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Title:
PROCESS FOR THE PREPARATION OF STANDARDIZED COMPOSITION OF ARJUNOGLUCOSIDE FROM THE BARK OF TERMINALIA ARJUNA
Document Type and Number:
WIPO Patent Application WO/2018/237135
Kind Code:
A2
Abstract:
Disclosed is a novel process for the isolation of bioactive compounds from Terminalia arjuna. More specifically, the invention discloses a process for isolation and enrichment of bioactive compounds Arjunic acid, Arjunolic acid, Arjungenin, Arjunetin, Arjunoglucoside-I, Arjunoglucoside-II, and Catechin from the bark of Terminalia arjuna. The invention also discloses a composition standardized to contain 3% arjunoglucosides isolated from the bark of Terminalia arjuna.

Inventors:
MAJEED MUHAMMED (US)
NAGABHUSHANAM KALYANAM (US)
BHAT BEENA (IN)
Application Number:
PCT/US2018/038738
Publication Date:
December 27, 2018
Filing Date:
June 21, 2018
Export Citation:
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Assignee:
MAJEED MUHAMMED (US)
NAGABHUSHANAM KALYANAM (US)
BHAT BEENA (IN)
International Classes:
A61K39/12; C12N7/00
Attorney, Agent or Firm:
NAGABHUSHANAM, Kalyanam (US)
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Claims:
We claim,

I . A process for the isolation of standardized composition of arjunoglucoside from bark of Terminalia arjuna, said process comprising steps of:

(a) Charging Terminal ia arjuna bark powder into an extractor,

(b) Adding 3 volumes of ethanol or methanol to Termiitalia arjuna bark powder and reflux ing for 3 hours at 65 to 70 °C,

(c) Filtering the ethanol or methanol extract of step b) and concentrating under vacuum to get a brown coloured powder.

(d) Dissolving the powder obtained from step c) into 5 volumes of de-mineralize water and stirring thoroughly for 1 hr at 50-55°C to obtain a solution,

(e) Transferring the solution from step d) to separating funnel and extracting with 2 volumes of solvent, 6 times, and separating the aqueous and organic layer,

(f) Collecting and combining the solvent fractions 1 -6 from step e and concentrating under vacuum to obtain brown coloured powder,

(g) Loading the powder of step f) into a silica gel and e!uting with chloroform: methanol,

(h) Identifying the compounds from step g) as Arjimic acid represented by STR#1 , Arjunolic acid represented by STR#2, Aijungenin represented by STR#3, Arjunoglucoside-I represented by STR?#4, Arjunoglucoside-II represented by STR#5, Arjunetin represented by STR#6, and catechin represented by STR#7.

The process as in claim 1 . wherein the solvent of step e) is selected from the group consisting of acetic acid, acetone, acetonitrile, benzene, carbon disulfide, carbon tetrachloride, chloroform, cyclohexane, 1 ,2-dichJoroethane, dichloromethane, diethyl ether, 1 ,2-dimethoxyethane, dimethylsulfoxide, 1 ,4-dioxane, ethanoi, ethyl acetate, hexane, isopropanol, methanol, methyl ethyl ketone, N.N-dimethylformamide, nitromethane, n-Propanol, pentane, pyridine, tetrahydrofuran, toluene, water or combinations thereof.

3. Tlie method as in claim 1 , wherein the solvent of step e) is preferably ethyl acetate.

4. A process for enriching arjunoglucoside II, said process comprising steps of:

(a) Charging TermhiaUa arjuna bark powder into an extractor,

(b) Adding 3 volumes of ethanol or methanol to Termhialki. arjuna bark powder and refluxing for 3 hours at 65 to 70 °C,

(c) Filtering the ethanol or methanol extract of step b) and concentrating under vacuum to get a brown coloured powder, Assay of arjunoglucoside-II by HPLC = 0.55% w/w

(d) Dissolving the powder obtained from step c) into 5 volumes of de-mineralize water and stirring thoroughly for 1 In at 50-55°C to obtain a solution,

(e) Transferring the solution from step d) to separating funnel and extracting with 2 volumes of solvent, 6 times, and separating the aqueous and organic layer,

(f) Collecting and combining the solvent fractions 1 -6 from step e and concentrating under vacuum to obtain brown coloured powder. Assay of arjunoglucoside-II by HPLC = 20- 21 % w/w.

5. The process as in claim 4, wherein the solvent of step e) is selected from the group consisting of acetic acid, acetone, acetonitrile, benzene, carbon disulfide, carbon tetrachloride, chloroform, cyclohexane, 1 ,2-diehloroethane, dichloromethane, diethyl ether, 1,2-dimethoxyethane, dimethylsuifoxide, 1 ,4-dioxane, ethanol, ethyl acetate, hexane, isopropanol, methanol, methylethyl ketone. N , N -di methyl formam ide, nitromethane, n-Propanol, pentane, pyridine, tetrahydrofuran, toluene, water or combinations thereof.

