Patent Application: US-201113695911-A

Abstract:
this invention provides the methods of separation and identification of a novel type of piperidine flavan alkaloids from an african herbal tea , the leaves of combretum micranthum commonly known as kinkeliba , and the procedures for preparing the total piperidine flavan alkaloids . in particular , this invention relates to the use of the plant extract that may contain tpfa as anti - diabetic agents in treatment of metabolic disorders and other applications related to this new chemical structure and derivatives thereof .

Description:
in one aspect the present invention provides a piperidine - flavan alkaloid compound of formula ( i ): one of r 1 and r 2 is hydrogen , and the other of r 1 and r 2 is an optionally substituted 2 - piperidinyl group characterized by formula ( a ): r 3 , r 4 and r 5 are each independently selected from the group consisting of hydrogen , hydroxyl , and c 1 - c 4 alkoxy ; r 6 , r 7 , and r 8 are each independently hydrogen , c 1 - c 4 alkyl , or r 11 c ( o )—; r 9 is hydrogen , c 1 - c 4 alkyl , or r 12 c ( o )—; r 10 at each occurrence is independently hydrogen , c 1 - c 4 alkyl , or oxo (═ o ); r 11 is hydrogen or c 1 - c 4 alkyl ; and r 12 is hydrogen , c 1 - c 4 alkyl , or or 11 . in one embodiment , the present invention provides a piperidine - flavan alkaloid compound of formula ( i ), or a pharmaceutically acceptable salt thereof , wherein r 6 through r 10 are each hydrogen . in another embodiment , the present invention provides a piperidine - flavan alkaloid compound of formula ( i ), namely kinkéloid a 1 , or a pharmaceutically acceptable salt thereof , wherein : r 2 , r 3 , r 4 , and r 5 are each h ; and in another embodiment , the present invention provides a piperidine - flavan alkaloid compound of formula ( i ), namely kinkéloid a 2 , or a pharmaceutically acceptable salt thereof , wherein : r 1 , r 3 , r 4 , r 5 are each h ; and in another embodiment , the present invention provides a piperidine - flavan alkaloid compound of formula ( i ), namely kinkéloid b 1 , or a pharmaceutically acceptable salt thereof , wherein : in another embodiment , the present invention provides a piperidine - flavan alkaloid compound of formula ( i ), namely kinkéloid b 2 , or a pharmaceutically acceptable salt thereof , wherein : in another embodiment , the present invention provides a piperidine - flavan alkaloid compound of formula ( i ), namely kinkéloid c 1 , or a pharmaceutically acceptable salt thereof , wherein : in another embodiment , the present invention provides a piperidine - flavan alkaloid compound of formula ( i ), namely kinkéloid c 2 , or a pharmaceutically acceptable salt thereof , wherein : in another embodiment , the present invention provides a piperidine - flavan alkaloid compound of formula ( i ), namely kinkéloid d 1 , or a pharmaceutically acceptable salt thereof , wherein : r 2 , r 3 , r 4 , and r 5 are each oh ; in another embodiment , the present invention provides a piperidine - flavan alkaloid compound of formula ( i ), namely kinkéloid d 2 , or a pharmaceutically acceptable salt thereof , wherein : r 1 , r 3 , r 4 , and r 5 are each oh ; and in another aspect the present invention provides a pharmaceutical composition derived from a species of the kinkéliba ( combretum micranthum ) family , the composition comprising at least one piperidine - flavan alkaloid compound of formula ( i ): one of r 1 and r 2 is hydrogen , and the other of r 1 and r 2 is an optionally substituted 2 - piperidinyl group characterized by formula ( a ): r 3 , r 4 and r 5 are each independently selected from the group consisting of hydrogen , hydroxyl , and c 1 - c 4 alkoxy ; r 6 , r 7 , and r 8 are each independently hydrogen , c 1 - c 4 alkyl , or r 11 c ( o )—; r 9 is hydrogen , c 1 - c 4 alkyl , or r 12 c ( o )—; r 10 at each occurrence is independently hydrogen , c 1 - c 4 alkyl , or oxo (═ o ); r 11 is hydrogen or c 1 - c 4 alkyl ; and r 12 is hydrogen , c 1 - c 4 alkyl , or or 11 . in one embodiment , the present invention provides a pharmaceutical composition derived from a species of the kinkéliba ( combretum micranthum ) family , the composition comprising at least one piperidine - flavan