TWI384993B - Loquat leaf cell extracts useful for hepatoprotection and/or reducing fat and preparation methods and uses of the same - Google Patents

Description

Extract for use in protecting liver and/or fat-reducing temporal lobe cells, and preparation method and use thereof

The present invention relates to the use of an extract of temporal lobe cells for protecting the liver and/or reducing fat, and a method of preparing the extract.

The scientific name of Essence is Eriobotrya japonica . The ancient Chinese name is Luzhi, also known as Jinmao or Luzhi. It is a genus of apple subfamily in rose, which is an evergreen small tree. The present study shows that the temporal lobe has a therapeutic effect of lowering body fat, anti-inflammatory, anti-tumor, hypoglycemic, and ameliorating abnormal metabolism. For a description, refer to Japanese Patent Laid-Open No. 2006104182, the disclosure of which is hereby incorporated by reference.

In addition, it has been confirmed by literature that triterpene acid components in loquat leaves have anti-inflammatory and anti-tumor activities (please refer to Bio1. Pharm. Bu11. 28(10), 1995-1999 (2005)); In US Pat. No. 6,908,632 B1, it is disclosed that corosolic acid (a triterpenic acid) has the function of regulating blood sugar. It is known from the above literature that the triterpenic acid component in sputum is effective for many diseases.

It has been found that triterpenic acid (such as tomentic acid, hyptadienic acid, maslinic acid and corosolic acid) can also be used in other plants besides mites. Obtained and has other biological activities. For example, in U.S. Patent No. 6,784,206 B2, it is taught that corosolic acid in the leaves of Lagerstroemia speciosa or Banaba can be used for weight loss and blood sugar regulation; Vochysia divergens The acid component of Potentilla chinensis can be used to slow down neuropathic or inflammatory pain (please refer to the European Journal of Pharmacology , 453 (2002), 203-208); in addition, behenic acid has the effect of lowering blood sugar (please refer to Biol. Pharm) Bull. 30(11) 2075-2078 (2007)).

Although triterpenic acid can be extracted from different plants, its content in natural plants is extremely low. If triterpenic acid is to be produced from natural plants, in order to meet market demand, a large number of plants must be planted and need to be complicated. The purification step and the use of a large amount of solvent, activated carbon and purified resin increase the manufacturing cost.

The industry has actively developed methods for the preparation of triterpenic acid, such as the use of tissue culture of temporal lobe cells (see Phytochemistry , 59, 315-323 (2002)), but its effect of increasing triterpenic acid content is limited. In WO 2007/004827 A1, a method for preparing corosolic acid is taught in which a high content of corosolic acid is obtained in a two-stage culture of eucalyptus leaves or leaves of Lagerstroemia speciosa. However, this method requires screening a cell strain with high productivity and adding an inducer to the culture solution, and requires a two-stage culture step, which is not only complicated in process but also high in manufacturing cost, and can only enhance the corosolic acid. The content is not beneficial to the increase of other triterpenic acid content. Therefore, a method for producing triterpenic acid conveniently and in large quantities is urgently required.

The present invention is directed to the above-mentioned needs, and the inventors of the present invention can greatly increase the content of triterpenic acid in the cultured loquat leaf cells by adding the loquat cell cell raffinate to the culture medium of the loquat leaf cells. In addition, the inventors of the present invention have further found that the extract of the temporal lobe cells has the effect of protecting the liver, and the active ingredient of the potentillanic acid and the salicylic acid in the extract can also be used for fat reduction.

A first object of the present invention is to provide an extract for protecting a temporal lobe cell of the liver, which comprises a compound of the following formula (1):

Wherein R ₁ and R ₂ are each independently hydrogen or methyl; R ₃ and R ₄ are each independently hydrogen, methyl or hydroxy; R ₅ is bonded to and formed by carbon atoms at positions indicated by Nos. 1 and 2. The structure of a six-membered ring of the following formula (2) or a five-membered ring of the following formula (3):

A second object of the present invention is to provide a method of preparing an extract of the above-mentioned temporal lobe cells.

A third object of the present invention is to provide a pharmaceutical composition comprising an effective amount of the above extract.

A fourth object of the present invention is to provide an application of the above-described extract of temporal lobe cells for the manufacture of a medicament for protecting the liver.

A fifth object of the present invention is to provide a pharmaceutical composition for reducing fat and/or protecting the liver comprising an effective amount of a compound of the following formula (4) and/or formula (5):

A sixth object of the present invention is to provide an extract for a reduced-fat temporal lobe cell comprising a compound having the above formula (4) and/or formula (5).

A seventh object of the present invention is to provide a process for the preparation of a compound of the above formula (4) and/or formula (5).

