RU2799555C1 - Method of isolation of new glycosides from pterocarpus marsupium and their therapeutic effects - Google Patents

Abstract

FIELD: pharmaceutical industry.

SUBSTANCE: invention relates to a composition for activating adenosine monophosphate-activated protein kinase (AMPK) and for inhibiting gluconeogenesis in mammalian cells. Composition for activating adenosine monophosphate-activated protein kinase (AMPK) and for inhibiting gluconeogenesis in mammalian cells containing at least 5 wt.% Pterocarpus marsupium extract, standardized to contain at least 0.5 wt.% pterocarposide (STR#1) and not less than 0.5 wt.% sabioside (STR#2), and the composition obtained by a method comprising the following steps: a) loading the powder of the Pterocarpus marsupium tree into the extractor, b) extracting with methanol to obtain an oleoresin, c) dissolving the oleoresin from step b) in water and extracting with toluene to obtain an aqueous layer and toluene layer, d) additional washing of the toluene layer from step c) with water to further obtain an aqueous fraction and a toluene fraction, e) mixing the aqueous fractions from step c) and step d) and spray drying to obtain an aqueous extract, f) fractionating the aqueous fraction from step e) with ethyl acetate to obtain an aqueous fraction and an ethyl acetate fraction, g) passing the ethyl acetate fraction from step f) through a solvent gradient column using 60–120 mesh silica gel to obtain enriched fraction 1 and fraction 2, h) passing enriched fraction 1 from step g) through an RP-18 isocratic silica gel column, then through an LH-20 column (Sephadex^®), and crystallization using a solvent at a temperature of -5 to 0°C to obtain a compound that is identified as the pterocarposide represented by STR#1, i) passing enriched fraction 2 from step g) through an RP-18 isocratic silica gel column, then through an LH-20 column (Sephadex^®) and crystallization using methanol at room temperature to obtain a compound that is identified as the sabioside represented by STR#2, j) loading the aqueous extract of Pterocarpus marsupium from step e) into the extractor, k) adding demineralized water to the extract, stirring for 3–4 hours at 65 to 70°C and leaving the solution for 8–10 hours to precipitate the insolubles, l) filtering the solution from step k) to remove the insolubles and obtain a clear filtrate, m) checking the insolubles from step l) for the presence of pterocarposide (STR#1) or sabioside (STR#2), discarding them if they are present in small amounts, n) collecting the filtrate from step l) and solvent extraction twice to obtain an aqueous layer and a solvent layer, o) concentrating the solvent layer from step n) recovering the solvent, p) extracting the aqueous layer from step n) with butanol three times and combining the butanol fractions, q) concentrating the butanol fractions and dissolving in water to standardize to a solution with a total dissolved solids content of 30%, r) spraying drying the solution from step q) obtaining compositions containing at least 5 wt.% Pterocarpus marsupium extract, standardized to contain at least 0.5 wt.% pterocarposide and not less than 0.5 wt.% sabioside represented by STR#1 and STR#2, respectively.

EFFECT: above composition is effective for activating adenosine monophosphate-activated protein kinase (AMPK) and for inhibiting gluconeogenesis in mammalian cells.

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Description

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The invention is a non-provisional application for U.S. Patent No. 62700446, filed July 19, 2018.

PRIOR ART

FIELD OF TECHNOLOGY

[001] The invention generally relates to active molecules from Pterocarpus marsupium. More specifically, the present invention relates to a method for isolating novel C-glycosides from Pterocarpus marsupium and their therapeutic effects.

DESCRIPTION OF THE PRIOR ART

[002] Pterocarpus marsupium is a deciduous tree native to parts of India, Nepal and Sri Lanka. It contains many flavonoids, glycosides, catechins, stilbenoids and tannins with therapeutic properties. Pterocarpus marsupium is reported to have a positive effect in the treatment of diarrhea, toothache, fever, urinary tract and skin infections. (S. S. Handa et al., Pterocarposide, an isoaurone C-glycoside from Pterocarpus marsupium, Tetrahedron Letters 41 (2000) 1579-1581). C-glycosides isolated from Pterocarpus marsupium are reported to have antihyperglycemic activity. However, most of the C-glycosides from this plant have yet to be identified to unlock its full therapeutic potential.