alkaloid compound of formula ( i ), wherein : in another embodiment , the present invention provides a pharmaceutical composition derived from a species of the kinkéliba ( combretum micranthum ) family , the composition comprising at least one piperidine - flavan alkaloid compound of formula ( i ), wherein : in another embodiment , the present invention provides a pharmaceutical composition derived from a species of the kinkéliba ( combretum micranthum ) family , the composition comprising at least one piperidine - flavan alkaloid compound of formula ( i ), wherein r 6 through r 10 are each hydrogen . in another embodiment , the present invention provides a pharmaceutical composition derived from a species of the kinkéliba ( combretum micranthum ) family , the composition comprising at least one piperidine - flavan alkaloid compound of formula ( i ), wherein the piperidine - flavan alkaloid compound is selected from the group consisting of kinkéloids a 1 , a 2 , b 1 , b 2 , c 1 , c 2 , d 1 , and d 2 . in another aspect the present invention provides a method of separating piperidine flavan alkaloids from leaves of combretum micranthum ( fam . combretaceae ), commonly known as kinkéliba , comprising extracting the leaves with one or more organic solvents . in a preferred embodiment , the present invention provides a method of separating piperidine flavan alkaloids from leaves of combretum micranthum ( fam . combretaceae ), commonly known as kinkéliba , comprising extracting the leaves with a c 1 - c 4 alkyl alcohol , more preferably methanol or ethanol . in another aspect the present invention provides a method for preparing total piperidine flavan alkaloids ( tpfa ) from the leaves of combretum micranthum , comprising : a ) extracting the leaves with an organic solvent to form a crude extract ; and b ) treating the crude extract with at least one of the following methods : iii ) acid - base precipitation in combination with extraction by an organic solvent , and in one preferred embodiment , the present invention provides a method for preparing total piperidine flavan alkaloids ( tpfa ) from the leaves of combretum micranthum , wherein said organic solvent in step a ) is an alcohol . in another preferred embodiment , the present invention provides a method for preparing total piperidine flavan alkaloids ( tpfa ) from the leaves of combretum micranthum , wherein said organic solvent in step a ) is ethanol or methanol . in another preferred embodiment , the present invention provides a method for preparing total piperidine flavan alkaloids ( tpfa ) from the leaves of combretum micranthum , wherein said solvent systems in method i ) comprises at least one non - polar solvent and at least one polar solvent . in another preferred embodiment , the present invention provides a method for preparing total piperidine flavan alkaloids ( tpfa ) from the leaves of combretum micranthum , wherein the acid - base precipitation in said method ii ), iii ) or iv ) comprises steps of ( a ) dissolving the crude extract with an acidic aqueous solution ; ( b ) filtering to remove insoluble nonpolar components ; and ( c ) adjusting ph of the filtrate solution with a base so that precipitates are formed . in another preferred embodiment , the present invention provides a method for preparing total piperidine flavan alkaloids ( tpfa ) from the leaves of combretum micranthum , wherein said organic solvent in method ii ) comprises an alcohol , more preferably n - butanol . in another preferred embodiment , the present invention provides a method for preparing total piperidine flavan alkaloids ( tpfa ) from the leaves of combretum micranthum , wherein said solvent fractionation in method iii ) comprises using at least one chlorinated hydrocarbon and one alcohol , more preferably said chlorinated hydrocarbon being chloroform , and said alcohol being n - butanol . in another preferred embodiment , the present invention provides a method for preparing total piperidine flavan alkaloids ( tpfa ) from the leaves of combretum micranthum , further separating the kinkéloid alkaloid compounds by silica gel chromatography . in another aspect the present invention provides a composition comprising an enriched tpfa