A METHOD OF ISOLATION OF ARJUNETIN FROM TERMINALIA ARJUNA BARK

Abstract

The present invention provides a method of isolating arjunetin from the bark of Terminalia arjuna. The invention provides a simplified, cost-effective method and scalable method for isolation of arjunetin from the Terminalia bark. The present invention encompasses an ethanolic extract of arjunetin that comprises arjunetin with 95% purity. The ethanolic extract of arjunetin is obtained from solvent extraction without using column chromatography. Arjunetin has various therapeutic applications, anti-diabetic, anti-inflammatory, anti-cancer, and anti-viral drugs.

Specification

Description:FIELD OF THE INVENTION
The present invention is related to a method of isolating arjunetin from the bark of Terminalia arjuna. The invention provides a simplified, cost effective method for isolation of Arjunetin from the Terminalia arjuna bark. Arjunetin has various therapeutic applications including for anti-diabetic, anti-inflammatory, anti-cancer, and anti-viral drugs.

BACKGROUND OF THE INVENTION
Medicinal plants are a promising source of new drugs to tackle this problem. Terminalia Arjuna (TA) (Family: Combretaceae), a tree native to the Indian subcontinent, is extensively used in many traditional forms of medicine for the treatment of hypertension and coronary heart diseases. It is known to confer cardioprotective properties such as strengthening heart muscles and improving the functioning of cardiac muscles, thereby alleviating heart failure, angina and hypercholesterolemia. Stem and bark of this plant exhibit various other therapeutic properties such as expectorant, anti-dysenteric, purgative, laxative and have also been used to treat leucoderma, anemia, hyperhidrosis, asthma, and tumors. In addition, it has also been reported that the bark of T. arjuna also possesses good anticancer, antiviral, and antimicrobial activities .It has been hypothesized that the cardioprotective activity of TA bark extracts is likely by the activation of endogenous antioxidant molecules by triterpenes, namely arjunolic acid. Plant-derived phytochemicals can offer a new direction in developing methods for disease control. The major advantage of these traditional systems of medicine such as Ayurveda, Siddha, or Unani is that, they depend on natively available flora and has been in use for treatment of almost all kinds of maladies for centuries. Stem and bark of the tree Terminalia arjuna Wight & Arn. (Combretaceae) has been documented to exhibit therapeutic properties like cardiotonic, anticancer, antiviral, antibacterial, antifungal, hypercholesterolemia, hypolipidemic, and anti-coagulant.
Terminalia arjuna, also known as arjuna, is a renowned medicinal plant that has been utilized in ancient Ayurvedic medicine. The bark of Terminalia arjuna is known to contain numerous bioactive compounds (more than 45 compounds) that hold potential for treating a variety of ailments. These compounds can be harnessed for their therapeutic properties in the management of various diseases. The isolation of individual pure compounds from complex crude bark extracts poses a significant challenge.
The isolation method for obtaining the active ingredients of a plant is a crucial process in the field of botanical research. Numerous methods have been documented in the literature for extracting bioactive compounds from the bark of Terminalia arjuna.
The isolation of pure single compounds from plant extracts containing multiple compounds without column chromatography is a highly challenging process. The present invention has described the isolation of pure compounds without the time-consuming use of column chromatography.
The existing methods involve the use of time-consuming column chromatography for the purification of compounds. These methods involve preparations of various Terminalia Arjuna solvent extracts and their corresponding enzyme inhibitory units. However, there are currently no reports available on the isolation of a pure compound from these extracts in bulk scale methods.
The present invention provides a method to obtain a pure single compound without the need for column chromatography. This breakthrough eliminates the laborious and time-consuming process typically used to isolate arjunetin from the bark of T. arjuna. The method yields arjunetin with high purity and provides easy scalability for industrial production.