An eighth object of the present invention is to provide an application for the use of a compound of the above formula (4) and/or formula (5) or a pharmaceutically acceptable salt or ester thereof for the manufacture of a medicament for reducing fat and/or protecting the liver.

A ninth object of the present invention is to provide an use of an extract of eucalyptus cells for producing a fat reducing agent, wherein the extract comprises a compound having the above formula (4) and/or formula (5).

The detailed description of the present invention and the preferred embodiments thereof will be described in the following description.

The inventors of the present invention have carried out numerous in vitro cell experiments and in vivo animal experiments, and found that the extract of the temporal lobe cells has the effect of protecting the liver, and the active ingredient thereof is a compound of the following formula (1):

Wherein R ₁ and R ₂ are each independently hydrogen or methyl; R ₃ and R ₄ are each independently hydrogen, methyl or hydroxy; R ₅ is bonded to and formed by carbon atoms at positions indicated by Nos. 1 and 2. The structure of a six-membered ring of the following formula (2) or a five-membered ring of the following formula (3):

In particular, the extract of the temporal lobe cells of the present invention comprises one or more of the following triterpenic acid compounds:

Among them, the compounds of the formulae (4) to (7) are respectively a sulphuric acid, a potentic acid, a behenic acid and a corosolic acid.

More specifically, the extract of the temporal lobe cells of the present invention comprises a compound of the formula (4) and/or the formula (5).

In this article, the term "protecting the liver" covers the protection of liver damage caused by chemicals. For example, the extract of the present invention can be used to protect liver damage caused by chemicals such as carbon tetrachloride, ethanol, and thioacetamide or to inhibit liver fibrosis caused by liver damage.

The present invention also provides a method for preparing an extract of loquat leaf cells, comprising: a) cultivating a loquat leaf cell in a culture solution, wherein the culture solution comprises a loquat leaf cell raffinate and the loquat leaf cell; b) extraction of leaf cells cultured in step a).

Wherein the temporal lobe cell line in step a) is obtained by the following steps: 1) taking a leaf of a seedling with a wound, and growing a callus tissue cell to the wound; and 2) removing the callus Organize cells and incubate them. In the foregoing step 1), the leaves of the seedlings of the seedlings can be wounded in any manner. For example, the seedlings of the seedlings with the wound can be obtained by cutting, tearing or shearing, preferably by cutting. The method of stroke; afterwards, the callus can be grown in the wound of the leaves of the seedlings of the seedlings by any means known to those skilled in the art, for example, by using a solid medium and adding appropriate nitrogen sources, carbon sources, and plant growth. Regulators, cell division regulating hormones (eg, 6-benzyl adenine (BA), kinetin, or zeatin) or cell growth regulating hormones (eg, alpha-naphthylacetic acid (NAA), 2 , 4-dichlorophenoxyacetic acid (2,4-D) or indole acetic acid (IAA), etc., cultured for about 1 month, see Avanzados et al. , Plant Cell Culture Protocols, Methods in Molecular Biology , vol. 318, p. 59-70, the disclosure of which is incorporated herein by reference.

It is known that the callus cell line is a cell mass differentiated from plant tissue and has the potential to regenerate into a new plant having the same traits and genetic characteristics as the original plant. If the callus cells are cultured on a suitable medium, the roots or shoots can be formed through the organogenesis pathway, and further cultured, and finally the new alfalfa seedlings can be grown; if the cell suspension culture method is used to cultivate the callus Tissue cells, which can divide more new temporal lobe cells, see Ma et al. , Leaf callus induction and suspension culture establishment in lychee ( Litchi chinensis Sonn.) cv. Huaizhi, Acta Physiologiae Plantarum, vol. 31, p. 401-405, the disclosure of which is incorporated herein by reference. In some embodiments of the method of the present invention, the temporal lobe cells are obtained by cell suspension culture.

The loquat cell raffinate used in step a) of the method of the invention is obtained by the following steps: I) taking a leaf cell and extracting it with a concentration of from about 80% by volume to about 100% by volume of ethanol.枇杷 leaf cells; II) concentrating the extracted product of step I) to obtain a first concentrate; III) dissolving the first concentrate using a concentration of from about 40% by volume to about 60% by volume of methanol, collecting a first An undissolved portion; and IV) dissolving the first undissolved portion using a concentration of from about 70% by volume to about 95% by volume of methanol, and collecting a second undissolved portion to produce a loquat cell cell raffinate.

Wherein, the temporal lobe cells in the above step I) can also be obtained by the following steps: 1) taking a leaf of a seedling with a wound, and growing the callus cells to the wound; and 2) removing the callus The cells are incubated. Relevant details are as described above in the incubation step for the temporal lobe cells in step a).