6. The method as in claim 4, w herein the solvent of step e) is preferably ethyl acetate.

7. A composition comprising atleast 3% arjuuoglucosides wherein said composition comprises Arjunic acid represented by STR^l , Arjunolic acid represented by STR#2, Arjungenin represented by STR#3, Arjunoglucoside-I represented by STR#4, Arjunoglucoside-II represented by STR#5, Arjunetin represented by STR#6, and catechin represented by STR#7.

Description:
PROCESS FOR THE PREPARATION OF STANDARDIZED COMPOSITION OF ARJIJNOGLUCOSIDE FROM THE BARK OF TermmaUa arjuna

CROSS-REFERENCE TO RELATED APPLICATIONS

This is PCT filing of US provisional application no. 62522801 , filed on 21 June 201 7.

BACKGROUND OF THE INVENTION

Field of the invention

[Para001] The present invention in general relates to bioactive compounds from TermmaUa arjuna. More specifically the present invention discloses a novel process for the preparation of standardized composition of arjunogiucoside from bark of TermmaUa arjuna. The standardized composition disclosed in the present invention includes hitherto undisclosed molecules that contribute to the biological effects of said composition.

Description of prior art

[Para002] TermmaUa arjuna or arjuna is a well known medicinal plant used in the ancient Ayurvedic medicine. The bark of TermmaUa arjuna reported to contain many bioactive compounds, which can be tapped for use in the treatment of many diseases. The following prior art describe the important biological effects of Terminalia arjuna.

1 . Pawar & Bhutani, Effect of oieanane triterpenoids from Terminalia arjima-a. cardioprotective ding on the process of respiratory oxyburst, Phytomedicine, 2005;25:391 -393

2. Sandhu et al., Effects of Withania somnifera (Ashwagandha) and TermmaUa arjuna (Arjuna) on physical performance and cardiorespiratory endurance in healthy young adults, int J Ayurveda Res, 2030; 3 (3): 144-149.

3. Zafar et al., Terminalia arjuna: Alternative Treatment for Cardiovascular Diseases, hit. J.

Pharm. Sci. Rev. Res., 2015;35(2): 52-56.

4. Pate! et al.. Development and Validation of Stability Indicating Assay for Arjuna Caplets and Stabi lity Studies with Concurrent Phytochemical investigations, Inventi Rapid; Pianta Activa, 2015; 1 : 1 -5. 5. Chandra Sekhar et al., Terminal ia arjima bark extract attenuates picrotoxin-induced behavioral changes by activation of serotonergic, dopaminergic, GABAergic and antioxidant systems, Chinese Journal of Natural Medicines, 2017, 15(8): 584-596

[Para003] Arjunoglucoside, a compound obtained from bark of Termina!ia arjima is well documented in !iteratures for its cardio-protective activity. Other than its cardioprotective property, TerminaUa arjima and its bark extract reported for wide range of activities such as anticancer, hepatoprotective, antiviral, antioxidant, anti -asthmatic, anti-fertility, anti-diabetic, wound healing, anti-platelet and anticoagulant, anti-bacterial and anti-fungal activity. (Saxena et al.. Cytotoxic agents from TerminaUa arjima, Planta Med. 2007:73( 14): 1486-90).

[Para004] There are many processes disclosed in the literature for the isolation of Arjunoglucoside and its derivatives from bark of TerminaUa arjima and other species of TerminaUa. Anbalangan et al. (A. Anbaiagan, Phytochemical and pharmacological studies on TerminaUa arjima (Roxb.) weight & Arnot and Borreria Hispida (Linn. ) K. Schum, Ph. D

on 13 June 201 8) reported a methanolic extract of stem bark of TerminaUa arjima chxomatographed over silica gei benzene and eluted with solvents of increasing polarity viz., benzene, chloroform and methanol. Total 6 compounds were identified from methanolic extract of stem bark of TerminaUa arjima such as 3-oxo-olean-l 2-ene-28-oic acid, Methyl maslinate, Hederagenin Methyl ester, Hederagenin, Maslinic acid and Arjimolic acid. The other processes for the isolation of arjunoglucosides are described in the following prior ail documents:

1. Tsuyuki et al., A new triterpene compound from TerminaUa arjima. Arjunoglucoside III .

Bulletin of the chemical society of Japan, 1979; 52( 10); 3127-3128

2. Honda et al., Arjimgenin, Arjunglucoside 1, and Arjunglucoside II. A New Triterpene and New Triterpene Giucosides from TerminaUa arjima. Bulletin of the Chemical Society of Japan, 1976;49( 1 1 ):3213-3218.

