HK1187538A - Substantially coumarin-free extract of cinnamon and its production method - Google Patents

Description

Substantially coumarin-free cinnamon extract and process for its preparation

Technical Field

The present invention relates to a process for the preparation of essentially coumarin-free extracts of cinnamomum Burmanii (cinmamoum Burmanii). The invention also relates to pharmaceutical compositions and dietary supplements comprising essentially coumarin-free extract of cinnamomum burmanii.

Background

Yin xiang is a native plant of indonesia and its potential as a medicine is known empirically. The yin-fragrant is commonly referred to as cinnamon. Cinnamon products have been sold for some time now as food additives and are indicated for the treatment of various diseases. However, despite its therapeutic effect, it has recently been known that most cinnamon products extracted from cinnamon plants contain high levels of coumarins.

Coumarins are natural benzopyranone compounds found in several plants including cinnamon, coumarins, vanilla and cuscuta maxima (herbs). It has been known since 1882 and used as a component in perfume production. For medical use, coumarin is a precursor in the production of anticoagulants. However, coumarin can become toxic to the liver, especially after prolonged use.

The European Safety Agency (ESA) recommends a maximum daily uptake (TDI) of 0.1mg coumarin per kg body weight, which can be taken daily for life without risk to health.

To date, there is no extract of cinnamomum burmannii on the market with low levels of coumarin. One commonly uses an alternative source of cinnamon, including cinnamon stannum (cinnamyl), because of its low coumarin levels and is considered safe. Cinnamon from stannum, however, is more expensive and thus uneconomical for large industrial applications. Therefore, there is a need to develop an extract of cinnamomum burmanii substantially free of coumarin. The present invention teaches a process for the preparation of an extract of cinnamomum burmanii which is substantially free of coumarin and therefore can be safely taken over a long period of time.

Disclosure of Invention

In preferred embodiments, the subject matter and/or aspects of the present teachings will be explained. The illustrated embodiments are intended for an understanding of the present invention and do not limit the possibilities of other embodiments that can be derived from the practice of the invention. The subject matter and/or aspects of the present teachings will be implemented from the detailed elements or combinations of the claims that follow.

To achieve the solution and in accordance with the subject matter of the present invention, as described in the embodiments and broadly described herein, a first aspect of the present invention relates to a process for preparing a substantially coumarin-free cinnamomum burmanii extract by an extraction process followed by a fractionation process based on the relative solubilities of two different immiscible liquids. This process is currently known as liquid-liquid extraction.

A second aspect of the invention relates to a substantially coumarin-free extract of cinnamomum burmanii, wherein the level of coumarin in said extract is equal to or less than 0.39mg coumarin/kg extract, used as a single component or in combination.

A third aspect of the invention relates to a pharmaceutical composition or formulation comprising as a single component or in combination an extract of cinnamomum burmanii substantially free of coumarin, wherein the level of coumarin in said extract is equal to or less than 0.39mg coumarin/kg extract.

Drawings

The following drawings, which are incorporated in and constitute a part of the specification of this application, illustrate one or more embodiments of the invention. The following drawings serve to explain the principles of the present teachings.

Figure 1 is a thin layer chromatography of coumarin standards.

FIG. 2 is a thin layer chromatography of crude extract of cinnamomum burmanii extracted with water and its substantially coumarin-free extract prepared using chloroform, n-butanol, ethyl acetate and n-hexane. Note that:

1 is a coumarin standard substance, and the coumarin standard substance is,

2 and 3 are crude extracts of cinnamomum burmannii which are extracted by water,

4 and 5 are crude extracts of cinnamomum burmannii fractionated with chloroform;

6 and 7 are crude extracts of cinnamomum burmannii fractionated with n-butanol;

8 and 9 are crude extracts of cinnamomum burmannii fractionated with ethyl acetate; and

10 and 11 are crude extracts of cinnamomum burmannii fractionated using n-hexane.

FIG. 3 is a thin layer chromatography of crude extract of cinnamomum burmanii extracted with methanol and its substantially coumarin-free extract prepared with chloroform, n-butanol, ethyl acetate and n-hexane. Note that:

1 is a coumarin standard substance, and the coumarin standard substance is,

2 and 3 are crude extracts of cinnamomum burmannii extracted by methanol,

4 and 5 are crude extracts of cinnamomum burmannii fractionated with chloroform;

6 and 7 are crude extracts of cinnamomum burmannii fractionated with n-butanol;

8 and 9 are crude extracts of cinnamomum burmannii fractionated with ethyl acetate; and

10 and 11 are crude extracts of cinnamomum burmannii fractionated using n-hexane.