Further, the ethanol concentration of the step I) is preferably from about 90% by volume to about 98% by volume; the methanol concentration of the step III) is preferably from about 45% by volume to about 55% by volume; and the methanol concentration of the step IV) is preferably It is from about 80% by volume to about 90% by volume. In some embodiments of the method of the present invention, when a concentration of about 50% by volume and about 85% by volume of methanol is used in steps III) and IV), respectively, a particularly satisfactory concentration of temporal lobe cell fraction can be obtained. In other words, the loquat cell raffinate prepared by the step of this particular partial aspect does not substantially contain about 50% by volume of the methanol soluble fraction and about 85% by volume of the methanol soluble fraction.

The content of the loquat cell cell raffinate in step a) is from about 0.5 ppm to about 20 ppm, preferably from about 1 ppm to about 5 ppm, most preferably about 1 ppm, based on the total amount of the culture solution. If the content of the loquat cell cell raffinate is less than 0.5 ppm, the effect of stimulating/inducing triterpenic acid production is limited; and if the content of the loquat leaf cell raffinate is higher than 20 ppm, the growth of the temporal lobe cells is inhibited, thereby Affect the yield of triterpenic acid.

According to the method of the present invention for preparing a eucalyptus cell extract, after the culturing procedure of step a) is completed, the extraction procedure of step b) is followed. Further, step b) substantially comprises the steps of: b1) extracting the leaf cells cultured in step a) using ethanol having a concentration of from about 80% by volume to about 100% by volume; b2) extracting the step b1) a product to obtain a second concentrate; b3) dissolving the second concentrate using a concentration of from about 40% by volume to about 60% by volume of methanol, collecting a third undissolved portion; b4) using a concentration of about 70% by volume Up to about 95% by volume of methanol dissolves the third undissolved portion and is filtered to collect the filtrate; and b5) the filtrate is concentrated as needed.

In some embodiments of the method of the present invention, the ethanol concentration of the above step b1) is preferably from about 90% by volume to about 98% by volume; and the methanol concentration of the step b3) is preferably from about 45% by volume to about 55% by volume; And the methanol concentration of step b4) is preferably from about 80% by volume to about 90% by volume. Further, the extraction and/or dissolution steps in the step b) may be repeated as many times as necessary to separate the active ingredient from the ineffective ingredient in the temporal lobe cells as much as possible, and extract all the active ingredients as much as possible.

In general, depending on the application form of the extract of the temporal lobe cells, the extract of the temporal lobe cells obtained in the step b) can be further dried as needed to facilitate transportation, storage or utilization. For example, a drying step (eg, concentration under reduced pressure and/or gas introduction) may be utilized to remove organic solvents remaining in the extract during the process. In one embodiment of the present invention, it is dried by concentration under reduced pressure to obtain an extract of white leafy cells of the loquat leaf.

In one embodiment of the present invention, the extract of the temporal lobe cells of the present invention is obtained by the step b) comprising the following procedure: the cultured temporal lobe cells are extracted with about 95% by volume of ethanol, filtered by a filter paper, and collected. The filtrate was concentrated under reduced pressure to obtain a concentrate. The concentrate was dissolved using about 50% by volume of methanol, filtered through a filter paper, and the undissolved portion was collected. The undissolved portion was dissolved using about 85% by volume of methanol, filtered through a filter paper, and the filtrate was collected → The filtrate was concentrated under reduced pressure to obtain an extract of eucalyptus cells. The above operation is as shown in Fig. 1. The extract of the eucalyptus cells obtained by the embodiment has substantially no about 50% by volume of a methanol soluble component and about 85% by volume of a methanol insoluble component, and contains a particularly satisfactory content of triterpenic acid. Active ingredient.

In particular, the method of the present invention stimulates/induces temporal lobe cells to produce high levels of triterpenic acid by adding eucalyptus cell raffinate to the culture solution, without the need to culture or screen a cell strain having high productivity through two-stage culture. . Compared to the prior art, the method of the present invention can produce triterpenic acid in large quantities economically and conveniently. In general, the triterpenic acid component is present in an amount of at least about 60% by weight, preferably at least about 75% by weight, more preferably at least about 80% by weight, most preferably at least about 85 weight percent, based on the dry weight of the extract of the temporal lobe cells. %.

The invention also relates to a pharmaceutical composition for protecting the liver comprising an extract of an effective amount of temporal lobe cells which can be obtained by the method of the invention. In the pharmaceutical composition for protecting the liver of the present invention, the content of the extract of the temporal lobe cells is not absolutely critical as long as it provides the benefit of the liver to be protected.

The pharmaceutical composition according to the present invention may be administered in any convenient manner, for example, but not limited thereto, and may be administered orally, subcutaneously or intravenously. Further, the pharmaceutical composition of the present invention can be used alone or in combination with a medicinal adjuvant, and can be practically used in veterinary and human medicine.