[003] Adenosine monophosphate-activated protein kinase (AMPK) has been known for many years as a central metabolic regulator for inhibiting energy-intensive pathways as well as activating compensatory energy production programs. The AMPK enzyme is activated when the energy status of the cell changes, such as muscle contraction and hypoxia. AMPK can be pharmacologically activated with the compound 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and the antidiabetic agent metformin. AMPK plays an important role in stimulating glucose uptake by the muscles through these physiological and pharmacological stimuli. AMPK activation during myocardial ischemia increases glucose uptake and glycolysis, and also increases fatty acid oxidation during reperfusion. The following articles cover the role of AMPK activation.

1. Ha J, Guan KL, Kim J, AMPK and autophagy in glucose/glycogen metabolism, Mol Aspects Med. Dec 2015; 46:46-62.

2. N. Musi and L. J. Goodyear, AMP-activated protein kinase and muscle glucose uptake, Acta Physiol Scand 2003, 178, 337-345.

3. Sambandam N, Lopaschuk GD, AMP-activated protein kinase (AMPK) control of fatty acid and glucose metabolism in the ischemic heart, Prog Lipid Res. May 2003; 42(3):238-56.

[004] Currently, AMPK is a therapeutic target for the treatment of metabolic disorders such as diabetes, obesity, etc. Inhibition of gluconeogenesis is also important in reducing the formation of ketone bodies in people with diabetes, which may otherwise be detrimental (Blackshear et al., The effects of inhibition of gluconeogenesis on ketogenesis in starved and diabetic rats, Biochemical Journal 1975, 148 (3): 353-362).

[005] Previous studies have successfully identified flavonoids and glycosides from Pterocarpus marsupium (Bezuidenhoudt et al., Flavonoid Analogues from Pterocarpus Species Phytochemistry. Vol. 26. No. 2 pp. 531-535. 1987), but failed to isolate some of the C -glycosides in their pure form in order to find out their biological activity. The present invention discloses a method for identifying new C-glycosides from Pterocarpus marsupium and their therapeutic effect.

[006] The main object of the invention is to disclose a method for isolating C-glycosides, which are pterocarposide (CAS number 264876-26-8) and sabioside (CAS number 108351-24-2) from Pterocarpus marsupium.

[007] Another object of the invention is to disclose a composition containing the C-glycosides pterocarposide (STR#1) and sabioside (STR#2) isolated from Pterocarpus marsupium and its therapeutic potential for AMPK activation and inhibition of gluconeogenesis.

[008] The present invention solves the above problems and provides associated benefits.

BRIEF DESCRIPTION OF THE INVENTION

[009] In a preferred embodiment, the present invention discloses a method for isolating pterocarposide (STR#1) and sabioside (STR#2) C-glycosides from Pterocarpus marsupium.

[0010] In a related embodiment, the invention discloses a composition containing at least 5 wt.% Pterocarpus marsupium extract standardized to contain at least 0.5 wt.% pterocarposide (STR#1) and at least 0.5 wt. % sabioside (STR#2).

[0011] In another preferred embodiment, the invention discloses a method for activating AMPK in mammalian cells, comprising the step of bringing the mammalian cells into contact with a composition containing at least 5 wt.% Pterocarpus marsupium extract, standardized to contain at least 0.5 wt. .% pterocarposide (STR#1) and not less than 0.5 wt.% sabioside (STR#2) to cause the effect of AMPK activation.

[0012] In another preferred embodiment, the invention discloses a method for inhibiting gluconeogenesis in mammalian cells, wherein said method comprises the steps of contacting the mammalian cells with a composition containing at least 5% by weight of a Pterocarpus marsupium extract standardized to contain at least 0 .5 wt.% pterocarposide (STR#1) and not less than 0.5 wt.% sabioside (STR#2) to cause the effect of reducing the formation of glucose.

[0013] Other features and advantages of the present invention will be apparent from the following more detailed description of the invention, taken in conjunction with the accompanying drawings illustrating, by way of example, the principle of the invention.

