BACKGROUND OF THE INVENTION Technical Field The present invention relates to an efficient isolation method of amygdalin and neoamygdalin using Reversed phase-HPLC and an efficient condition for inhibition of the racemization of amygdalin isolated from Persicae semen or Armeniacae semen.

Background Art The present invention relates to an optimum extraction method for obtaining maximum extraction yield of amygdalin having anti-cancer activity.

Cancer is a subject to overcome with first priority with a view to extending human life span. Currently, the ratio of cancer occurrence has been increasing by about 5% year by year because of the increased ratio of old people and environmental deterioration. The number of dead people taken with cancer disease was 6,000, 000 in 1997, which falls under 12% of total world-wide mortality. 100,000 number of patients taken with cancer have been newly found and 50,000 number of patients died every year.

The increasing rate of cancer patient is 10%, the number of cancer patient is 120,000 and cancers occur in the order of stomach cancer (21%), liver cancer (12%), lung cancer (11%) in men, and cervical cancer (20%), stomach cancer (16%) and breast cancer (13%) in women.

Most of anti-cancer agent show anti-cancer activity caused by the inhibition of

nucleic acid synthesis, i. e. , it intervenes various metabolic pathway. However, those anti-cancer agent acts on not only cancer cells selectively but also normal cells, which gives rise to various side effect such as bone marrow malfunction, gastrointestinal disorder and alopecia by causing damage to organic cell of which cell division is active.

Anti-cancer agent can be classified into six categories according to their biochemical mechanism.

Anti-cancer agent can be classified with a chemotherapy agent and a biological therapy agent; A chemotherapy agent is highly reactive and is synthetic chemical substance such as an alkylating agent to transform DNA structure or to cleave DNA sequence; An anti-metabolite to inhibit metabolic pathway in multiplying cancer cells; An antibiotic having anti-cancer activity isolated from various microorganism, natural product and hormonal drug. A biological therapy agent can be classified with several agents; An immuno-therapy agent to remove caner cells by way of enhancing immune response of patients taken with cancer using by therapeutic vaccine, monoclonal antibody and cytokine; A mitotic inhibitor, for example, vinca alkaloid, to obstruct the cell differentiation in metaphase of mitosis as a drug specific to cell division cycle; A gene therapy agent which is administered to the patient having cancer disease, caused by gene deficiency or gene mutation, for treating cancer by the mechanism of controlling abnormal gene and producing therapeutic protein in cell or tissue; An anti-cancer agent containing antisense, nucleic acid having oncogene- inhibitory activity with selectively binding to oncogene; and an angiogenesis-inhibiting agent.

Metabolism therapy for example, Laetril therapy as an alternative therapy other than above-described drug therapy has been known to alternative medicine field and developed at the basis that human disease comes from improper harmonization of

human metabolism and therefore, if it has been normalized, all the disease caused by it can be treated. Laetril is made for intravenous injection comprising amygdalin glycoside and is a bitter substance named as Vitamin 17 being found in a seed of apricot, a seed of apple and bitter substance of natural almond. However, it should be carefully prepared in order not to be hydrolyzed, which produces HCN gas in metabolic pathway.

Generally, the studies on D-amygdalin (D-mandelonitrile-,-D-gentiobioside) well known as laetril has been developed actively as an anti-cancer drug till now. It is reported that neoamygdalin (D-mandelonitrile--D-gentiobioside) which does not have anti-cancer activity, is produced by the racemization of amygdalin in water solution, and therefore, it has been needed to search suitable condition for inhibiting the racemization of D-amygdalin to neoamydalin in water solution.

A racemic mixture is optically inactive and consists of equivalent amount of a pair of stereoisomer. Actually isoamygdalin consists of 50% of amygdalin and 50% of neoamygdalin in nature.

As an indicator to predict the inhibition degree of racemization of D- amygdalin, the complete separation of isoamydalin is important and therefore, a lot of researchers have studied to separate isomers of amygdalin. It has been reported about the isolation of isoamygdalin by gas column chromatography which is practical, however, it has disadvantage of using the derivatives of sample instead of sample itself (Takayama et al.; J. Chromatogr., 197, pp240-245,1980).