Furthermore, the present invention relates to the use of an extract of eucalyptus cells or an extract of eucalyptus cells prepared by the method of the present invention for the manufacture of a medicament which can be used to prepare an agent for protecting the liver in any suitable form. For example, when an extract of eucalyptus cells is used to manufacture an agent for protecting the liver, it is usually used in an amount of about 25 to about 90 mg per kilogram of body weight per day (4) and/or formula (5). The compound, preferably from about 30 to about 75 mg per kg of body weight, is a compound of formula (4) and/or formula (5).

Wherein, in preparing an agent suitable for oral administration, the extract of the temporal lobe cell of the present invention may be mixed with an adjuvant suitable for the project without adversely affecting the activity of the extract, for example, a solvent, an oil solvent, Diluent, stabilizer, absorption retarder, disintegrating agent, emulsifier, binder, lubricant, moisture absorbent, and the like. For example, the solvent may be selected from the group consisting of water and sucrose solutions, the diluent may be selected from the group consisting of lactose, starch, and microcrystalline cellulose, the absorption delaying agent may be selected from the group consisting of chitosan and aglycosyl polysaccharide, and the lubricant may be selected from the group consisting of Magnesium, an oily solvent may be selected from plant or animal oils such as olive oil, sunflower oil and cod liver oil. Here, a suitable oral administration form can be prepared by a conventional method, for example, a tablet, a capsule, a granule, a powder, a flow extract, a solution, a syrup, a suspension, an emulsion, an expectorant, and the like.

For pharmaceutical agents having a form suitable for subcutaneous or intravenous administration, the extract of the temporal lobe cells is mixed with materials such as solubilizers, emulsifiers, or other adjuvants as needed for the purpose of the preparation. Such as intravenous infusion, emulsion intravenous infusion, injection, dry powder injection, suspension injection, and dry powder suspension injection and so on. Solvents which may be employed are, for example, water, physiological saline solutions, alcohols (e.g., ethanol, propanol, or glycerol, etc.), sugar solutions (e.g., glucose or mannitol solutions), or combinations of the foregoing.

Optionally, in addition to the adjuvant component of the above-mentioned available pharmaceutical preparations, additives such as a flavoring agent, a toner, and a coloring agent may be added to improve the mouthfeel and visual feeling when the obtained medicament is taken; Preservatives, preservatives, antibacterial agents, antifungal agents, etc., to improve the storage properties of the resulting agents.

Furthermore, it is possible to further enhance the efficacy of the pharmaceutical composition (or pharmaceutical agent) of the present invention or to increase the flexibility and blending degree of the formulation of the pharmaceutical composition (or pharmaceutical agent) of the present invention, and one or more other active ingredients. As long as the other active ingredient does not adversely affect the benefit of the extract of the temporal lobe cells.

On the other hand, as shown in the examples below, the inventors of the present invention have unexpectedly discovered that the extract of eucalyptus cells has the following formula (4) and the potentic acid compound of the formula (5) has fat-reducing and / or protect the liver, such as reducing body fat accumulation or reducing blood triglyceride levels, it can be especially used to treat hypertriglyceridemia or weight loss, including weight loss, weight control, obesity and so on. Without being bound by theory, Xianxinshan adienoic acid or potentic acid can achieve the effect of reducing fat by inhibiting the formation of fat cells. As used herein, the term "fat-lowering" refers to reducing body fat and/or blood fat in the blood.

Accordingly, the present invention is also directed to a pharmaceutical composition for reducing fat and/or protecting the liver comprising an effective amount of a compound of the following formula (4) and/or formula (5):

Or a pharmaceutically acceptable salt or ester thereof; and an extract of a cell for reducing fat, which also comprises a compound of formula (4) and/or formula (5).

In natural loquat leaves, the content of the salicylic acid (i.e., the compound of formula (4)) is extremely low, even undetectable, and is also limited in the prior art. However, according to the method for preparing an extract of eucalyptus cells according to the present invention, a high content of an extract of sulphuric acid can be obtained, as confirmed in the following examples. According to the present invention, the extract of the loquat leaf cells typically contains at least about 3% by weight, preferably at least about 4% by weight, more preferably at least about 5% by weight, of the salicylic acid, based on the dry weight of the extract. Furthermore, the extract of the temporal lobe cells of the present invention typically contains at least about 20% by weight, preferably at least about 30% by weight, more preferably at least about 40% by weight, of the potentillanic acid, based on the dry weight of the extract.