extract derived from the leaves of combretum micranthum . in another aspect the present invention provides a method for treatment of a disease or condition related to glucose metabolism , comprising administering to a subject in need thereof a therapeutically effective amount of any piperidine - flavan alkaloid compound or composition thereof described above . in one embodiment , the present invention provides a method for treatment of a disease or condition related to glucose metabolism , comprising administering to a subject in need thereof a therapeutically effective amount of any piperidine - flavan alkaloid compound or composition thereof described above , wherein said treatment has an effect selected from the group consisting of : v ) lowering the enzyme that is necessary for the production of glucose in the liver . in another aspect the present invention provides a method for treatment of a disease or condition related to glucose metabolism , comprising administering to a subject in need thereof a therapeutically amount of tpfa derived from leaves of combretum micranthum ( fam . combretaceae ). in another aspect the present invention provides use of a piperidine - flavan alkaloid compound as described above for manufacture of a medicament for treatment of a disease or condition related to glucose metabolism . in yet another aspect the present invention provides use of a total piperidine - flavan alkaloid ( tpfa ) extract derived from the leaves of combretum micranthum ( fam . combretaceae ), for manufacture of a medicament for treatment of a disease or condition related to glucose metabolism . total crude extract of the kinkeliba leaves was prepared by different methods of aqueous ethanol extraction and methanol extraction . sun - dried kinkéliba leaves from leen , pout , senegal ( near thiès ) were ground to a fine powder with a perten laboratory mill 3100 . the kinkéliba leaves were extracted twice with 100 % ethanol and a third time with 80 % ethanol ( v / v ) by maceration for 24 hours ( extraction method i ) or extracted three times with 100 % methanol by reflux for 2 hrs ( extraction method ii ). each step was tested by lc - ms to ensure a complete extraction , and after 3 extractions , the level of alkaloids had dropped to 10 % of the initial amount . the filtrations from both extraction methods were dried giving a total weight of 24 . 26 % and 35 . 27 % using aqueous ethanol extraction and methanol extraction , respectively , from the starting material . tpfa was prepared by several methods including , but not limited to , solvent fractionation using different polar solvent systems , acid - base precipitation , acid - base precipitation with n - butanol extraction and acid - base precipitation with chloroform and n - butanol fractionation . during solvent fractionation , the crude extract was dissolved in water and partitioned between hexane , chloroform , ethyl acetate , and n - butanol , along with the remaining water fraction . lc - ms analysis indicated that the alkaloids were focused in the n - butanol fraction . during acid - base precipitation , the crude extract was dissolved in 3 % acetic acid in water and filtered by vacuum to separate the non - polar components or non - alkaloids that did not dissolve . the acidic solution was brought from ph 3 to ph 9 by the addition of nh 4 oh ( 38 % in water ) and the solution was allowed to precipitate and settle for 60 min before filtering by vacuum . the filtrate was collected and washed with distilled water until it ran neutral while the precipitate was dissolved in methanol and dried to obtain the total alkaloid . during acid - base precipitation with n - butanol extraction , the crude extract was dissolved in 3 % acetic acid in water and filtered by vacuum to separate the non - polar components that did not dissolve . the acidic solution was brought from ph 3 to ph 9 by the addition of nh 4 oh ( 38 % in water ) and the solution was allowed to precipitate and settle for 60 min before extraction with n - butanol to obtain the total alkaloids . during acid - base precipitation with chloroform and n - butanol fractionation , the crude extract was then dissolved in 3 % acetic acid in water and filtered by vacuum to separate the non - polar components that did not dissolve . the acidic solution was brought from ph 3 to ph 9 by the addition of nh 4 oh ( 38 % in water ) and the solution was allowed to precipitate and settle for 60 min before fractionation with chloroform and n - butanol . the total alkaloids were focused in n - butanol part . the invention is described more fully by way of the following non - limiting examples . air - dried kinkéliba leaves , 1 . 