FIG. 4 is a thin layer chromatography of crude extract of cinnamomum burmanii extracted with ethanol and its polar extracts prepared with chloroform, n-butanol, ethyl acetate and n-hexane. Note that:

1 is a coumarin standard substance, and the coumarin standard substance is,

2 and 3 are crude extracts of the cinnamomum burmannii which are extracted by ethanol,

4 and 5 are crude extracts of cinnamomum burmannii fractionated with chloroform;

6 and 7 are crude extracts of cinnamomum burmannii fractionated with n-butanol;

8 and 9 are crude extracts of cinnamomum burmannii fractionated with ethyl acetate; and

10 and 11 are crude extracts of cinnamomum burmannii fractionated using n-hexane.

FIG. 5 is a thin layer chromatography of crude extract of cinnamomum burmanii extracted with acetone and its substantially coumarin-free extract prepared with chloroform, n-butanol, ethyl acetate and n-hexane. Note that:

1 is a coumarin standard substance, and the coumarin standard substance is,

2 and 3 are crude extracts of cinnamomum burmannii extracted by using acetone,

4 and 5 are crude extracts of cinnamomum burmannii fractionated with chloroform;

6 and 7 are crude extracts of cinnamomum burmannii fractionated with n-butanol;

8 and 9 are crude extracts of cinnamomum burmannii fractionated with ethyl acetate; and

10 and 11 are crude extracts of cinnamomum burmannii fractionated using n-hexane.

FIG. 6 is a high performance liquid chromatography of a standard coumarin compound.

FIG. 7 is a high performance liquid chromatography of crude extract of cinnamomum burmannii extracted with water.

FIG. 8 is a high performance liquid chromatography of crude extract of cinnamomum burmanii extracted with methanol.

FIG. 9 is high performance liquid chromatography of crude extract of cinnamomum burmanii extracted with ethanol.

FIG. 10 is a high performance liquid chromatography of crude extract of cinnamomum burmanii extracted with acetone.

FIG. 11 is a high performance liquid chromatography of essentially coumarin-free extract of Cinnamomum burmannii extracted with water and fractionated with chloroform.

FIG. 12 is a high performance liquid chromatography of essentially coumarin-free extract of Cinnamomum burmannii extracted with methanol and fractionated with chloroform.

FIG. 13 is a high performance liquid chromatography of essentially coumarin-free extract of Cinnamomum burmannii extracted with ethanol and fractionated with chloroform.

FIG. 14 is a high performance liquid chromatography of essentially coumarin-free extract of Cinnamomum burmannii extracted with acetone and fractionated with chloroform.

FIG. 15 is a high performance liquid chromatography of essentially coumarin-free extract of Cinnamomum burmannii extracted with water and fractionated with n-butanol.

FIG. 16 is a high performance liquid chromatography of essentially coumarin-free extract of Cinnamomum burmannii extracted with methanol and fractionated with n-butanol.

FIG. 17 is a high performance liquid chromatography of essentially coumarin-free extract of Cinnamomum burmannii extracted with ethanol and fractionated with n-butanol.

FIG. 18 is a high performance liquid chromatography of essentially coumarin-free extract of Cinnamomum burmannii extracted with acetone and fractionated with n-butanol.

FIG. 19 is a high performance liquid chromatography of essentially coumarin-free extract of Cinnamomum burmannii extracted with water and fractionated with ethyl acetate.

FIG. 20 is a high performance liquid chromatography of essentially coumarin-free extract of Cinnamomum burmannii extracted with methanol and fractionated with ethyl acetate.

FIG. 21 is a high performance liquid chromatography of essentially coumarin-free extract of Cinnamomum burmannii extracted with ethanol and fractionated with ethyl acetate.

FIG. 22 is a high performance liquid chromatography of essentially coumarin-free extract of Cinnamomum burmannii extracted with acetone and fractionated with ethyl acetate.

FIG. 23 is a high performance liquid chromatography of essentially coumarin-free extract of Cinnamomum burmannii extracted with water and fractionated with n-hexane.

FIG. 24 is a high performance liquid chromatography of essentially coumarin-free extract of Cinnamomum burmannii extracted with methanol and fractionated with n-hexane.

FIG. 25 is a high performance liquid chromatography of essentially coumarin-free extract of Cinnamomum burmannii extracted with ethanol and fractionated with n-hexane.

FIG. 26 is a high performance liquid chromatography of essentially coumarin-free extract of Cinnamomum burmannii extracted with acetone and fractionated with n-hexane.