Wherein, after the extract of the eucalyptus cell is obtained, a purification step can be used to obtain the sulphuric acid or the potentic acid, for example, by liquid phase partitioning or column chromatography. In one embodiment of the present invention, purification is carried out by high performance liquid chromatography to obtain the standard compounds of seledioic acid and potentic acid.

In the pharmaceutical composition for reducing fat and/or protecting the liver of the present invention, the content of the compound of the formula (4) and/or the formula (5) is not absolutely critical as long as it provides the desired fat loss and/or Or protect the liver's benefits.

Furthermore, the present invention also relates to the use of a compound of the formula (4) and/or formula (5) or a pharmaceutically acceptable salt or ester thereof or the use of a eucalyptus leaf comprising a compound of the formula (4) and/or formula (5) The use of an extract of cells for the manufacture of a medicament for use in reducing fat and/or protecting the liver.

Similar to the above-mentioned agent (or pharmaceutical composition) for protecting the liver, the agent for reducing fat can also be prepared in any suitable form, and can be adversely affected without having an influence on the formula (4) (ie, salicylic acid). And / or an adjuvant or other active ingredient of the compound of the formula (5) (ie, potentic acid) is mixed to increase the applicability of the medicament.

In general, when an extract containing eucalyptus cells containing a compound of the formula (4) and/or the formula (5) is used for the manufacture of a medicament for fat reduction, it is usually about 75 per kilogram of body weight per person per day. Up to about 180 mg of the compound of formula (4) and/or formula (5), preferably from about 90 to about 150 mg per kg of body weight of the compound of formula (4) and/or formula (5).

The invention is further illustrated by the following specific embodiments. The embodiments are provided for illustrative purposes only and are not intended to limit the scope of the invention.

[Examples] [Example 1] Preparation of extract of loquat leaf cells

(1) Establishing sterile seedlings

From the orchard of Toutunkeng, Taichung County, the seeds of the mature carp ( Eriobotrya japonica Lindl . ) were collected and rinsed with water for 30 minutes. The seeds were soaked in 70% ethanol for about 1 minute, then immersed in a 1% sodium hypochlorite solution containing 0.01% Tween 20, placed in an ultrasonic oscillator, surface sterilized for about 15 minutes, and then transferred to an aseptic workstation. The seeds were washed three to four times with sterile water, and the seeds were cultured in MS medium (Murashige-Skoog medium) containing 30 g/liter of sucrose. Two to three weeks later, the seeds began to germinate, and the leaves of the seedlings two months after the germination were collected for use as test materials.

(2) Inducing callus tissue

The leaves of the seedlings were cut into about 0.3 cm and transferred to a solid medium (MS basic formula containing 2.5 ppm of 6-benzyl adenine (BA, 6-benzyladenine), 1 ppm of α-naphthylacetic acid (NAA, α- Naphthaleneacetic acid), 3% (30 g/L) sucrose and 0.3% (3 g/L) crystal culture (gelrite). One month later, a pale yellow callus grew from the cut wound.

(3) cultivating temporal lobe cells

Approximately 60 grams of the above pale yellow callus (which can be differentiated into newer temporal lobe cells) was sieved through a 590 micron sieve to disperse the callus cells to a certain size and then transferred to a culture containing 4,000 ml. Culture (BioFlo110 Bioreactor, New Brunswick Scientific, USA) in a liquid (MS basic formula containing 2.5 ppm BA, 1 ppm NAA and 3% (30 g/L) sucrose) (culture conditions: aeration of 0.3 Vvm (gas volume flow per unit of liquid volume per minute), stirring speed of 40 rpm, temperature of 24 to 26 ° C). A new medium was subcultured every ten days, and each time it was subculture, 500 ml of the old suspension cell solution (i.e., the culture solution) was left, and then 4,500 ml of the new culture solution was added. Transfer another 4,500 ml of old suspension cell solution (containing about 320 g of fresh cell weight) into a 25.5 liter medium (MS base containing 2.5 ppm BA, 1 ppm NAA, and 3% (30 g/L) sucrose) A bioreactor (30 liter stainless steel bottle) was added with 1 ppm of the loquat cell cell raffinate to the culture to continue to culture the differentiated temporal lobe cells. After 18 days, the culture was terminated.

(4) Preparation of loquat cell cell raffinate

The cultured eucalyptus cells were obtained in a separate culture batch, dried (about 5,400 g before drying, about 320 g after drying), and extracted by refluxing with 95% by volume of ethanol three times, and then filtered by filter paper. The filtrate was collected and concentrated under reduced pressure. The concentrate was dissolved in 50% by volume of methanol, dissolved twice, and then filtered through a filter paper to obtain an undissolved portion. Thereafter, the undissolved portion was dissolved again with 85% by volume of methanol, dissolved three times, filtered with a filter paper, and the undissolved portion was collected to obtain a loquat cell cell raffinate.