04 kg , were extracted using above method ( extraction method i ) to obtain 252 . 3 g crude extract . the extract was then dissolved in water and partitioned between hexane ( 3 × 1 . 8 l ), chloroform ( 3 × 1 . 8 l ), ethyl acetate ( 3 × 2 l ), and n - butanol ( 3 × 1 . 5 l ), with the remaining water fraction totaling approximately 1 . 5 l . it was determined by lc - ms that the alkaloids were focused in the n - butanol fraction with a total weight of 80 . 66 g calculating to 7 . 75 % from the starting materials . this fraction has been applied for further separation of the pure flavan alkaloids and biological study . air - dried kinkéliba leaves , 98 . 39 g , were extracted using above method ( extraction method ii ) to obtain 34 . 7 g crude extract . the extract was then dissolved in approximately 800 ml of 3 % acetic acid in water and filtered by vacuum to separate the non - polar components or non - alkaloids that did not dissolve . the acidic solution was brought from ph 3 to ph 9 by the addition of nh 4 oh ( 38 % in water ) and the solution was allowed to precipitate and settle for 60 min before filtering by vacuum . the filtrate was collected and washed with distilled water until it ran neutral while the precipitate was dissolved in methanol and dried . the total alkaloid extract weighed 10 . 8840 g calculating to 11 . 06 % from the starting materials . all the steps , from the total extract to the total alkaloid extract were calibrated to the same concentration and analyzed by lc - ms to confirm the total alkaloid extraction technique . air - dried kinkéliba leaves , 102 . 93 g , were extracted using above method ( extraction method ii ) to obtain 36 . 3 g crude extract . the extract was then dissolved in approximately 800 ml of 3 % acetic acid in water and filtered by vacuum to separate the non - polar components that did not dissolve . the acidic solution was brought from ph 3 to ph 9 by the addition of nh 4 oh ( 38 % in water ) and the solution was allowed to precipitate and settle for 60 min before extraction with n - butanol ( 3 × 1 l ) to extract the precipitated alkaloids . the total alkaloid fraction was dried and weighed 17 . 85 g calculating to 17 . 34 % from the starting materials . all the steps , from the total extract to the total alkaloid extract were calibrated to the same concentration and analyzed by lc - ms to confirm the total alkaloid extraction technique . air - dried kinkéliba leaves , 98 . 68 g , were extracted using above method ( extraction method ii ) to obtain 34 . 8 g crude extract . the extract was then dissolved in approximately 800 ml of 3 % acetic acid in water and filtered by vacuum to separate the non - polar components that did not dissolve . the acidic solution was brought from ph 3 to ph 9 by the addition of nh 4 oh ( 38 % in water ) and the solution was allowed to precipitate and settle for 60 min before fractionation with chloroform ( 3 × 1 l ) and n - butanol ( 3 × 1 l ). the total alkaloid fraction of n - butanol part was dried to obtain 11 . 4450 g calculating to 11 . 60 % from the starting materials . all the steps , from the total extract to the total alkaloid extract were calibrated to the same concentration and analyzed by lc - ms to confirm the total alkaloid extraction technique . fig2 illustrates the chemical profile of the total flavan alkaloids prepared by the above 4 procedures using lc - ms , and fig3 shows the total ion ms spectra of the 4 flavan alkaloids , kinkéloid a , kinkéloid b , kinkéloid c and kinkéloid d . the total alkaloid fractions were cleaned up and concentrated by running the fractions through a sephadex lh - 20 column , hydration and elution with 100 % methanol . the eluted fractions from the sephadex column were analyzed by lc - ms and the fractions that contain only alkaloids were combined and dried , calculating to approximately 32 . 