Detailed Description

The present invention is discussed in detail by providing examples, which do not limit the scope of the present invention to the illustrated examples.

The present invention is mainly divided into two main steps, including, in the first step, extracting cinnamomum burmanii with a first solvent including, but not limited to, water and/or various organic solvents including, but not limited to, alcohols and acetone to prepare a crude extract; in a second step, the crude extract is fractionated using other organic solvents immiscible with the first solvent, including but not limited to chloroform, n-butanol, ethyl acetate or n-hexane, to obtain an extract substantially free of coumarin. Analytical observation was carried out by Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC).

According to the teachings of the present invention, the term "substantially coumarin-free" means that the level of coumarin is below the maximum daily ingestible (TDI) limit recommended by the European Safety Agency (ESA). The raw material is a natural material which is not subjected to any extraction method; the crude extract is an extract which is not subjected to a fractionation method; and an extract that is substantially free of coumarin is an extract that has been subjected to an extraction process followed by a fractionation process whereby the level of coumarin is below the maximum daily ingestible amount (TDI) limit recommended by the European Safety Agency (ESA).

Various solvents including water, alcohols, acetone, or combinations thereof may be used to perform the extraction process in accordance with the teachings of the present invention. After the extraction process using any solvent, the content of coumarin varies depending on the quality of the raw material and the solubility of coumarin in the solvent used.

After the extraction process, the fractionation process is repeated until the level of coumarin content reaches a specific target.

A. Extraction process using water and removal of coumarin therefrom

An essentially coumarin-free extract of cinnamomum burmannii according to the teachings of the present invention is prepared using a water-based extraction process. Coumarin is highly soluble in hot water, so that ordinary water extraction leads to a high content of coumarin in the yin-fragrant extract solution.

The extraction method using water starts with grinding any part of the cinnamomum burmanii plant, preferably the bark part. Then, the bark is immersed in hot water for 1 to 2 hours, wherein the temperature of the hot water is within the range of 50 to 100 ℃, and the volume of the hot water is 5 to 15 times of the volume of the ground raw material of the bark of the cinnamomum burmannii. The filtration process is performed to obtain a filtrate, which is then concentrated by evaporating the filtrate to reach a viscosity in the range of 40-100 cps. The viscosity of the concentrate was measured with a BrookfieldsDV-E viscometer with spindle #61 at 27 ℃ at 100rpm for 2 minutes. Thus obtaining a crude extract of cinnamomum burmannii.

Then, to reduce the content of coumarin in the crude extract, the concentrate is fractionated on the basis of relative solubility by liquid-liquid extraction (LLE) using organic solvents including, but not limited to, chloroform, n-butanol, ethyl acetate and/or n-hexane. The ratio of crude extract to fractionating solvent is about 1: 1 to 1: 2. The mixture was gently shaken to avoid the formation of an emulsion. LLE is repeated until the crude extract of cinnamomum burmanii is substantially free of coumarin. The content of coumarin was analyzed by Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC). TLC and HPLC will be explained in further detail later. Finally, the essentially coumarin-free extract of cinnamomum burmannii is dried.

B. Extraction process using alcohols and removal of coumarins therefrom

Alternatively, an essentially coumarin-free extract of cinnamomum burmanii according to the teachings of the present invention may also be prepared by an extraction process using alcohols. Coumarins are readily soluble in alcohols, resulting in alcohol-based cinnamomum burmanii extracts containing high levels of coumarins.

Alcohols useful in accordance with the teachings of the present invention include, but are not limited to, methanol, ethanol, and butanol. Ethanol and methanol are preferred. The extraction process using alcohol starts with grinding any part of the cinnamomum burmanii, preferably the bark part; and then soaking the raw materials in ethanol or methanol with the volume 5-15 times that of the raw materials for 1-2 hours. In this example, the alcohol content in the water does not exceed 70%. Performing a filtration process to obtain a filtrate, and then concentrating by evaporating the filtrate to reach a viscosity in the range of 40-100 cp. The viscosity of the concentrate was measured with a Brookfield DV-E viscometer with spindle #61 at 27 ℃ at 100rpm for 2 minutes. Thus obtaining a crude extract of cinnamomum burmannii.

Then, to reduce the content of coumarin in the crude extract, the concentrate is fractionated by liquid-liquid extraction (LLE) using organic solvents including, but not limited to, chloroform, n-butanol, ethyl acetate and/or n-hexane. The ratio of crude extract to fractionating solvent is about 1: 1 to 1: 2. The mixture was gently shaken to avoid the formation of an emulsion. LLE was repeated until the crude extract was substantially free of coumarin. The content of coumarin was analyzed by Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC). The essentially coumarin-free extract of cinnamomum burmannii is then dried.