(5) Preparation of temporal lobe cell extract

The cultured loquat leaf cells were obtained in a separate culture batch, dried at 60 ° C (about 5,400 g before drying, about 320 g after drying), and refluxed with 10 liters of 95 vol% ethanol. After three times, it was filtered through a filter paper, and the filtrate was collected and concentrated under reduced pressure. The concentrate was dissolved in 5 liters of 50 vol% methanol, dissolved twice, and then filtered through a filter paper to obtain an undissolved portion. Thereafter, the undissolved portion was dissolved with 10 liters of 85 vol% methanol, dissolved three times, filtered through a filter paper, and the filtrate was collected and concentrated under reduced pressure to obtain about 53 g of a white powdery eucalyptus cell extract. The loquat leaf cell extract obtained by the above procedure does not substantially contain 50% by volume of a methanol soluble component and 85% by volume of a methanol insoluble component.

(6) Purification of triterpenic acid compounds

An extract of about 1 gram of white powdered eucalyptus cells was placed on a silicone gel (LiChroprep RP-18, E. Merck, 40 to 63 microns) and dispensed at a methanol to water ratio of 8:2 to 10:0. , using preparative high performance liquid chromatography (HPLC, pump: Shimadzu LC-8A (Japan, Kyoto); mobile phase: 85 vol% methanol; flow rate: 3 ml / min; column: YMC, J' The Sphere series ODS-H80 column (10 mm inner diameter, 250 mm long, 5 micron particle size) is purified to give two triterpenic acid compounds.

The purified compound was analyzed by mass spectrometry (Jeol GCmate, Tokyo, Japan) and NMR ( ¹ H, ¹³ C, DEPT, COSY, HMQC, HMBC, Jeol 500 MHz, Tokyo, Japan), wherein the main component was confirmed to be a mountain diene Acid (hyptadienic acid, compound of formula (4)) and tonicic acid (compound of formula (5)).

Saponic acid (7.2 mg): ¹ H-NMR (pyridine-d ₅ ): δ 0.98, 1.08, 1.15, 1.17, 1.42, 1.70 (each 3H, s, H-23 to H-29), 1.10 ( 3H, d , J = 6.6 Hz, H-30), 3.04 (1H, s , H-18), 4.44, 4.56 (each 1H, d , J = 14.9 Hz, H-1), 5.60 (1H, t , J = 3.4 Hz, H-12). ¹³ C-NMR (pyridine-d ₅ ): δ 16.7 (C-30), 17.6 (C-6), 18.8 (C-26), 18.9 (C-25) , 21.7 (C-24), 25.3 (C-27), 26.9 (C-21), 27.0 (C-11), 27.1 (C-16), 27.1 (C-29), 29.6 (C-15), 30.1 (C-23), 34.5 (C-7), 38.4 (C-22), 41.9 (C-4), 42.3 (2C, C-8, C-14), 42.4 (C-9), 43.6 ( C-20), 48.2 (C-17), 50.9 (C-10), 54.7 (C-18), 60.8 (C-1), 63.6 (C-5), 72.6 (C-19), 128.1 (C -12), 133.6 (C-3), 140.2 (C-13), 156.7 (C-2), 180.6 (C-28).

Potentillanic acid (25.3 mg): ¹ H-NMR (pyridine-d ₅ ): δ 0.92 (3H, s, H-24), δ 1.00 (3H, s, H-25), δ 1.12 ( 3H, s, H-26), δ 1.12 (3H, s, H-30), δ 1.28 (3H, s, H-23), δ 1.43 (3H, s, H-29), δ1. 65(3H, s, H-27), 1.74 (1H, t, J = 12.7 Hz, H-1), 1.90 (1H, dd, J = 12.0, 3.8 Hz, H-1), δ 2.34 (1H) , td, J = 13.2, 4.1 Hz, H-15), δ 3.05 (3H, s, H-18), δ 3.14 (1H, td, J = 13.1, 4.1 Hz, H-16), δ 3. 77(3H, s, H-3), δ 4.31 (1H, dt, J = 10, 2.6 Hz, H-2), δ 5.59 (1H, s, H-12). ¹³ C-NMR (pyridine -d ₅ ): δ17.1 (C-30), 17.2 (C-25), 19.1 (C-6), 22.8 (C-24), 17.7 (C-26), 24.5 (C-11), 25.1 (C-27), 26.9 (C-16), 27.4 (C-21), 27.6 (C-29), 29.7 (2C, C-15, C-23), 34.0 (C-7), 39.0 (C -22), 39.1 (C-10), 39.3 (C-4), 41.1 (C-8), 42.6 (C-1), 42.8 (C-20), 43.3 (C-14), 48.1 (C- 9), 48.7 (C-17), 49.2 (C-5), 55.1 (C-18), 66.6 (C-2), 73.2 (C-19), 79.8 (C-3), 128.5 (C-12) ), 140.4 (C-13), 181.2 (C-28).