07 % of the starting material . the concentrated alkaloid mixture was separated into the separated alkaloids by preparative hplc with a microsorb c18 column ( varian , 10 μm , 41 . 4 × 250 mm ). the mobile phase was 0 . 1 % formic acid in water ( a ) and in methanol ( b ) at a gradient of 10 % b at 0 min , 10 % b at 20 min , 60 % b at 170 min , and 60 % at 200 min . the flow rate was set to 18 ml / min and the fractions were collected at 1 min / tube from 50 to 170 min following injection . the eluted fractions were analyzed by lc - ms and the appropriate fractions were combined and dried to give 3 of flavan alkaloids , kinkéloid b , kinkéloid c , and kinkéloid d ( fig1 ). kinkéloid a , the alkaloid in lowest concentration , could not be collected separate from b due to peak tailing , so a fraction containing both kinkéloid a & amp ; b was separated by preparative tlc with silica at 1000 μm thickness and a developing solvent of 20 % methanol in chloroform plus 0 . 1 % triethylamine . the kinkéloid a sample was cleaned up by analytical lc with an inertsil column ( ods - 3 , 3 μm , 4 . 6 × 250 mm ). the mobile phase was 0 . 1 % formic acid in water ( a ) and in methanol ( b ) at a gradient of 20 % b at 0 min , 50 % b at 20 min , and 50 % at 25 min at a flow rate of 0 . 8 ml / min . the eluted fractions were combined and dried to give the 4th new type of flavan alkaloid , kinkéloid a ( fig1 ). the spectroscopic assignments of all four kinkéloid structures ( fig1 ) are listed in table 1 , without differentiation between c - 6 and c - 8 . the ms spectra of the four kinkéloids ( fig3 ) illustrates the molecular weights to be 341 , 357 , 373 and 389 , indicating a series of compounds that differ by a single hydroxyl in each case with the oxygen accounting for the 16 mass unit difference . at the outset , from hrms , two possible formulas were deliberated for kinkéloid b from hrms , c 20 h 23 no 5 and c 18 h 21 n 4 o 4 + , but the positive ion esi - ms gives an [ m + h ] + ion at m / z 358 which would not be possible for a positively charged compound . this leaves c 20 h 23 no 5 as the molecular formula of kinkéloid b with kinkéloid a , c 20 h 23 no 4 , kinkéloid c , c 20 h 23 no 6 , and kinkéloid d , c 20 h 23 no 7 . the skeleton was not easily determined because the samples isolated were still mixtures of isomers , both structural , c - 6 and c - 8 attachment of the piperidine , and stereoisomers at both chiral centers ( or three chiral centers in the case of kinkéloid d ). the initial analysis of the 13 c spectrum for kinkéloid b yielded an oxygenated methine , at 79 . 57 ppm , as well as an oxygenated methyl or nitrogenated methine , at 54 . 79 ppm . nine signals were found in the unsaturated / aromatic range , 96 . 03 - 146 . 49 ppm and three more signals are compatible with oxygenated aromatic carbons from 155 . 00 to 158 . 03 ppm . finally , a collection of five saturated carbons were found between 20 and 30 ppm and a signal at 46 . 82 ppm that could be a methylene attached to nitrogen . the proton spectrum showed a collection of messy signals , integrating to 12h , in the range of 1 . 5 - 4 . 0 ppm , a doublet at 4 . 54 ppm , 1h , a singlet at 6 . 07 ppm , 1h , a pair of doublets of doublets at 6 . 75 ppm , 2h , and a doublet at 6 . 85 ppm , 1h . the assistance of 2 - d spectra , hsqc , cosy and hmbc , was required to connect the flavan skeleton and piperidine substituent in a manner reasonable to fit the data . according to the hsqc spectrum , the three protons ( dd at 6 . 72 and 6 . 78 ppm and d at 6 . 85 ppm ) correlated to the three aromatic carbons from 114 to 118 ppm . this , with the splitting pattern in the proton spectrum , indicated a 3 , 4 - dihydroxy phenyl substituent . in keeping with the molecular formula c 20 h 23 no 5 , a dihydroxy phenyl ring leaves three oxygens not yet accounted for as well as six more aromatic carbons and the structure is now comparable to 3 ′, 4 ′, 5 , 7 - tetrahydroxyflavan . due to the different chemical environment between c - 6 and c - 8 of the flavan and alkaloid nmr signals , a flavan structure with a nitrogen - containing substituent attached to ring a was explored . the cosy spectrum shows the saturated carbons between 20 and 30 ppm all seem to coordinate with each other but a closer look , combined with hsqc assignments , separates the three saturated carbons of the flavan from the five saturated carbons of the piperidine . the protons at 1 . 