SUMMARY
One aspect of the invention is a method of isolating arjunetin with high purity from the bark of Terminalia arjuna, the method comprising the steps of:
a) Drying and grinding the bark of Terminalia arjuna to obtain a bark powder ;
b) Subjecting the bark powder in step a. to a first solvent extraction to yield a first fraction and bark powder remaining after first solvent extraction;
c) Subjecting the bark powder in step b. to a second solvent extraction to yield a second fraction and bark powder remaining after second solvent extraction;
d) Subjecting the bark powder in step c. to a third solvent extraction to yield a third fraction and wherein the third solvent is ethanol;
e) Subjecting the third fraction in step d. to filtration followed by vacuum drying to yield a crude ethanol extract; and
f) Isolating arjunetin compound from the crude ethanol extract powder in step e.
In one aspect, the first solvent is hexane or petroleum ether. In one aspect, the second solvent is chloroform, ethyl acetate or dichloromethane. In one aspect, the order of polarity increases from the first to the third solvent.
In one aspect, the first, second and third solvent extraction is done at a temperature of 60-70°C.
In one aspect, the solvent extraction with step b. and c. is done for 65-80 hours at a temperature of 60-70 °C.
In one aspect, the extraction with the third solvent is done for 65-80 hours at a temperature of 60-70 °C.
In one aspect the crude ethanol extract obtained in step e. comprises 95% pure arjunetin. (wt/wt)
In one aspect, the arjunetin isolated in step f. has a yield of 10%.In one aspect, the crude ethanol extract obtained in step e. is further subjected to column chromatography to yield arjunetin with 99% purity.
In one aspect, the column chromatography is done in a single step. In one aspect, the amount of bark powder in first, second and third solvent extraction is in the range of 0.3-0.7 kg/L.
One aspect of the invention is an ethanol extract of arjunetin wherein the amount of arjun tin is 95%.
BRIEF DESCRIPTION OF FIGURES:
Figure 1. shows Structure of Arjunetin
Figure 2. shows Schematic representation of extraction of various fractions from bark powder of T.arjuna.
Figure 3. shows Schematic representation of extraction of crude ethanol fractions from bark powder of T.arjuna.
Figure 4. shows Schematic representation of isolation arjunetin from ethanol extract of bark powder of T.arjuna.
Figure 5. shows RP-HPLC spectrum of arjunetin
Figure 6. shows UV-Visible spectrum of arjunetin in methanol
Figure 7. shows IR spectrum of arjunetin
Figure 8. shows 1H NMR spectrum of arjunetin (DMSO-d6)
Figure 9. shows 13C NMR spectrum of arjunetin (DMSO-d6)
Figure 10. shows ESI-MS mass spectrum of arjunetin isolated from T. arjuna bark ethanol extract
Figure 11. shows ESI-MS mass spectral fragmentation analysis of arjunetin isolated from T. arjuna bark ethanol extract
DETAILED DESCRIPTION OF THE INVENTION
In the following detailed description, a reference is made to the accompanying drawings that form a part hereof, and in which the specific embodiments that may be practiced is shown by way of illustration. These embodiments are described in sufficient detail to enable those skilled in the art to practice the embodiments and it is to be understood that other changes may be made without departing from the scope of the embodiments. The following detailed description is therefore not to be taken in a limiting sense.
The present invention encompasses a method of isolating arjunetin from the bark of Terminalia arjuna in a cost effective, efficient manner, economically feasible and scalable manner.
The methods described in the prior art for isolation of active compounds including arjunetin from Terminalia suffer from a number of disadvantages. The biggest disadvantage is the low yield of arjunetin (0.002%). A significant disadvantage of the prior methods utilization of a tedious column chromatographic method for separation of arjunetin. The existing methods entailed the sequential extraction of powdered Terminalia arjuna bark using hexane, chloroform, and ethanol in a chemical extractor.
The present invention utilizes lesser chemicals, and steps thereby enhancing the efficiency and scalability of arjunetin isolation.
This present invention plays a vital role in unlocking the therapeutic potential of plants and is essential for the development of new and innovative products in the natural health industry. By utilizing advanced extraction techniques, researchers are able to efficiently extract and concentrate these valuable compounds, ensuring their potency and efficacy.
ADVANTAGES OF THE INVENTION

• Preparation of pure single compound arjunetin from the bark of Terminalia arjuna with high yield in a scalable and economically feasible method.
• High purity of (95% pure) of arjunetin without the use of a column chromatography, solely utilizing a chemical reactor.
• Efficient, time-saving, and cost-effective approach for arjunetin isolation
• Isolating Arjunetin and its derivatives from the bark of Terminalia arjuna, with potential applications as anti-diabetic, anti-inflammatory, anti-cancer, and anti-viral drugs.
• Anti-diabetic activity of Arjunetin isolated from ethanol extract: Arjunetin at concentrations ranging from 0.730 to 2.289 µg/ml showed dose-dependent stimulation of glucose uptake.
• Anti-inflammatory activity of arjunetin isolated from ethanol extract: Arjunetin at concentrations ranging from 0.730 to 2.289 µg/ml showed dose-dependent reduction of NO production compared with control.
• Anti-HIV activity of arjunetin isolated from ethanol extract: IC50 3.673 µg/ml
• Anti-cancer activity of Arjunetin isolated from ethanol extract: The IC50 value of Arjunetin on MCF-7 cell line was found to be 10.807 µg/ml