BRIEF DESCRIPTION OF GRAPHICS

[0014] FIG. 1 is a Western blot image showing AMPK activation in H4IIE cells using a pterocarposide composition.

[0015] FIG. 2 is a graphical representation showing the increase in pAMPK expression in HepG2 cells using a pterocarposide composition.

[0016] FIG. 3 is a graphical representation showing the reduction in glucose production in H4IIE cells using a pterocarpozoid composition.

DESCRIPTION OF THE MOST PREFERRED EMBODIMENTS FOR CARRYING OUT THE INVENTION

[0017] In a most preferred embodiment, the invention discloses a method for isolating C-glycosides from Pterocarpus marsupium, said method comprising the steps of:

a) loading the Pterocarpus marsupium tree powder into the extractor,

b) solvent extraction to obtain oleoresin,

c) dissolving the oleoresin from step b) in water and solvent extraction to obtain an aqueous layer and a solvent layer,

d) further washing the solvent layer from step c) with water to further obtain an aqueous fraction and a solvent fraction,

e) mixing the aqueous fractions from step c) and step d) and spray drying to obtain an aqueous extract,

f) fractionating the aqueous fraction from step e) with a solvent to obtain an aqueous fraction and a solvent fraction,

g) passing the solvent fraction from step f) through a solvent gradient column using 60-120 mesh silica gel to obtain enriched fraction 1 and fraction 2,

h) passing enriched fraction 1 from step g) through an RP-18 isocratic silica gel column, then through an LH-20 column (Sephadex ^® ), and crystallization using a solvent at a temperature of -5 to 0°C to obtain the compound, which is identified as pterocarposide presented by STR#1,

i) passing enriched fraction 2 from step g) through an isocratic RP-18 silica gel column, then through an LH-20 column ( ^Sephadex® ), and crystallization using a solvent at room temperature to obtain a compound which is identified as the sabioside represented by STR# 2.

[0018] In a related embodiment, the solvent is selected from the group consisting of, but not limited to, methanol, ethanol, butanol, ethyl acetate, chloroform, toluene, acetone, and hexane.

[0019] In another preferred embodiment, the invention discloses a composition containing at least 5 wt.% Pterocarpus marsupium extract, standardized to contain at least 0.5 wt.% pterocarposide (STR#1) and not less than 0.5 wt. .% sabioside (STR#2), where the specified composition is obtained by a method including the steps:

a) loading the Pterocarpus marsupium tree powder into the extractor,

b) solvent extraction to obtain oleoresin,

c) dissolving the oleoresin from step b) in water and solvent extraction to obtain an aqueous layer and a solvent layer,

d) further washing the solvent layer from step c) with water to further obtain an aqueous fraction and a solvent fraction,

e) mixing the aqueous fractions from step c) and step d) and spray drying to obtain an aqueous extract,

f) fractionating the aqueous fraction from step e) with a solvent to obtain an aqueous fraction and a solvent fraction,

g) passing the solvent fraction from step f) through a solvent gradient column using 60-120 mesh silica gel to obtain enriched fraction 1 and fraction 2,

h) passing enriched fraction 1 from step g) through an RP-18 isocratic silica gel column, then through an LH-20 column (Sephadex ^® ), and crystallization using a solvent at a temperature of -5 to 0°C to obtain the compound, which is identified as pterocarposide presented by STR#1,

i) passing enriched fraction 2 from step g) through an isocratic RP-18 silica gel column, then through an LH-20 column ( ^Sephadex® ), and crystallization using a solvent at room temperature to obtain a compound which is identified as the sabioside represented by STR# 2,

j) loading the aqueous extract of Pterocarpus marsupium from step e) into the extractor,

k) adding demineralized water to the extract and stirring for 3-4 hours at a temperature of 65 to 70°C and leaving the solution for 8-10 hours to precipitate insoluble substances,

l) filtering the solution from step k) to remove insoluble substances and obtain a clean filtrate,

m) checking insoluble substances from step l) for the presence of pterocarposide (STR#1) or sabioside (STR#2), discarding them if they are present in small quantities,