C. Extraction process using acetone and removal of coumarin therefrom

Still alternatively, an essentially coumarin-free extract of cinnamomum burmanii according to the teachings of the present invention may also be prepared by an extraction process using acetone.

The extraction process starts with grinding any part of the cinnamomum burmanii plant, preferably the bark part, and then soaking in acetone in a volume of 5-15 times the volume of the raw material for 1-2 hours. In this example, water was used having an acetone content of not more than 70%. The filtration process is performed to obtain a filtrate, which is then concentrated by evaporating the filtrate to reach a viscosity in the range of 40-100 cps. The viscosity of the concentrate was measured with a Brookfield DV-E viscometer with spindle #61 at 27 ℃ at 100rpm for 2 minutes. Thus obtaining a crude extract of cinnamomum burmannii.

The concentrate is then fractionated by liquid-liquid extraction (LLE) using organic solvents including, but not limited to, chloroform, n-butanol, ethyl acetate and/or n-hexane. The ratio of crude extract to fractionating solvent is about 1: 1 to 1: 2. The mixture was gently shaken to avoid the formation of an emulsion. LLE is repeated until the crude extract of cinnamomum burmanii is substantially free of coumarin. The content of coumarin was analyzed by Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC). Similar to the extraction process using water and alcohols, the essentially coumarin-free extract of cinnamomum burmanii is then dried.

D. Thin Layer Chromatography (TLC) was used to analyze coumarin content

This experiment was performed to determine that the content of the cinnamomum burmanii extract is substantially free of coumarin. The yin-fragrant extract obtained from several different extraction methods as described above was analyzed on Thin Layer Chromatography (TLC) plates. Crude extract and essentially coumarin-free cinnamomum burmanii extract were prepared at a concentration of 4000ppm and compared to coumarin standards at the same concentration. Elution was performed using toluene and ethyl acetate (93:7) as mobile phases. The chromatogram was then observed under UV at 254nm and 366nm and under visible light.

The points for the standard coumarin compounds are shown at Rf of 0.42. Lines 2 and 3 of each of figures 4, 5 and 6 show that only the crude extract prepared prior to liquid-liquid extractive fractionation has the same Rf as the standard coumarin compound. This can be clearly seen in the 254nm UV observed eluate. On the other hand, as shown by lines 4 to 11 in each of fig. 4, 5 and 6, the substantially coumarin-free cinnamomum burmanii extract, which is a product of the fractionation method using liquid-liquid extraction, did not show any point in the vicinity of rf0.42 representing the site of the coumarin compound. This indicates that the cinnamomum burmannii extract prepared according to the teachings of the present invention is substantially free of coumarin.

E. High Performance Liquid Chromatography (HPLC) analysis of coumarin content

Analysis was performed using High Performance Liquid Chromatography (HPLC) Alliance2695 with detector photodiode array Alliance2889 to quantitatively determine the content of coumarin in substantially coumarin-free extract of cinnamomum burmanii.

The essentially coumarin-free extract of cinnamomum burmannii was dried and then analyzed by HPLC. In this analysis, a crude extract and an essentially coumarin-free extract of cinnamomum burmanii were prepared at a concentration of 4000ppm and compared to a coumarin standard at a concentration of 50 ppm. The analytical procedure was carried out using a mobile phase of water acetonitrile (60:40) which was observed at a residence time of 12 minutes at 274 nm.

The results show that the peak for the standard coumarin compound can be seen at a residence time of 5.099. Crude extract of cinnamomum burmanii obtained from extraction method using water, alcohol and acetone shows a peak representing coumarin compound. Specifically, as shown in fig. 7, the crude extract obtained from the aqueous extraction showed a peak at about 5.012 minutes. In the crude extract obtained from the alcohol extraction, peaks at about 5.012 minutes for ethanol and 5.007 minutes for methanol were shown. The crude extract obtained from acetone extraction showed a peak at about 5.011 minutes.

Contrary to this finding, according to the teachings of the present invention, the extract of cinnamomum burmanii, which has been fractionated using organic solvents chloroform or n-butanol or ethyl acetate and/or n-hexane, does not show any peak representing coumarin. See FIGS. 13-26. Thus, the extract of cinnamomum burmannii prepared according to the teachings of the present invention is substantially free of coumarin.

The concentration of coumarin in the crude and substantially coumarin-free extracts can be calculated by comparing the peak area of a known concentration (50ppm) of coumarin standard with the peak areas of the crude and substantially coumarin-free extracts shown at the same residence time as the coumarin standard.