3. Permit et al., A New Aromatic Compound from the Stem Bark of Tenninalia catappa, Nat Prod Commun. 2015; 10(6): 1005-1007.

[Para005] Though the isolation of arjunoglucoside and its derivatives are already reported in the literature, inventors fust time reported novel process for the preparation of standardized composition of arjimoglucoside from bark of Terminaiia arjuna. The novel process for isolation aijunoglucoside and its derivatives is both economically viable and industrially scalable.

[Para006] The principle objective of the invention is to disclose a novel process for the isolation and preparation of standardized composition of ai junoglucoside from bark of Terminaiia arjwia.

[Para007] It is another objective of invention to disclose the novel process for isolation and enrichment of arjunoglucosides.

J Para008| It is another objective of invention to disclose a composition standardized to contain 3% arjunoglucosides.

[Para009] The present invention fulfils the aforesaid objectives and provides further related advantages.

SUMMARY OF THE INVENTION

[Para010] The present invention pertains to a novel process for the isolation of bioactive compounds from Terminaiia arjwia. More specifically, the invention discloses a process for isolation and enrichment of bioactive compounds Arjunic acid, Arjimolic acid, Arjungenin, Arjimetin, Arjunogiucoside-I, Arjunoglucoside-Il, Catechin and Gallocateehin from the bark of Terminaiia arjwia. The invention also discloses a composition standardized to contain 3% arjunoglucosides isolated from the bark of Terminaiia arjuna.

BRIEF DESCRIPTION OF THE DRAWINGS

[Para011] Fig. la shows the proton NMR spectrum for the identification of Arjunic acid isolated from the bark of Terminaiia arjuna.

[Para012] Fig. lb shows the carbon NMR spectrum for the identification of Arjunic acid isolated from the bark of Terminaiia arjuna.

[Para013] Fig, 2a shows the proton NMR spectrum for the identification of Arjunoiic acid isolated from the bark of Tenninalia arjwia. [Para014] Fig, 2b shows the carbon NMR spectrum for the identification of Arjunoiic acid isolated from the bark of Termmalia arjuna.

[Para015] Fig, 3a shows the proton NMR spectrum for the identification of Arjungenin isolated from the bark of Termmalia arjuna.

[Para016] Fig. 3b shows the carbon NMR spectrum for the identification of Arjungenin isolated from the bark of Terminaiia arjuna.

[Para017] Fig. 4a shows the proton NMR spectrum for the identification of Arjunoghicoside-1 isolated from the bark of Terminaiia arjuna.

[Para018] Fig, 4b shows the carbon NMR spectrum for the identification of Arjunogliicoside-I isolated from the bark of Termina!ia arjuna.

[Para019] Fig. 5a shows the proton NMR spectrum for the identification of Arjunoghicoside-II isolated from the bark of Terminal la arjuna.

[Para020] Fig. 5b shows the carbon NMR spectrum for the identification of Aijiinogiucoside-I ! isolated from the bark of Termmalia arjuna.

[Para021] Fig. 6a shows the HPLC chromotogram for the identification of Arjunogiucoside-II methanoiic extract isolated from the bark of Terminaiia arjuna.

[Para022] Fig. 6b shows the HPLC chromotogram for the identification of enriched Arjimoglucoside-II isolated from the bark of Termmalia arjwia.

[Para023] Fig. 7a shows the proton NMR spectrum for the identification of Arjunetin isolated from the bark of Terminaiia arjuna.

[Para024] Fig. 7b shows the carbon NMR spectaim for the identification of Arjunetin isolated from the bark of Terminalki arjwia.

[Para025] Fig. 8a shows the proton NMR spectrum for the identification of catechin isolated from the bark of Terminaiia arjuna. [Para014] Fig. 8b shows the carbon NMR spectrum for the identification of catechiii isolated from the bark of Terminalia arjuna.

DESCRIPTION OF MOST PREFFERED EMBODIMENTS

[Para014] In the most preferred embodiment, the invention discloses a novel process for the isolation of standardized composition of arjunogiucoside from bark of Terminalia arjuna, said process comprising steps of:

(a) Charging Terminalia arjuna bark powder into an extractor,

(b) Adding 3 volumes of ethanoi or methanol to Terminal ia arjuna bark powder and reflux ing for 3 hours at 65 to 70 °C,

(c) Filtering the ethanoi or methanol extract of step b) and concentrating under vacuum to get a brown coloured powder,

(d) Dissolving the powder obtained from step c) into 5 volumes of de-mineralize water and stirring thoroughly for 1 hr at 50-55ºC to obtain a solution..