n) collecting the filtrate from step l) and solvent extraction twice to obtain an aqueous layer and a solvent layer,

o) concentrating the solvent layer from step n) to recover the solvent,

p) extracting the aqueous layer from step n) with solvent three times and combining the solvent fractions,

q) concentration of solvent fractions and dissolution in water for standardization to a solution with a total dissolved solids content of 30%,

r) spray-drying the solution from step q) to obtain a composition containing at least 5% by weight of Pterocarpus marsupium extract, standardized to contain at least 0.5% by weight of pterocarposide and at least 0.5% by weight of sabioside, represented by STR#1 and STR#2, respectively.

[0020] In a related embodiment, the solvent is selected from the group consisting of, but not limited to, methanol, ethanol, butanol, ethyl acetate, chloroform, toluene, acetone, and hexane.

[0021] In another preferred embodiment, the invention discloses a composition containing at least 5 wt.% Pterocarpus marsupium extract, standardized to contain at least 0.5 wt.% pterocarposide (STR#1) and not less than 0.5 wt. .% sabioside (STR#2).

[0022] In another preferred embodiment, the invention discloses a method for activating AMPK in mammalian cells, comprising the step of bringing the mammalian cells into contact with a composition containing at least 5 wt.% Pterocarpus marsupium extract, standardized to contain at least 0.5 wt. .% pterocarposide (STR#1) and not less than 0.5 wt.% sabioside (STR#2) to cause the effect of AMPK activation. In a related embodiment, the mammalian cells are human cells.

[0023] In another preferred embodiment, the invention discloses a composition containing at least 5 wt.% Pterocarpus marsupium extract, standardized to contain at least 0.5 wt.% pterocarposide (STR#1) and not less than 0.5 wt. .% sabioside (STR#2), for use in AMPK activation in mammalian cells.

[0024] In another preferred embodiment, the invention discloses a method for inhibiting gluconeogenesis in mammalian cells, wherein said method comprises the steps of contacting the mammalian cells with a composition containing at least 5% by weight of a Pterocarpus marsupium extract, standardized to contain at least 0 .5 wt.% pterocarposide (STR#1) and not less than 0.5 wt.% sabioside (STR#2) to cause the effect of reducing the formation of glucose. In a related embodiment, the mammalian cells are human cells.

[0025] In another preferred embodiment, the invention discloses a composition containing at least 5 wt.% Pterocarpus marsupium extract standardized to contain at least 0.5 wt.% pterocarposide (STR#1) and at least 0.5 wt. .% sabioside (STR#2), for use in the inhibition of gluconeogenesis in mammalian cells.

[0026] The following sections of this description consist of illustrative examples of the most preferred embodiments of the present invention.

[0027] EXAMPLES

[0028] Example 1: C-glycoside composition and method for its preparation

[0029] C-glycosides from Pterocarpus marsupium are isolated and identified using the following steps:

a) loading the Pterocarpus marsupium tree powder into the extractor,

b) extraction with methanol to obtain oleoresin,

c) dissolving the oleoresin from step b) in water and extracting with toluene to obtain an aqueous layer and a toluene layer,

d) further washing the toluene layer from step c) with water to further obtain an aqueous fraction and a toluene fraction,

e) mixing the aqueous fractions from step c) and step d) and spray drying to obtain an aqueous extract,

f) fractionating the aqueous fraction from step e) with ethyl acetate to obtain an aqueous fraction and an ethyl acetate fraction,

g) passing the ethyl acetate fraction from step f) through a solvent gradient column using 60-120 mesh silica gel to obtain enriched fraction 1 and fraction 2,

h) passing enriched fraction 1 from step g) through an isocratic RP-18 silica gel column, then through an LH-20 ( ^Sephadex® ) column and crystallization using methanol at -5 to 0°C to obtain a compound which is identified as pterocarposide represented by STR#1,

i) passing enriched fraction 2 from step g) through an RP-18 isocratic silica gel column, then through an LH-20 ( ^Sephadex® ) column and crystallization using acetone at room temperature to obtain a compound which is identified as the sabioside represented by STR#2 .