DEFINITIONS:

As used herewith, the term "arjunetin" a triterpenoid glycosides compound (CAS No. 31297-79-7) found in various plants including in the bark and roots of the Terminalia arjuna (belongs to the family of Combretaceae tree). Arjunetin appears as pale yellow crystals and is soluble in alcohols and organic solvents, with slight solubility in water
As used herewith, the term "solvent extraction" refers to a process used to isolate a specific compound from a raw material by utilizing a solvent. The process is initiated often by mixing the raw material, which contains the compound of interest, with an appropriate solvent such as a hydrocarbon or alcohol. This mixture is then separated into two distinct phases: the solvent phase, which contains the dissolved compound of interest, and the remaining raw material. Various methods, including filtration or centrifugation, may be employed to achieve this separation. Finally, the compound of interest is recovered from the solvent, often through evaporation or another separation technique. This method is extensively used in industries like pharmaceuticals, food processing, and metallurgy due to its efficiency in extracting valuable compounds
In solvent extraction, a specific "solvent fraction" such as a "hexane fraction" refers to the part of a mixture that dissolves in hexane, a non-polar solvent. During the extraction process, a mixture containing the desired compound is combined with hexane, which selectively dissolves the non-polar compounds. This results in two layers: the hexane layer (organic phase) containing the dissolved non-polar compounds, and the aqueous layer (if water is used as the other solvent) containing the remaining components. The hexane fraction is the layer that includes hexane and the dissolved non-polar compounds. This fraction can then be further processed to isolate and purify the desired compounds.
The first, second and third solvent as used herewith include hexane (99% pure hexane) (CAS No. 110-54-3), chloroform (CAS No. 67-66-3), ethanol (CAS No. 64-17-5).
As used herewith, the term "yield" refers to the amount or proportion of a compound obtained from a particular process or reaction. It is a measure of the efficiency and effectiveness of the process. Yield may be expressed in various ways, such as the percentage of the theoretical maximum amount of product, the weight or volume of the product obtained, or the number of units produced. The yield would indicate how much of the desired compound is produced relative to the starting materials used.
As used herewith, the term "purity" refers of a compound refers to the extent to which a compound is free from impurities or contaminants. A pure compound is composed of only one type of molecule or element. The purity of a compound can be determined by measuring the amount of impurities present, with higher purity indicating fewer impurities. Pure compounds have specific melting and boiling points, and the presence of impurities can cause these points to vary. Advanced techniques like spectroscopy are often used to detect even small amounts of impurities, providing a more accurate measure of purity.
EMBODIMENTS
The current invention encompasses a method of isolating arjunetin from the bark of terminalia arjuna tree.
The foregoing description of the specific embodiments willfully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such as specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modifications. However, all such modifications are deemed to be within the scope of the claims.
The scope of the embodiments will be ascertained by the claims to be submitted at the time of filing a complete specification.
One embodiment of the invention is a method of isolating arjunetin with high purity from the bark of Terminalia arjuna, the method comprising the steps of:
a. Drying and grinding the bark of Terminalia arjuna to obtain a bark powder;