87 and 2 . 13 ppm ( c - 3 ) correlate to the protons at 2 . 62 ppm ( c - 4 ) as well as the proton at 5 . 0 ppm ( c - 2 ). conversely , the protons of c - 6 ″, 1 . 87 and 2 . 31 ppm , show correlations to protons at 1 . 62 and 1 . 87 ppm ( c - 4 ″ and c - 5 ″) and the doublet at 4 . 54 ppm ( c - 1 ″). additionally , c - 4 ″ and - 5 ″ protons correlate to c - 3 ″ at 2 . 97 and 3 . 4 ppm . the hsqc spectrum corrected the assignment of an oxygenated methyl at 54 . 79 ppm to a nitrogenated methine and the downfield shift is explained by attachment to an aromatic ring . this nitrogenated methine and the nitrogenated methylene , at 46 . 82 ppm , can be connected to three of the five saturated 13 c signals between 20 and 30 ppm ( c - 4 ″, - 5 ″ and - 6 ″) composing a 2 - piperindinyl substituent attached to the aromatic ring a at the methine . a similar flavonoid alkaloid compound with a 2 - piperidinyl substituent was used for comparison to confirm this piperidine moiety ; ahond et al . isolated n - demethylcapitavine ( 10 ). the reported 13 c spectral data matches the piperidine assignments of the kinkéloids at 53 . 2 , 46 . 5 , 23 . 5 , 22 . 6 , and 27 . 9 ppm for c - 1 ″- c - 6 ″, respectively , confirming the piperidinyl moiety . the high - resolution mass spectrometric data of kinkéloid b ( fig4 ) was utilized to confirm placement of the piperidine on ring a of the flavan . a major fragment of m / z 222 , a fragment that all the alkaloids display , was determined to be the molecular formula of c 12 h 16 no 3 and needed to be formed by a very consistent fracture . because all the alkaloids demonstrate this major fragment , the hrms data of kinkéloid b can be confidently used for the elucidation of the other kinkéloids . the fragment , c 12 h 16 no 3 at m / z 222 , is attributed to the proposed mass spectrometric retro - diels - alder ( rda ) fragmentation for the kinkéloid structure , presented in fig5 , accounting for this major ion . this rda fragmentation of the flavan is common for flavonoid compounds and strengthens the argument for the piperidine attachment to ring a . at this point the varying number of hydroxyl substituents could be assigned to ring b of the flavan , providing mono -, di -, or tri - substituted phenyls , and c - 3 to give a flavan - 3 - ol . these hydroxyl substituents , at any of these positions , would stay with the lost fragment ensuring a consistent m / z 222 fragment in the esi - ms spectrum of each kinkéloid . the hmbc spectrum of kinkéloid b confirmed the connection of ring b to c - 2 with correlations between the protons of c - 2 ′ and - 6 ′ and the c - 2 signal and vice versa . correlations between the protons of c - 4 ( 2 . 62 ppm ) and c - 2 , c - 5 and c - 10 validate the flavan structure , specifically the saturated ring c . additionally , the hmbc spectrum revealed a couple correlations to confirm the attachment of the piperidine moiety to c - 6 of the flavan structure . the proton at 4 . 54 ppm ( c - 1 ″) shows correlations to the carbons at 29 . 53 , 102 . 96 and 155 . 65 ppm which correspond to c - 6 ″, c - 6 and c - 5 , respectively . this confirms , specifically h - 1 ″ to c - 5 , that the piperidine is attached to c - 6 . an additional hmbc spectrum , on a different sample , was used to demonstrate the correlations would vary for a mixture of both structural isomers . the hmbc spectrum of this sample produces correlations between the singlet at 6 . 04 ppm ( c - 8 ) and the carbons at 155 . 10 and 158 . 33 ppm ( c - 7 and - 9 ), confirming piperidine attachment at c - 6 , as well as correlations between the proton at 5 . 90 ppm ( c - 6 ) and the carbon at 155 . 75 ( c - 5 ), confirming the other isomer with attachment at c - 8 . at this point , the skeleton of these alkaloid compounds is confirmed as a piperidine flavan compounds with a 2 - piperidine moiety attached at c - 6 and c - 8 . kinkéloid c , c 20 h 23 no 6 , is the 3 ′, 4 ′, 5 ′, 5 , 7 - pentahydroxyflavan structure ; the aromatic region of ring b was simplified compared to kinkéloid b because of the identical environment for the ring b carbons and protons . the three oxygenated aromatic carbons at 147 . 