[0030] The stereochemistry of the isolated pterocarposide (STR#1) and sabioside (STR#2) is shown below:

[0031] Pterocarposide

CAS number 264876-26-8

Molecular formula: C ₂₁ H ₂₀ O ₉

Chemical name: (3E)-7-β-D-glucopyranosyl-6-hydroxy-3-[(4-hydroxyphenyl)methylene]-2(3H)-benzofuranone.

[0032] Sabioside

CAS number 108351-24-2

Molecular formula: C ₂₁ H ₂₀ O ₁₀

Chemical name: 8-β-D-glucopyranosyl-3,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one.

[0033] In addition, a water-soluble composition containing at least 5 wt.% P. marsupim extract was prepared and standardized to contain at least 0.5 wt.% pterocarposide (STR#1) and at least 0.5 wt.% sabioside (STR#2). The steps for obtaining the composition are given below:

a) loading the aqueous extract of Pterocarpus marsupium into the extractor,

b) adding demineralized water to the extract, and stirring for 3-4 hours at a temperature of 65 to 70°C, and leaving the solution for 8-10 hours to precipitate insoluble substances,

c) filtering the solution from step b) to remove insoluble substances and obtain a clean filtrate,

d) checking the insolubles from step c) for the presence of pterocarposide (STR#1) or sabioside (STR#2), discarding them if present in trace amounts,

e) collecting the filtrate from step c) and solvent extraction twice to obtain an aqueous layer and a solvent layer,

f) concentrating the solvent layer from step e) to recover the solvent,

g) extracting the aqueous layer from step e) with butanol three times and combining the butanol fractions,

h) concentration of butanol fractions and dissolution in water for standardization to a solution with a total dissolved solids content of 30%,

i) spray drying the solution from step h) to obtain a composition containing at least 5 wt.% P. marsupim extract containing pterocarposide and sabioside represented by STR#1 and STR#2, respectively.

[0034] Example 2: Activation of AMPK

[0035] Conducted several experiments on cells of rat hepatoma H4IIE and human hepatoma cells HepG2. Confluent plates of H4IIE or HepG2 cells were treated with a formulation containing pterocarposide (STR#1) and sabioside (STR#2) (pterocarposide based formulation) or a positive control, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), at the following doses.

[0036] Doses of the pterocarposide composition: 0.05, 0.1, 0.5 and 1 μM.

[0037] AICAR (positive control): 2 mM.

[0038] Cells were lysed and proteins were separated on 4-20% SDS-PAGE gel and transferred to nitrocellulose. AMPK activation was detected by Western blotting with pAMPK (Thrl72) and pACC (Ser79). AMPK and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were used as controls.

[0039] Results: The composition containing pterocarposide (STR#1) and sabioside (STR#2) dose-dependently increased the phosphorylation status of AMPK (Thr172) with maximum phosphorylation observed between concentrations of 0.1-0.5 μM (Fig. 1) . The composition also dose-dependently increased acetyl-CoA carboxylase (ACC) phosphorylation. This is the first demonstration that a composition containing pterocarposide (STR#1) and sabioside (STR#2) activates AMPK. Increased ACC phosphorylation may indicate that the composition may inhibit fatty acid synthesis and potentially promote fatty acid oxidation. Similar results were also observed in HepG2 cells (FIG. 2).

[0040] Example 2: Inhibition of Glucose Formation

[0041] Confluent dishes with H4IIE were treated with 0.1 μm or 0.5 μm of a composition containing pterocarposide (STR#1) and sabioside (STR#2) to study its effect on dexamethasone-induced glucose formation. H4IIE cells were treated with 500 nM dexamethasone and 0.1 mM 8-CTP-cAMP (Dex/cAMP), various concentrations of a pterocarposide formulation, or 5 nM insulin in glucose formation buffer (DMEM (Dulbecco's Modified Eagle's Medium) without glucose , pH 7.4, containing 20 mM sodium lactate and 2 mM sodium pyruvate, no phenol red) for 5 hours.