And some of studies on the separation method of amygdalin using high performance liquid chromatography, has been tried till now however, the method also dose not provide the complete separation of amygdalin (Smith et al.; J. Chromatogr Sci.,

22, pp94-98, 1984 : Cairns et al.; Anal. Chem., 50, pp317-322, 1978 : Dybowski et al.; Anal. Proc. (London), 18, pp316-318, 1978).

Present inventors have developed reversed-phase HPLC method using octadecylsillane (C18) column to separate D-amygdalin and neoamygdalin with near complete separation level and further, have endeavored to solve the problem occurred in the isolation of amygdalin from crude drug such that considerable amount of D- amygdalin is converted to neoamygdalin since the extraction solvent in extracting crude drug is hot water in usual. In order to overcome above described problem, present inventors confirmed the optimum condition which could inhibit the production of neoamygdalin by changing its acidity and on the basis of above described study, we accomplished the optimum extraction condition by applying the condition on Persicae semen.

Persicae semen, seed of Prunus persica Batsch, Prunus persica var. davidiana Maximowicz belonged to Rosaceae has been used as a lubricant or an anti-platelet agglutination agent long years ago in Asian countries. It has been reported that Persicae semen contains about 3.6% of amygdalin group compounds, 0.4% of essential oils, 45% of fatty oils such as olefin type glycerin, linolenic glycerin and other trace components, for example, palmitic acid, stearic acid, choline, acetylcholine, emulsin and so on (B. S.

Chung and M. K. Shin; HyangyakDaesaJeon, Youngrim Co. , pp632,1998).

In the result, prevent inventors finally found the extraction condition in order to inhibit the conversion of amygdalin to neoamygdalin in acidic condition such as citric acid, acetic acid and ascorbic acid etc, and completed the present invention by developing reversed-phase HPLC method.

SUMMARY OF THE INVENTION The present invention provides the optimum extraction method for maximum extraction yield of amygdalin being contained in Persicae semen or Armeniacae semen.

Disclosure of the invention Accordingly, it is an object of the present invention to provide an extraction and separation method of amygdalin from Persicae semen or Armeniacae semen characterized in extracting under acidic condition.

Through above described isolation method, the conversion of amygdalin to neoamygdalin can be inhibited by above described acidic condition.

Preferably, above-described acid is at least one selected from the group consisting of citric acid, acetic acid, ascorbic acid or the mixture thereof, more preferably 0. 05-5% citric acid.

Also, additionally it is another object of the present invention to provide a separation method of neoamygdalin using Reversed-phase High Performance Liquid Chromatography (Reversed-phase HPLC) in the mixture of amygdalin and neoamygdalin.

Above described separation method is characterized in using sodium phosphate buffer containing butanol or acetonitrile, preferably, 5-6% acetonitrile, as a mobile phase.

Also, above described separation method is characterized in using octasillane

(C8) or octadecylsillane (C18) column, preferably octadecylsillane (C18) column as a column in Reversed-phase HPLC.

Also, above described separation method is characterized in using M720UV detector set at 214nm as a detector in Reversed-phase HPLC.

Additionally, above described separation method is characterized in adopting the column flow rate ranging from 1 m/min to 5 m/min, preferably 1 m/min detector in Reversed-phase HPLC.

An inventive separation method using Reversed-phase HPLC and inhibiting method of the conversion of neoamygdalin from the mixture of amygdalin and neoamygdalin of the present invention may be prepared in accordance with the following preferred embodiment.

Hereinafter, the present invention is described in detail.

The amygdalin-containing water fraction is prepared from Persicae semen or Armeniacae semen and heated at 100 °C for 3-120 mins. Acidic solution selected from the group consisting of 0. 001-5% citric acid, acetic acid, ascorbic acid and the mixture thereof is added thereto and the solution is heated with reflux extraction at 100 °C for 3-120 mins to be compared using by HPLC.

And also, standard amygdalin is left alone for 1-3 hrs, preferably 2 hrs at room temperature using water soluble ammonia to prepare the mixture of amygdalin and neoamygdalin. The mixture is subjected to HPLC in condition that sodium phosphate buffer solution containing 5-10%, preferably 6% acetonitrile, is used as a mobile phase,

octasillane (C8) or octadecylsillane (C18), preferably octadecylsillane (C18) filled column, as a column at the column flow rate ranging 1 mQ/min to 5 mQ/min.