15 ( c - 3 - and - 5 ′) and 133 . 90 ppm ( c - 4 ′) and the upfield aromatic carbons at 106 . 16 ( c - 2 ′ and - 6 ′) confirm a trihydroxy phenyl ring b . kinkéloid a , c 20 h 23 no 4 , is the 4 ′, 5 , 7 - trihydroxyflavan kinkéloid ; the 1 h and 13 c spectral shifts and observed splitting confirms this structure . the carbon signals for ring b show overlapping signals at 128 . 45 ppm for c - 2 ′ and - 6 ′ and 116 . 32 ppm for c - 3 ′ and - 5 ′ and the oxygenated aromatic carbon is at 158 . 00 ppm . the corresponding proton signals are a doublet for c - 2 ′ and - 6 ′ with an expected j - value of 7 . 8 hz for ortho splitting and a triplet of 8 . 7 hz for c - 3 ′ and - 5 ′ which can be attributed to the ortho and meta splitting . finally , kinkéloid d is the 3 ′, 4 ′, 5 ′, 5 , 7 - pentahydroxyflavan - 3 - ol kinkéloid . this was confirmed by the 13 c spectrum displaying one less aliphatic carbon at ˜ 20 ppm which was replaced with a carbon at ˜ 66 ppm in the oxygenated carbon region ; additionally , the aliphatic region of the 1 h spectrum was somewhat simplified , due to the oxygenation of c - 3 and subsequent reduced splitting . a singlet at 3 . 34 ppm corresponded to the oxygenated proton at c - 3 , this then adjusts the proton and carbon signals for c - 4 . all animal experiments were performed according to procedures approved by the rutgers institutional animal care and use committee . ten - week - old male c57bl / 6j mice were purchased from the jackson laboratory ( bar harbor , me .) and maintained on a high - fat diet containing 60 % fat - derived calories ( d12492 , research diets , new brunswick , n . j .) with 12 hr light and dark cycles . the animals were randomized into 4 groups of 10 animals each . the control group was gavaged daily with the vehicle solution alone ( 5 % dmso ) and three treatment groups were gavaged with 200 mg / kg of treatment of tpfa . plasma glucose levels were measured at week 0 and 4 in sub - mandibular vein blood samples using a glucometer ( accu - chek advantage , roche diagnostics , indianapolis , ind .). a 6 hr fast was necessary to allow blood glucose concentrations to arrive at the basal level , plasma glucose concentrations were measured immediately before and 3 and 6 hr following the treatments of metformin and tpfa . this fasting glucose level was repeated at week 6 for each group to measure the full effect of the kinkéliba treatment on basal glucose levels . at week 7 , an oral glucose tolerance test ( ogtt ) was performed . for ogtt , the mice were fasted overnight ( 16 hrs ) and gavaged with 2 g / kg glucose solution . glucose levels were measured using a glucometer at 0 , 40 , 80 , and 130 min after glucose administration . at the end of the study , mice were dosed with treatment solutions at four hours before they were euthanized . liver and visceral fats were removed and weighed . the adipose tissue weights were normalized to final body weight as g / 100 g body weight . a section of the liver was collected and stored at − 80 ° c . until used ; total rna was extracted from the livers using trizol reagent ( invitrogen ), following the manufacturer &# 39 ; s instructions . the rna was treated with dnasei ( invitrogen ), following the manufacturer &# 39 ; s guidelines , to remove any traces of dna contamination . the cdnas were synthesized with 2 . 5 μg of rna for each sample , using stratascript reverse transcriptase ( stratagene ), following the manufacturer &# 39 ; s protocol . the synthesized cdnas were treated as samples from the cell culture for quantitative pcr to determine the expression of pepck in the treated animals versus the control . for the results of the animal study , statistical analyses of the experimental observations , expressed as means ± sem , can be assumed to be one - way anova followed with a tukey &# 39 ; s multiple means comparison test , unless otherwise indicated . treatments were considered significantly different if p & lt ; 0 . 