[0042] Cells were washed with Dulbecco's PBS (Dulbecco's phosphate-buffered saline) and then incubated for 3 hours in glucose formation buffer with the same concentrations of Dex/cAMP, insulin and pterocarposide composition. Glucose production was assessed by measuring the glucose concentration in the medium, as described in Wang et al (2000) with modifications, using a glucose assay kit (HK) (Sigma Chemicals).

[0043] Results: Treatment with the pterocarposide formulation (0.1 and 0.5 μM) inhibited dexamethasone-induced glucose production in H4IIE cells to the same extent as insulin (100 nM). The results are presented as mg of formed glucose ± SEM (Fig. 3). Corrections for cell number were made based on protein concentration, which was estimated using the Bradford Protein Assay Reagent from Bio-Rad (Bio-Rad, Hercules, Calif.). (Wang, J. C., Stafford, J. M., Scott, D. K., Sutherland, C., Granner, D. K. (2000). The molecular physiology of hepatic nuclear factor 3 in the regulation of gluconeogenesis. The Journal of Biological Chemistry 275: 14717-14721) .

[0044] Although the invention has been described with reference to the preferred embodiment, it should be apparent to those skilled in the art that the invention is not limited thereto. On the contrary, the scope of the invention is to be interpreted only in conjunction with the appended claims.

Claims (23)

1. Composition for activation of adenosine monophosphate-activated protein kinase (AMPK) and inhibition of gluconeogenesis in mammalian cells, containing at least 5 wt. % Pterocarpus marsupium extract, standardized to contain at least 0.5 wt. % pterocarposide (STR#1) and not less than 0.5 wt. % Sabioside (STR#2):

moreover, the composition is obtained by a method comprising the steps: a) loading the Pterocarpus marsupium tree powder into the extractor, b) extraction with methanol to obtain oleoresin, c) dissolving the oleoresin from step b) in water and extracting with toluene to obtain an aqueous layer and a toluene layer, d) further washing the toluene layer from step c) with water to further obtain an aqueous fraction and a toluene fraction, e) mixing the aqueous fractions from step c) and step d) and spray drying to obtain an aqueous extract, f) fractionating the aqueous fraction from step e) with ethyl acetate to obtain an aqueous fraction and an ethyl acetate fraction, g) passing the ethyl acetate fraction from step f) through a solvent gradient column using 60-120 mesh silica gel to obtain enriched fraction 1 and fraction 2, h) passing enriched fraction 1 from step g) through an isocratic RP-18 silica gel column, then through an LH-20 ( ^Sephadex® ) column and crystallization using a solvent at -5 to 0°C to obtain a compound which is identified as pterocarposide represented by STR#1, i) passing enriched fraction 2 from step g) through an isocratic RP-18 silica gel column, then through an LH-20 ( ^Sephadex® ) column and crystallization using methanol at room temperature to obtain a compound which is identified as the sabioside represented by STR#2 , j) loading the aqueous extract of Pterocarpus marsupium from step e) into the extractor, k) adding demineralized water to the extract, stirring for 3-4 hours at a temperature of 65 to 70°C and leaving the solution for 8-10 hours to precipitate insoluble substances, l) filtering the solution from step k) to remove insoluble substances and obtain a clean filtrate, m) checking insoluble substances from step l) for the presence of pterocarposide (STR#1) or sabioside (STR#2), discarding them if they are present in small quantities, n) collecting the filtrate from step l) and solvent extraction twice to obtain an aqueous layer and a solvent layer, o) concentrating the solvent layer from step n) to recover the solvent, p) extracting the aqueous layer from step n) with butanol three times and combining the butanol fractions, q) concentration of butanol fractions and dissolution in water for standardization to a solution with a total dissolved solids content of 30%, r) spray drying the solution from stage q) to obtain a composition containing at least 5 wt. % Pterocarpus marsupium extract, standardized to contain at least 0.5 wt. % pterocarposide and not less than 0.5 wt. % sabioside represented by STR#1 and STR#2, respectively. 2. The composition of claim 1 wherein the solvent is selected from the group consisting of methanol, ethanol, butanol, ethyl acetate, chloroform, toluene, acetone and hexane. 3. The composition of claim. 1, where the mammalian cells are human cells.

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