(7) Determination of the content of triterpenic acid in the extract of temporal lobe cells

The triterpenic acid content of the eucalyptus cell extract was determined by HPLC. The conditions used for HPLC were as follows: the pump system Shimadzu LC-10ATvp; the refractive index detector Shimadzu RID-10A; the column column HyPURITY C-18 column (the inner diameter was 4.6 mm, the length was 250 mm, and the particle size was 5 μm). The solvent system was methanol/0.15 vol% aqueous acetic acid, methanol: 0.15 vol% aqueous acetic acid=85:15 by volume, flow rate was 0.5 ml/min, and temperature was 35 °C. As shown in Table 1, the content of the salicylic acid was about 5% by weight based on the dry weight of the extract of the loquat leaf cells, and the content of the potentic acid was about 39% by weight.

As shown in Table 2, the content of the triterpenic acid component in the original plant is not high, and the content which is enhanced by the tissue culture method is also limited, so it is uneconomical to use the loquat leaf as the source of the triterpenic acid. However, compared with the prior art, by using the method for preparing the extract of the temporal lobe cells of the present invention, a high content of four triterpenic acid components can be obtained, and the total content is as high as 173.9 mg / g (on a dry cell basis). .

Furthermore, it is worth noting that, according to the disclosure of WO 2007/004827 A1, temporal lobe cells can only be cultured in a 250 ml container, and the preparation method of the present invention can be applied to containers of a size of 150 liters or more.

[Example 2] Extract of temporal lobe cells protects liver of mice Experiment A, extract of temporal lobe cells inhibits liver inflammation and liver fibrosis and reduces liver damage

Thirty-six ICR mice (purchased from Lesco Biotech) were divided into four groups, one group being the control group, and the other three groups were induced with liver damage and liver fibrosis in mice by carbon tetrachloride. Mice of the experimental group were administered 0.5% by weight of carboxymethylcellulose (CMC) vehicle or eucalyptus cell extract (70 or 200 mg/kg body weight), respectively.

On the other hand, induction of liver fibrosis in mice was performed. ICR mice were given twice weekly carbon tetrachloride (10% by volume in olive oil (0.1 ml/10 g body weight)) for eight weeks. During the induction period, mouse extracts were administered daily. At the end of induction, blood was collected from the peritoneal vein of the mouse for anesthesia (carbon anesthesia) for determination of plasma biochemical values. In addition, the liver of the mouse was quickly removed and washed with ice physiological saline. Thereafter, the liver was divided into four portions and each was immersed in 10% by volume of neutral formalin for pathological sectioning. One of them was dried at a temperature of 100 ° C for determination of the degree of fibrosis of the liver, and the other was stored at -80 ° C for determination of glutathione.

In addition, after obtaining blood from the mice, they were centrifuged at 4,700 rpm for 15 minutes and measured using an automatic biochemical analyzer (Cobas Mira, Roche, Rotkreuz, Switzerland) and a commercially available reagent (Roche Diagnostics, Mannheim, Germany). The activity of glutamate transaminase (ALT) and aspartate transaminase (AST) in mouse plasma. Among them, glutamic acid aminotransferase and glutamic acid oxalic acid transaminase are indicators of liver inflammation, and the results are shown in Table 3.

The determination of proteins in liver tissue was performed according to the method of Lowry, and bovine plasma protein was used as a standard (see, for example, Lowry et al . Protein measurement with the folin phenol reagent. J. Biol . Chem . 1951; 193:262-275). The contents of this document are hereby incorporated by reference. The results are shown in Table 4.

The determination of glutamine sulphate in liver tissue is based on the method of Hissin, which can be found in Hessin et al . A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal . Biochem . 1976; 74: 214-226, The contents are hereby incorporated by reference. Among them, weigh 0.5 g of liver tissue, add 5 ml of 1.15 wt% potassium chloride solution, homogenize with a homogenizer, take 1 ml of homogenate, then add 1 ml of 10 wt% trichloroacetic acid, After mixing uniformly, it was centrifuged at 3000 g for 15 minutes. Next, take 0.01 ml of the supernatant and add 0.18 ml of phosphate-EDTA buffer and 0.01 ml of σ-phthaldehyde (1 mg/ml methanol) solution. After mixing uniformly, the fluorescence was detected at a wavelength of 420 nm and the excitation light wavelength was 350 nm. The content of glutamine-based sulfur is represented by "micromol/gram structure", and the results are shown in Table 4.

