the tannins were found in the fractions eluting at lower MeOH concentrations while the saponins tended to elute at higher MeOH concentrations from the C18 column. Initially it was decided to limit this investigation to those fractions which eluted with 70, 80 and 90% MeOH. However it became apparent that a large number of compounds existed in each of these fractions.
• FIG 26 shows that, at the conditions employed for the separation and employing the three wavelengths shown, the extract could be separated into 5 distinct fractions. Varying the gradient conditions did not result in further separation of the peaks and therefore these five peaks were collected as indicated for further purification steps. Table 6 presents the yields obtained and preliminary 1 H-NMR results which indicate that no pure compounds were isolated at this point. Fraction F70.2. Fraction F70.1 was not further investigated as 1 H-NMR suggested that a considerable carryover from fraction F60 was present.
• fraction F70.2.2 No further separation of fraction F70.2.2 was possible using C18 or C8 columns with MeOH or MeCN in either H 2 0 or 1% TFA as the mobile phase.
• preliminary 1 H-NMR indicated the presence of aromatic signals. Separations using phenyl bonded silica are based on p interactions. Therefore a phenyl column (5 cm) was used to separate fraction F70.2.2 into eight fractions as shown in FIG 30 and Table 8.
• peaks F70.2.2.3, F70.2.2.6 and F70.2.2.7 were predominantly single compounds by 1 H-NMR.
• insufficient material was available to re-chromatograph the fractions in order to obtain enough pure compound for structural elucidation.
• Fraction 70.3 As was the case with F70.2, fraction F70.3 could not be satisfactorily l o separated using MeOH/1 % TFA mixtures. Again MeCN in 1 % TFA gradients were used and F70.3 was separated into seven fractions (FIG 32 and Table 10) using a semi-preparative C18 silica (25 cm). Again the separation could be monitored successfully at 233nm.
• FIG 4.18 shows chromatograms of fractions F70.3.5 and F70.3.7 indicating the peaks which contained pure compounds as shown by 1 H-NMR immediately after drying the samples. It should also be noticed that even though the peak
• Fraction F70.4. An initial separation of F70.4 was achieved using a 70%MeOH/1% TFA gradient on a C18 semi-preparative column (25 cm) (FIG 35 and Table 12). As can be seen fraction F70.4.2 consisted of a single, large peak which upon closer examination, by both HPLC and 1 H-NMR, was found to consist 5 of several compounds. Further separation of F70.4.2 was not obtained with C18 or C8 columns using MeOH or MeCN gradients and as 1 H-NMR showed the presence of aromatic protons in F70.4.2 a phenyl column (5 cm) was used.
• Standard sugar solutions used were ⁇ -D-glucuronic acid, ⁇ -D-fucose, ⁇ -D-glucose, V-L- arabinose, ⁇ -D-galactose, V-L-rhamnose, ⁇ -D-galacturonic acid and ⁇ -D- xylose.
• the TLC plates were developed with a phenol-sulphuric acid solution (dissolve 3g phenol and 5mL 97% H 2 S0 4 in 95 mL ethanol). The plates were dipped in the solution and heated at 110°C for until spots visualise (10-15 minutes). Instrumentation. The methods and instrumentation used for NMR and ESMS are presented in detail in Section B.
• Compound F70.3.6 was also subjected to fast atom bombardment (FAB) MS in both positive and negative ion mode (Kratos Concept ISQ High Resolution/Quadrupole Tandem Mass Spectrometer, Central Science Laboratory University of Kenya).
• the matrix used was meta-nitro benzyl alcohol (MNBA).
• Sub-fractions from 70.2.5 were analysed and assigned structures through the use of spectra, in particular the 1 H, 13 C and 1 H, 13 C-gHSQC (gHSQC), in a manner similar to that used above for fraction 70.3.60.
• the resultant structures fall within three categories, aglycones, monodesmosides and bidesmosides. The description of their structures begins with the aglycones.
• FTIR spectrum The FTIR of a thin film of neat compound is shown in FIG 43.
• the broad band centred at 3400 cm “1 indicates O-H stretch and the group of peaks between 2820 and 3000 cm “1 suggest C-H stretch.
• Ester carbonyl stretch was shown by the strong peaks at 1723 and 1709 cm “1 , while C-0 stretch was seen between 1275 and 1039 cm “1 .
• NMR methods Initially it was decided to use NMR solvents which were reported in the literature, principally methanol (MeOH-d 4 ) and pyridine-c 5 . However it soon became apparent that both solvents presented problems with the saponins isolated in the current work.
• the 1 H-NMR spectrum of compound F70.3.6 in DMSO-Qe can be seen in FIG 44.