(e) Transferring the solution from step d) to separating funnel and extracting with 2 volumes of solvent, 6 times, and separating the aqueous and organic layer,

(f) Collecting and combining the solvent fractions 1 -6 from step e and concentrating under vacuum to obtain brown coloured powder,

(g) Loading the powder of step f) into a silica gel and eluting with chloroform; methanol,

(h) Identifying the compounds from step g) as Arjuiiic acid represented by STR#1, Arjunolic acid represented by STR#2, Arjungenin represented by STR#3, Arjunoglucoside-I represented by STRM, Arjunoglucoside-II represented by STR#5, Arjunetin represented by STR#6, and catechin represented by STR#7.

[Para014] In a related embodiment, the solvent of step e) is selected from the group consisting of acetic acid, acetone, acetonitrile, benzene, carbon disulfide, carbon tetrachloride, chloroform, cyclohexane, 1,2-dichioroethane, dichloronie thane, diethyl ether, 1,2-dimethoxyethane, dimethy Isulfoxide, 1 ,4-dioxane, ethanol, ethyl acetate, hexane, isopropanol, methanol, methylethyl ketone, N,N-dimethylformamide, nitromethane, n-Propanol, pentane, pyridine, tetrahydrofuran, toluene, water or combinations thereof. In another related embodiment, the solvent of step e ) is preferably ethyl acetate.

[Para029] In another preferred embodiment, the invention discloses a process for enriching arjunoglucoside II, said process comprising steps of:

(a) Charging Termhialia arjitna bark powder into an extractor,

(b) Adding 3 volumes of ethanol or methanol to Termhialia arjwia bark powder and reflux ing for 3 hours at 65 to 70 °C,

(c) Filtering the ethanol or methanol extract of step b) and concentrating under vacuum to get a brown coloured powder. Assay of arjunoglucoside-II by HPLC = 0.55% w/w

(d) Dissolving the pow der obtained from step c) into 5 volumes of de-mineralize water and stirring thoroughly for 1 hr at 50-55°C to obtain a solution,

(e) Transferring the solution from step d) to separating funnel and extracting with 2 volumes of solvent, 6 times, and separating the aqueous and organic layer,

(f) Collecting and combining the solvent fractions 1 -6 from step e and concentrating under vacuum to obtain brown coloured powder, Assay of arjunoglucoside-II by HPLC = 20- 2 1 % w/w.

[Para030] In another preferred embodiment, the invention discloses a composition comprising atleast 3% arjunoglucosides wherein said composition comprises Arjunic acid represented by STR#I , Arjunolic acid represented by STR#2, Arjungeain represented by STR#3, Arjunoglucoside-I represented by STR#4, Arjunoglucoside-II represented by STR#5, Arjunetin represented by STR#6, and catechin represented by STR#7. [Para031] The specific examples included herein below illustrate the aforesaid most preferred embodiments of the present invention.

[Para032] Example 1 : Process for isolating arjimoglueosides from the bark of Terminalia arjuna

[Para033] The present invention discloses a process for isolating arjimoglueosides from Terminalia arjuna. The process contains the fol lowing step of charging 2 kg of Terminalia arjuna bark powder into an extractor. Then added 3 volumes of methanol to Terminal hi arjuna bark powder and re fluxed for 3 hours at 65 to 70 °C. The solution was then filtered and concentrated under vacuum to get a brown coloured powder, Assay of arjunoglucoside-II by HPLC = 0.55% w/w. Yield approx . 500 g (25%). The powder was then dissolved into 5 volumes of de-mineralize water and stirred thoroughly for 1 hr at 50-55°C. The solution was then transferred to separating funnel and extracted with 2 volumes of ethyl acetate, 6 times, and the aqueous and organic layers were separated. The 6 ethyl acetate fractions were collected and concentrated under vacuum to obtain brown coloured powder, assay of arjunoglucoside-II by HPLC = 20-2 1 % w/w, yield approx . 40 g. The powder was then loaded into a si lica gel and eluted with chloroform: methanol. This fraction contains Arjimic acid, Arjunolic acid, Arjungenin, Arjunoglucoside-I & II , Arjunetin and catechin. The compounds were then identified using NMR as Arjimic acid ( Fig la and lb), Arjunolic acid (Fig 2a and 2b), Arjungenin (Fig. 3a and 3b), Arjunoglucoside-I (Fig. 4a and 4b), Arjunoglucoside-II (Fig. 5a. 5b, 6a and 6b), Arjunetin (Fig. 7a and 7b), and catechin (Fig. 8a and 8b).

[Para034] A composition containing atleast 3% arjimoglueosides comprising Arjimic acid, Arjunolic acid, Arjungenin, Arjunoglucoside-I, Arjunoglucoside-II, Arjunetin, and catechin was also formulated.

[Para035] While the invention has been described with reference to a preferred embodiment, it is to be clearly understood by those skilled in the art that the invention is not limited thereto. Rather, the scope of the invention is to be interpreted only in conjunction with the appended claims.