b. Subjecting the bark powder in step a. to a first solvent extraction to yield a first fraction and bark powder remaining after first solvent extraction;
c. Subjecting the bark powder in step b. to a second solvent extraction to yield a second fraction and bark powder remaining after second solvent extraction;
d. Subjecting the bark powder in step c. to a third solvent extraction to yield a third fraction and wherein the third solvent is ethanol;
e. Subjecting the third fraction in step d. to filtration followed by vacuum drying to yield a crude ethanol extract; and
f. Isolating arjunetin compound from the crude ethanol extract in step e.
In one embodiment, the first solvent is hexane or petroleum ether. In one embodiment, the first solvent is hexane.
In one embodiment the first fraction is a hexane fraction. In one embodiment the first fraction is a hexane fraction
In one embodiment, the second solvent is chloroform, ethyl acetate or dichloromethane. In one embodiment, the second solvent is chloroform. In one embodiment, the second fraction is a chloroform fraction.
In one embodiment, the order of polarity increases from the first to the third solvent.
In one embodiment, the bark of terminalia arjuna was air dried before grinding in step a.
In one embodiment, the bark of Terminalia arjuna is ground to uniform particles. In one embodiment, the size of the particles is in the range of 10-40 µm. In one embodiment, the size of the particles is in the range of 10-30 µm. In one embodiment, the size of the particles is 20 µm.
In one embodiment, the first, second and third solvent extraction is done at a temperature of 60-70 °C. In one embodiment, the first, second and third solvent extraction is done at a temperature of 62-67 °C. In one embodiment, the first, second and third solvent extraction is done at a temperature of 65 °C.
In one embodiment, the solvent extraction with step b. and c. is done for 65-80 hours at a temperature of 60-70 °C. In one embodiment, the solvent extraction with step b. and c. is done for 65-80 hours at a temperature of 65 °C.
In one embodiment, the extraction with the third solvent is done for 65-80 hours at a temperature of 60-70 °C.
In one embodiment, the bark powder remaining after the first, second and third solvent extraction is derived after filtration and vacuum drying of the solvent at the end of each stage of solvent extraction.
In one embodiment the crude ethanol extract obtained in step e. comprises at least 93% pure arjunetin (wt/wt). In one embodiment the crude ethanol extract obtained in step e. comprises at least 95% pure arjunetin (wt/wt). In one embodiment the crude ethanol extract obtained in step e. comprises 95% pure arjunetin (wt/wt).
In one embodiment, the arjunetin isolated in step f. has a yield of 10%.
In one embodiment, the crude ethanol extract obtained in step e. is in form of a powder. In one embodiment, the ethanol evaporation is done using Buchi rotary evaporator.
In one embodiment, the crude ethanol extract obtained in step e. is further subjected to column chromatography to yield arjunetin with 99% purity. In one embodiment, the water and methanol are used as column chromatography eluents wherein the ratio of water to methanol is in the range of 90:10 to 0:100.
In one embodiment, the column chromatography is done in a single step.
In one embodiment, the amount of bark powder in first, second and third solvent extraction is in the range of 0.3-0.7kg/L.
One embodiment of the invention is an ethanol extract of arjunetin wherein the amount of arjunetin is 95%.
In one embodiment, the ethanolic extract of arjunetin is obtained in step e.
In one embodiment, the ethanolic extract of arjunetin has catalase inhibitory activity. In one embodiment, the ethanolic extract of arjunetin has anti-inflammatory activity.
In one embodiment, the ethanol extract of arjunetin is stable for up to 2 years at a temperature of 25-30 °C. In one embodiment, the ethanol extract of arjunetin is stable for up to 1 years at a temperature of 25-30 °C.