• the spectrum can be broadly divided into three regions.
• the first most shielded region (*0.00 - *2.20) is predominantly associated with methyl signals.
• Six methyl singlets are evident in this region at chemical shifts of *0.71 (3H), *0.79 (3H), *0.84 (3H), *0.94 (3H), *0.97 (6H) and *1.33 (3H).
• the second region (*2.20 - *6.00) contains protons in close proximity to carbons bearing oxygen, notably four doublets characteristic of anomeric protons at *7.43, *7.52, *7.59, *7.66, *7.89 and *7.94.
• Benzoate at both C 21 and C 22 Compounds in this group each had a benzoate functionality at both C 2 ⁇ and C 22 .
• Four such compounds were isolated in the current project. Each of the four compounds were isolated as amorphous white substances and the weights were F70.4.3.5.2 (2.7 mg), F70.4.2.4.2 (4.9 mg), F80.6.4 (11.7 mg) and F80.6.7 (3.9 mg).
• Fraction F70.3.3 was isolated as 47.8mg of an amorphous white mass which was shown to contain several compounds by 1 D and 2D NMR. Several attempts to resolve these compounds resulted in the collection of 6.6mg of fraction F70.3.3.2.2, which still contained three compounds by 1 D and 2D NMR. However it was possible to assign the structure of one of these compounds using a similar approach as for the other compounds.
• F70.3.4.5 A small quantity (2.7 mg) of F70.3.4.5 was isolated as an amorphous white compound. The structure of F70.3.4.5 is shown in
• FIG 60 Compound F70.3.5.2: This compound was isolated as 12.2 mg of an amorphous white compound.
• bolus doses of each of the test compounds (F70.3.2 and F70.3.6), paw withdrawal thresholds (g) were normalized by subtraction of the individual baseline PWT values quantified immediately prior to drug administration.
• the area under the normalized PWT versus time (AUC) was calculated using the trapezoidal rule.
• Dose- o response curves were constructed by plotting the AUC values versus the i.v. dose for each of F70.3.2 and F70.3.6.
• FIG 65 shows that for the ipsilateral hindpaw, there was a dose- related increase in the antinociceptive potency of F70.3.2. By contrast, there was an absence of antinociception in the contralateral hindpaw.5 FIG.
• FIG. 66 shows that the ipsilateral hindpaw, there was a dose-related increase in the antinociceptive potency of F70.3.6 in the dose range, 0.002- 0.01 mg/kg. However, further increases in the dose magnitude resulted in a decreased rather than an increased antinociceptive response. Similar to compound F70.3.2, there was an absence of antinociception in the0 contralateral hindpaw.
• FIG 67 shows that rats where injected with saline only show that the experimental procedures themselves did not evoke significant antinociception The is a distinct increase and then plateau of the antinocieptive effect of compounds F70.3.6 and F70.3.2 using the above techniques, as shown in FIG. 68.
• FIG 69 shows that by 5 days post-FCA administration, the mean ( ⁇
• FIGS 70 and 71 show the structures of further compounds of the invention.
• FIG 70 shows compounds (1) to (7) characterized by inclusion of groups A, B, C and D as shown. These compounds have been prepared from water extracts of the dried bark of B acutangula as described previously and have had their structure assigned as also described previously. Similar comments apply to the compounds of FIG 71.
• Triterpenoids XI New triterpenoid sapogenins from the fruits of Barringtonia acutangula. Journal of Pharmaceutical Sciences 50(11 ): 937-940. 5 27. Barua, A.K., Dutta, S.P. and Das, B.C. (1968). Triterpenoids - XXIX. The structure of Barringtogenol B - A new triterpenoid sapogenin from Barringtonia acutangula Gaertn. Tetrahedron 24: 1113-1117. 28. Barua, A.K., Dutta, S.P. and Pal, S.K. (1967). Triterpenoids - XXX.
• Triterpenoids - XIX The constitution of Barringtogenol C - A new triterpenoid sapogenin from Barringtonia acutangula Gaertn. Tetrahedron 21; 381-387. 32. Barua, A.K. and Chakrabarti, P. (1964). Triterpenoids XVIII. The l o constitution of Barringtogenol C. Science and Culture 30(7): 332-334. 33. Barua, A.K., Chakraborti, S.K., Chakrabarti, P. and Maiti, P.C. (1963). Triterpenoids. Part XIV. Studies on the constitution of Barringtogenol C - A new triterpenoid sapogenin from Barringtonia acutangula Gaertn. Journal of the Indian Chemical Society 40(6):
• Triterpenoids - XXXI Studies on a triterpene isolated from Barringtonia acutangula Gaertn. Science and Culture 34(6): 259-260.

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