An inventive separation process can be explained in detail by following procedures: For example, the first step is heating amygdalin-containing solution for 3-120 mins at 100 °C to determine the degree of conversion rate from amygdalin to D- amygdalin and L-amygdalin; The second step is confirming the degree of conversion rate from amygdalin to D-amygdalin and L-amygdalin by subjecting similar procedure to above step excepting 0. 001-5% citric acid solution instead of above solution.

Also, it is an object of the present invention to provide an effective inhibiting condition of the conversion from amygdalin to neoamygdalin in HPLC.

Similarly, to explain specific condition of Reversed-phase HPLC method to isolate amygdalin from the mixture of amygdalin and neoamygdalin, exemplary embodiment of the present invention can be explained in detail by following procedures.

For example, the first step is obtaining the mixture of amygdalin and neoamygdalin characterizing that the solution of amygdalin and ammonia is left alone for 1-3 hrs, preferably about 2 hrs; The second step is setting separation tube as a stationary phase; The third step is setting a mobile phase to move a test sample to the separation tube; The forth step is detecting by flowing a test sample to a detector through the separation tube; The fifth step is controlling the flow rate of a mobile phase under 1000-4000psi of the pressure, the condition of which is in the scope of the present invention.

At the above second step, octasillane (C8) or octadecylsillane (C18)-filled separation tube column having suitable non-polarity is preferable. It is obvious to the person in the art that those non-polar tubes can be purchased commercially.

At the above third step, sodium phosphate buffer solution containing acetonitrile, preferably, 4-10% acetonitrile, more preferably 6% acetoniltrile can be used as a mobile phase since the solution can shorten the isolation period and accomplish complete dissolution rate.

At the above fourth step, the detector is set at 200-260nm since amygdalin and neoamygdalin can absorb the light at the wavelength of 214nm, but not at the above 260nm or the below 200nm because of little absorbance. It is obvious to the person in the art that above UV detector can be varied according to the degree of absorbance when exposed the light of constant wavelength in the test sample absorbing the light and can be purchased commercially.

At the above fifth step, the column flow rate is set to 1. 0-5. 0 InQ/min. If the column flow rate is above 5.0 m/min to cause the pressure of a mobile phase to too high, the machine can be out of order. Contrarily, if the column flow rate is below lml/min, the analysis time is too long. Therefore, the suitable analysis time should be adjusted to the condition that the column flow rate is 1. 0-5. 0 m/min, which makes the pressure of a mobile phase to 1000-4000psi.

Brief Description of the Drawings The above and other objects, features and other advantages of the present invention will more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which; Fig. la shows a chromatogram of D-amygdalin and neoamygdalin determined by reverse-phase HPLC with a sodium phosphate buffer (pH 3.8) containing 0. 3% 1-

butanol as a mobile phase; Fig. lb shows a chromatogram of D-amygdalin and neoamygdalin determined by reverse-phase HPLC with a sodium phosphate buffer (pH 3. 8) containing 6% acetonitrile, as a mobile phase; Fig. 2a shows a conversion rate of D-amygdalin to neoamygdalin 3 mins after heating; Fig. 2b shows a conversion rate of D-amygdalin to neoamygdalin 30 mins after heating; Fig. 2c shows a conversion rate of D-amygdalin to neoamygdalin 120 mins after heating; Fig. 3a shows an inhibiting rate of the conversion of amygdalin to neoamygdalin in case using 0. 001% citric acid; Fig. 3b shows an inhibiting rate of the conversion of amygdalin to neoamygdalin in case using 0.05% citric acid; Fig. 4a represents an inhibiting rate of the conversion of amygdalin to neoamygdalin in case using 0.005% citric acid when amygdalin was extracted from Persicae semen; Fig. 4b represents an inhibiting rate of the conversion of amygdalin to neoamygdalin in case using 0.05% citric acid when amygdalin was extracted from Persicae semen; Fig. 4c represents inhibiting rate of the conversion of amygdalin to neoamygdalin in using 0. 1% citric acid when amygdalin was extracted from Persicae semen.

BEST MODE FOR CARRING OUT THE INVENTION It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.