EXAMPLES
Collection of Plant Materials

The dry stem bark of Terminalia arjuna was obtained from the Centre of Traditional Medicine and Research (CTMR), Chennai, India, and collected from Pillaiyarpattai, Sri Karpagam Vinayager Temple Thalaviruksha, Sivagangai District, Tamil Nadu, India, and authenticated at Siddha Central Research Institute, Arumbakkam, Chennai, TamilNadu, India (voucher specimen no: T19081901A).

Example 1: Extraction and Isolation of Arjunetin from T.arjuna Bark using Chemical Reactor

2.5 kilograms of air-dried bark of TA was finely ground into a powder and then sequentially extracted with solvents of increasing polarity (hexane, chloroform, and ethanol, each solvent totalling 5 litters) for 72 hours using a chemical reactor. Following filtration, evaporation was carried out under vacuum conditions, and the resulting residues (ethanol extract powder) were stored at room temperature. The ethanol fraction (250 grams, 95% purity) was subjected to column chromatography over Sephadex LH 20, utilizing various concentrations of methanol and water as eluent, with a final elution of 100% methanol. This process yielded compound (Arjunetin) as a white crystalline solid, totalling 10-12 grams (99% purity). For a more detailed illustration, please refer to Figure 2-4.

No Methods TA Bark After Extraction Isolated pure Arjunetin after column chromatography
% Yield
1 Existing processes
2.5 Kg 100 g
(multiple compounds)
50 mg
0.002%
2 Method described in present invention
2.5 Kg 250 g
95% Pure Arjunetin
10 g

0.4%

Example 2: Characterization of Arjunetin

Arjunetin was obtained as colorless solid from ethanol fraction using 100% methanol as eluent. It showed UV λmax (methanol) at 423 nm. It exhibited characteristic IR absorption bands trisubstituted double bonds (1630 cm-1, 894 cm-1), hydroxyl (3355 cm-1), ester (1730 cm-1 ), methyl (1452 cm-1), gem dimethyl 1391 cm-1, C-O stretching frequency (1260, 1161, 1049 cm-1). On the basis of mass and 13C NMR spectra the molecular ion peak of isolated compound (1) was determined at m/z 673.4 [M+Na]+ C36H58NaO10, m/z 689 [M+K]+ , m/z 511 [M-162]+, m/z 527 [M-162-44]+, m/z 430 [M-162-44-34]+, m/z 413 [M-162-44-41]+ supported one glycoside, three removable hydroxyl group and one ester function in the molecule (Figures 5-11).

1H NMR spectrum of isolated compound exhibited signals of an olefinic proton at δH 7.0 (1H, brs, H-12) and seven methyl singlet's δH (0.12, 0.16, 0.19, 0.33, 0.36, 0.40, 0.57, 7XCH3, 21H), the spectrum showed three oxygen bearing methine protons at δH 4.47 (1H, brs, H-19), δH 4.66 (1H, s, H-3α), δH 4.73 (1H, d,J = 9.5 Hz, H-2β), δH 3.48 (1H, brs-H18), δH 3.32-3.89 (glucose hydrogen's). 13C NMR spectrum displayed 36 signals inclusive of seven methyl signals δC (13.9, 16.9, 17.0, 17.6, 23.5, 25.8, and 27.3), three secondary hydroxyl groups δC 69.7 (C-2), 75.6 (C-3), 77.9 (C-19), one trisubstituted double bond δC 121.8 (C-12), 144.2 (C-13), ester carbonyl δC 175.4 (C-28), glucose carbon δC 60.2, 60.8, 67.6, 72.5, 76.8, 94.26. The anomeric proton signals at δH 6.8 (1H, -H1') in the 1H NMR spectrum and anomeric carbon signals at δH 94.2 in the 13C NMR spectra of compound attributed to a β-glucose unit linked to the C28 carbonyl group of the genin through an ester bond (Tripathi, V.K et al., 1996), (Honda, T. et al., 2001), (Sunyana, J. et al., 2009). Fig 8-9. From the above, all spectroscopic data the isolated compound was named arjunetin (Figure 5-11).
, Claims:1. A method of isolating arjunetin with high purity from the bark of Terminalia arjuna, the method comprising the steps of:
a. Drying and grinding the bark of Terminalia arjuna to obtain a bark powder;
b. Subjecting the bark powder in step a. to a first solvent extraction to yield a first fraction and bark powder remaining after first solvent extraction;
c. Subjecting the bark powder in step b. to a second solvent extraction to yield a second fraction and bark powder remaining after second solvent extraction;
d. Subjecting the bark powder in step c. to a third solvent extraction to yield third fraction and wherein the third solvent is ethanol;
e. Subjecting the third fraction in step d. to filtration followed by vacuum drying to yield a crude ethanol extract; and
f. Isolating arjunetin compound from the crude ethanol extract in step e.
2. The method of claim 1, wherein the first solvent is hexane or petroleum ether.
3. The method of claim 1, wherein the second solvent is chloroform, ethyl acetate or dichloromethane.
4. The method of claim 1, wherein the order of polarity increases from the first to the third solvent.
5. The method of claim 1, wherein the first, second and third solvent extraction is done at a temperature of 60-70°C.
6. The method of claim 1, wherein the solvent extraction with step b. and c. is done for 65-80 hours at a temperature of 60-70 °C.
7. The method of claim 1, wherein the extraction with the third solvent is done for 65-80 hours at a temperature of 60-70 °C.
8. The method of claim 1, wherein the crude ethanol extract obtained in step e. comprises 95% pure arjunetin (wt/wt).
9. The method of claim 1, wherein the arjunetin isolated in step f. has a yield of 10%.
10. The method of claim 1, wherein the crude ethanol extract obtained in step e. is further subjected to column chromatography to yield arjunetin with 99% purity.
11. The method of claim 8, wherein water and methanol are used as column chromatography eluents wherein the ratio of water to methanol is in the range of 90:10 to 0:100.
12. The method of claim 10, wherein the column chromatography is done in a single step.
13. The method of claim 1, wherein the amount of bark powder in first, second and third solvent extraction is in the range of 0.3-0.7 kg/L.
14. An ethanol extract of arjunetin wherein the amount of arjuntein is 95%.

Documents