Table 1 content of coumarin in cinnamomum burmannii extract prepared according to the teachings of the present invention

Example (b): calculation of average maximum ingestible amount of yin-fragrant extract

Maximum daily intake of coumarin =0.1mg/kg body weight

Mean body weight =70kg

Maximum daily ingestible level of coumarin =7mg coumarin

The level of coumarin in the crude extract is in the range of 3.04 to 22.73ppm or 3.04 to 22.73mg coumarin/kg extract.

The maximum daily intakes of the extract are in the range equal to 2.302kg of extract (7mg coumarin)/(3.04 mg coumarin/kg of extract) to equal to 0.31kg of extract (7mg coumarin)/(22.73 mg coumarin/kg of extract).

The maximum level of coumarin in the substantially coumarin-free extract =0.39ppm =0.39mg coumarin/kg extract.

The minimum daily ingestible amount of extract =7mg coumarin/0.39 mg coumarin/kg extract =17.948kg extract.

The data shown in table 1 show that crude extract of cinnamomum burmanii prepared by extraction methods with water, alcohols and acetone (no more than 70% in water) contains high levels of coumarin. However, after fractionation of the crude extract by liquid-liquid extraction according to the teachings of the present invention, the coumarin content becomes very low, even below the detection limit. Furthermore, the data shown in the examples show that extracts substantially free of coumarin can be consumed within a wide range of quality without any physical damage, while the intake limits for crude extracts are very low. The values of the extracts showing substantial absence of coumarin are not much lower than the maximum daily intake (TDI) recommended by the European Safety Agency (ESA). Thus, substantially coumarin-free extracts prepared in accordance with the teachings of the present invention can be safely ingested daily and used for extended periods of time.

Furthermore, the present invention also contemplates the use of essentially coumarin-free cinnamomum burmanii extract as a single active ingredient or a combination thereof as a pharmaceutical composition or formulation for safe and long-term consumption on a daily basis.

F. Industrial applications

The extract or pharmaceutical dosage form of the essentially coumarin-free cinnamomum burmanii extract can be used on an industrial scale for the preparation of extracts, powder extracts and/or pharmaceutical dosage forms mainly for oral dosage forms, such as solid, semi-solid or liquid, or for the production of food and beverages.

Claims (7)

1. A process for the preparation of a substantially coumarin-free extract derived from cinnamomum burmanii, said process comprising the steps of:

(a) grinding the raw materials of the yin-fragrant,

(b) extracting the raw material in step (a) by impregnation with a first solvent to obtain a crude extract of cinnamomum burmanii,

(c) fractionating the extracted feedstock in step (b) by liquid-liquid extraction using a second solvent to obtain a substantially coumarin-free extract, wherein the second solvent is immiscible with the first solvent, and optionally,

(d) drying the substantially coumarin-free extract.

2. The process of claim 1, wherein the first solvent used in step (b) comprises water, alcohols, acetone, or combinations thereof.

3. The method of claim 1, wherein the ratio of said crude extract of step (b) to said second solvent used in step (c) is about 1: 1 to 1: 2.

4. The method of claim 1, wherein the substantially coumarin-free extract has a coumarin content of 0.39mg or less coumarin/kg extract.

5. A substantially coumarin-free extract derived from cinnamomum burmanii, said extract prepared by a process comprising the steps of:

(a) grinding the raw materials of the yin-fragrant plants,

(b) extracting the raw material in step (a) by impregnation with a first solvent to obtain a crude extract of cinnamomum burmanii,

(c) fractionating the extracted feedstock in step (b) by liquid-liquid extraction using a second solvent to obtain a substantially coumarin-free extract, wherein the second solvent is immiscible with the first solvent, and optionally,

(d) drying the substantially coumarin-free extract;

wherein the level of coumarin in said substantially coumarin-free extract is equal to or less than 0.39mg coumarin/kg extract.

6. A pharmaceutical composition or dietary supplement comprising the substantially coumarin-free cinnamomum burmanii extract according to claim 5 as a single component or in combination with suitable pharmaceutical excipients for the manufacture of pharmaceutical products in solid, semi-solid or liquid form or for the manufacture of food/beverages.

7. A pharmaceutical composition or dietary supplement comprising the substantially coumarin-free cinnamomum burmanii extract according to claim 5, for use in the manufacture of a pharmaceutical product or food supplement comprising a tablet, capsule, syrup, effervescent tablet, gel, ointment, emulsion or mouthwash formulation comprising vitamins.