The present invention is more specifically explained by the following examples.

However, it should be understood that the present invention is not limited to these examples in any manner.

EXAMPLES The following Reference Example, Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope.

Reference Example 1. High performance liquid chromatography apparatus The HPLC systems used were a M930 pump (Young Lin, Kyunggi, Korea) with an M720 UV detector (Young Lin) set at 214 nm. The column used was a Capcell Pak Cis, Type UG120 (4.6 mmX250 mm, 5 llm, Shiseido, Tokyo, Japan) at 35°C contained in a CT30 column oven (Young Lin, Kyunggi, Korea). The column flow rate was 1 m/min. The mobile phase was a 10 mM sodium phosphate buffer (pH 3. 8) containing 1-butanol or acetonitrile, NMR (Bruker, Advance-600, Germany) of 600MHz and mass spectrometer (JEOL, JMS-AX505WA) were used for analysis.

Example 1. Sample Preparation Analytic amygdalin (Tokyo Kasei Chemical Co. , Japan) was used as a standard solution and distilled water was refined with Pure system (Pure power III, Taiwan).

Acetonitrile was of HPLC grade. All other chemicals were of analytical reagent grade.

Neoamygdalin was prepared from D-amygdalin and aqueous ammonia as described by Fischer. lOmg of D-Amygdalin was added to 10 m of 0.005 M ammonia solution and the mixture (lOmg) was stored for 2 hrs at room temperature.

Neoamygdalin was purified by HPLC (Young Lin, Korea), the column flow rate was 5 m/min and the mobile phase was a 6% acetonitrile. The column used was a Octadecylsillane (Nucleosil, 100-5 C18 (250mmxlOmm I. D. ) at 35°C.

The physicochemical properties of separated neoamygdalin was determined by FAB-HR mass and'H-NMR (CD30D) spectroscopy. The molecular weights of D- amygdalin and neoamygdalin by FAB-HR mass spectroscopy were 458.1672 [M+l] and

458. 1674 [M+'], respectively. Proton NMR provides evidence of the presence of epimers by two distinctly different methine chemical shift values. Chemical shift values were 5.89 ppm for D-amygdalin and 6.07 ppm for neoamygdalin.

Experimental Example 1. Racemization of D-amygdalin by heating For confirming racemization of D-amygdalin by heating, solutions containing 0.6 mg/mA of amygdalin in 50mQ of distilled water were prepared and heated at 100 C for 3,30, and 120 mins under reflux. After reaction, the mixture was cooled at once and was filtered with disposable syringe filter unit of 0. 2u, m MFS 13. HPLC system was worked under the condition of Reference Example 1 and mobile phase used was 6% acetonitrile.

As a result of experiment, D-amygdalin started to convert to neoamygdalin 3mins after heating (Fig. 2a). About 30% and more than 50% of D-amygdalin, respectively, were converted to neoamygdalin at 30 mins and 120 mins after heating reaction (Fig. 2b and Fig. 2c).

It was found that D-amygdalin in aqueous solution was converted to neoamygdalin by heating alone. Because only D-amygdalin is effective against cancer, it was necessary to determine the conditions for inhibition of its conversion to neoamygdalin.

Experimental Example 2. Inhibition of the conversion of D-amygdalin to neoamygdalin with citric acid Citric acid and ascorbic acid were examined to investigate whether they could inhibit the conversion of D-amygdalin to neoamygdalin. Each of 50mA of 0.001% citric acid (pH 5.9) and 50mA of 0.05% citric acid (pH 4.3) was added in round flask, 30mg of D-amygdalin was heated at 100 C for 120 mins under reflux. After reaction, the mixture was cooled at once and was filtered with disposable syringe filter unit of 0. 2pm MFS 13.

HPLC system was worked under the condition of Reference Example 1 and mobile phase used was 6% acetonitrile.

As a result of the experiment, the conversion rate was more than 50% in 0.001% citric acid solution (pH 5.9) (Fig. 3a), the conversion rate to neoamygdalin in 0.05% citric acid solution (pH 4.3) were almost 0% (Fig. 3b). Therefore, 0. 05-'-1% citric acid solution prevent the conversion and glycolysis of D-amygdalin.