05 . the acute blood glucose levels were determined at weeks 0 and 4 ; there was a significant lowering of glucose levels by the sixth hour for the tpfk treated group compared to the control ( fig6 , left ). metformin is included here as a positive control and the data is illustrated as means ± sem , a two - way anova followed by bonferroni post test was performed with p & lt ; 0 . 05 indicated . at week 6 , the ambient glucose level was measured again and the results , over the 6 weeks , demonstrated the treatments were affecting the basal glucose levels . the control group showed an increase of fasting blood glucose by 11 . 21 % while the tpfa treated group all decreased 13 . 96 %. the actual data ( fig6 , right .) shows the control group starting at the lowest blood glucose but increasing throughout the experiment while the treatment group decreases from week 0 to week 6 indicating that the tpfa treatment group effectively lowers the basal glucose level , signifying an anti - diabetic effect . the oral glucose tolerance test was performed in week 7 of the experiment . the tpfa treatment group significantly improved glucose tolerance ( fig7 ) by lowering the peak , 40 min following the glucose challenge . additionally , the glucose levels at 80 min post glucose challenge were significantly lowered when compared to the control group , another indicator of improved glucose metabolism . the curves across 130 min experiment are the values as means ± sem , a two - way anova followed by bonferroni post test was performed with p & lt ; 0 . 05 and p & lt ; 0 . 001 indicated . the plasma insulin concentrations were measured for each animal across the treatment and control groups at the conclusion of the study . the plasma collection and testing resulted in nine viable samples per group after some samples hemolyzed and one animal died in week 6 . the tpfa treated group exhibits lower insulin levels than the control ( fig8 , right ). the liver tissue samples were tested for pepck gene expression to determine the actual affect of the treatment on hepatic glucose production ( 13 , 14 ); a decrease in this gene in the liver indicates antidiabetic activity . the result indicates that the tpfa treated group demonstrated significantly lowered levels of pepck ( fig8 ). these results indicate the ability of tpfa fraction to lower the enzyme that is necessary for the production of glucose in the liver . it will be evident to one skilled in the art that the present disclosure is not limited to the foregoing illustrative examples , and that it can be embodied in other specific forms without departing from the essential attributes thereof . it is therefore desired that the examples be considered in all respects as illustrative and not restrictive , reference being made to the appended claims , rather than to the foregoing examples , and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein . all references cited herein are incorporated by reference in their entirety herein . 1 . iwu , m . m ., handbook of african medicinal plants . 1993 , florida : crc press , inc . 464 . 2 . kerharo , j ., combrétacées , in la phannacopée sénégalaise tranditionnelle plantes médicinales et toxiques , j . g . adam , editor . 1974 , vigot fréres : paris . p . 341 - 360 . 3 . le grand , a ., les phytotherapes anti - infectieuses de la foret - savane , senegal ( afrique occidentale ) iii . un resume des substances phytochimiques et l &# 39 ; activite antimicrobienne de 43 species . journal of ethnopharmacology , 1989 . 25 : p . 315 - 338 . 4 . le grand , a . and p . a . wondergem , les phytotherapes anti - infectieuses de la foret - savane , senegal ( afrique occidentale ) i . un inventaire . journal of ethnopharmacology , 1987 . 21 : p . 109 - 125 . 5 . d &# 39 ; agostino , m ., et al ., flavonoids of combretum micranthum . fitoterapia , 1990 . 61 : p . 477 . 6 . tignokpa , m ., et al ., plantes medicinales populaires des marches de dakar ( senegal ). international journal of crude drug research , 1986 . 24 : p . 75 - 80 . 7 . olajide , o ., j . m . makinde , and d . t . okpako , evaluation of the anti - inflammatory property of the extract of combretum micranthum g . don . 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