Experimental Example 3. Application to natural products Each of 50mQ of 0.001% citric acid (pH 5.9) and 50mQ of 0.05% citric acid (pH 4.3) was added in round flask, 2g of Persicae semen (Chung Buk province, Moseangdang) officially approved in the Korean Pharmacopoeia (VII) was heated at 100 C for 120 mins under reflux. After extraction, was filtered with filter paper at once and moved to separated funnel. 50mQ of hexane was added thereto and hexane layer was removed after the extraction. The fractionation process was repeated 3 times. After diluting citric acid solution with two-fold of water, was filtered with disposable syringe filter unit of 0. 2am MFS 13. HPLC system was worked under the condition of Reference Example 1 and mobile phase used was 6% acetonitrile.

We applied these methods to the extraction process of D-amygdalin from Persicae semen. Therefore, we found out the inhibitory effects on the conversion to neoamygdalin with the percentage concentration of citric acid. The conversion rate to neoamygdalin in 0.005% citric acid solution was about 20% and pH was 5.7 (Fig. 4a), the conversion rate in 0.05% citric acid solution was about 10% and pH was 4.7 (Fig.

4b). In 0.05% citric acid solution, racemization of standard amygdalin was completely inhibited, but that of the amygdalin in Persicae semen was not completely inhibited because of the increase in pH due to various other constituents in Persicae semen. The conversion rate to neoamygdalin in 0. 1% citric acid solution was almost 0%, pH was 2.9 and about one-tenth of D-amygdalin is in the form of neoamygdalin (Fig. 4c). The

neoamygdalin originates from the native source of Persicae semen, as confirmed by the existence of neoamygdalin in cold methanol extract. Therefore, 0. 1-5% citric acid solution can prevent the conversion and glycolysis of D-amygdalin when D-amygdalin is extracted from Persicae semen.

Experimental Example 4. Separation of D-amygdalin and neoamygdalin The conditions of the mobile phase to separate D-amygdalin and neoamygdalin by reverse-phase HPLC were examined in 10 mM sodium phosphate buffer (pH 3.8) containing 1-butanol or acetonitrile. Sample was D-amygdalin and neoamygdalin prepared in Example 1. HPLC system was worked under the condition of Reference Example 1 and mobile phase used was sodium phosphate buffer containing 0. 2-0. 6% of 1-butanol (pH 3.8), 4-10% of acetonitrile (pH 3.8) and 4-10% of acetonitrile. The column flow rate was 1 m/inin.

As a result of experiment, complete baseline resolution was obtained using phosphate buffer containing 0. 3% 1-butanol, but it had the shortcoming of requiring 135 mins for separation (Fig la). The separation time became shorter by increasing the concentration of 1-butanol, but baseline resolution was not obtained.

When lOmM phosphate buffer (pH 3.8) containing 6% acetonitrile was used instead of 1-butanol, baseline resolution was obtained and the separation time was shortened to 45 mins (Fig. lb). When lOmM phosphate buffer (pH 3. 8) containing 4-5% acetonitrile was used, baseline resolution was obtained but separation time was lengthened, when lOmM phosphate buffer (pH 3.8) containing 7-10% acetonitrile was used, separation time was shortened but baseline resolution was not obtained. The result was almost similar in using 6% acetonitrile and lOmM phosphate buffer (pH 3.8) containing 6% acetonitrile, but baseline resolution was excellently obtained when lOmM phosphate buffer (pH 3. 8) containing 6% acetonitrile was used.

Therefore, it appeared that 10mM sodium phosphate buffer (pH 3.8) containing

6% acetonitrile was a suitable mobile phase and it was used it for the separation of D- amygdalin and neoamygdalin.

From above the results, we found out the optimum condition of inhibition of neoamygdalin conversion through racemization of D-amygdalin contained in Persicae Semen or Armeniacae Semen and the separation method thereof with HPLC.

INDUSTRIAL APPLICABILITY As described in the present invention, we confirmed the optimum condition of inhibition of neoamygdalin conversion through racemization of D-amygdalin contained in Persicae semen or Armeniacae semen, and provided the separating method of D- amygdalin and neoamygdalin using a reversed-phase HPLC. It can be used as the mass production method of D-amygdalin for anti-cancer agent.