Chronic hepatitis or liver damage can lead to liver fibrosis, and liver fibrosis is mainly caused by the proliferation of extracellular matrix collagen. Among them, hydroxyproline is a special amino acid in collagen, so it is determined in the liver. The amount of hydroxyproline can be used to estimate the extent of liver fibrosis. The determination of the content of collagen (or hydroxyproline) in liver tissue is based on the method of Neuman, which can be found in Neuman et al . The determination of hydroxyproline. J. Biol. Chem . 1950; 184: 299-306, The contents are hereby incorporated by reference. Among them, after the liver tissue was hydrolyzed, it was subjected to hydrogen peroxide oxidation, and then p -dimethylaminobenzoaldehyde ( p- dimethylaminobenzoaldehyde) was colored, and the absorbance was measured at a wavelength of 540 nm. The content of hydroxyproline was expressed in "micrograms per gram of tissue", and the results are shown in Table 4.

After the liver tissue was fixed with formalin, the wax was embedded and sliced, and staining was performed using two kinds of staining methods, one was general HE staining (hematoxylin and eosin stain), and the other was It is a special staining method for collagen, Sirius Red staining. The determination of liver fibrosis was performed using an image analysis system (Image-Pro Plus version 5.1, Media Cybernetics, Maryland, USA) to analyze the proportion of liver fibrosis. The above test results are shown in Fig. 2, Fig. 3 and Table 5.

As can be seen from Table 3, the extract of temporal lobe cells can inhibit the increase of plasma ALT and AST activity in mice caused by carbon tetrachloride. This result shows that the extract of temporal lobe cells can alleviate the liver damage caused by carbon tetrachloride. inflammation.

In addition, as can be seen from Table 4, the liver damage caused by carbon tetrachloride in mice causes a decrease in the protein content in the liver, and because of its ability to inhibit the synthesis of proteins in the liver, it causes a decrease in the protein content in the liver, and tetrachlorine. The oxidative stress or damage caused by carbonization consumes and reduces the content of glutamine-based sulfur in the liver, while Table 4 shows that the extract of the temporal lobe cells of the present invention can increase the content of protein and glutamine-based sulfur, indicating that it can alleviate Liver damage. Furthermore, as shown in Fig. 2, the pathological section also showed that the extract of the temporal lobe cells can improve the severe necrosis of hepatocytes caused by carbon tetrachloride.

The results in Table 4 also show that the extract of the temporal lobe cells can reduce the increase in the hydroxyproline content in the liver caused by carbon tetrachloride, and therefore, the extract of the temporal lobe cells can inhibit liver fibrosis. As can be seen from Fig. 3 and Table 5, the results of the pathological section also showed that the extract of the temporal lobe cells inhibited liver fibrosis.

Experiment B, extract of temporal lobe cells inhibits the performance of cell surface receptor CD14

Extracts from temporal lobe cells may reduce liver fibrosis by inhibiting inflammation. For this mechanism, see Salminea et al ., Terpenoids: natural inhibitors of NF-κB signaling with anti-inflammatory and anticancer potential. Cell Mol. Life Sci 2008; : 2979-2999, the contents of which is hereby incorporated by reference. Carbon tetrachloride causes an increase in lipopolysaccharide (LPS) in the blood. Lipopolysaccharide enters the liver and acts on the surface receptor CD14 of the liver macrophage (Kupffer cell) to activate The reservoir cells and promote the production of cytokines, causing inflammatory reactions leading to liver damage. For this mechanism, see Su, 2002. Lipopolysaccharides in liver injury: molecular mechanisms of Kupffer cell activation. Am. J. Physiol. 283, G256-G265, This document is hereby incorporated by reference. Therefore, immunostaining of the surface receptor CD14 was carried out, and it was observed whether the temporal lobe cell extract reduced liver fibrosis by inhibiting inflammatory action by observing the expression of CD14.

First, after the pathological section in the above experiment A was subjected to dewaxing and alcohol dehydration treatment, the endogenous peroxidase was removed with 3 vol% hydrogen peroxide, and then 5% by weight of milk was added, and reacted for 30 minutes to block the non- A combination of specificity. Next, the CD14 antibody was added, and the mixture was incubated at room temperature for 2 hours, then washed with a phosphate buffer solution (PBS), and a secondary antibody was added thereto, followed by incubation at room temperature for 30 minutes. Colorimetric assays were performed using immunodetection kits (purchased from BioGenex, San Ramon, Canada) and diaminobenzidine (DAB), stained with hematoxylin, dehydrated and mounted, and the results are shown in Figure 4. Shown.

As can be seen from Fig. 4, carbon tetrachloride can promote the performance of CD14, while the extract of temporal lobe cells can inhibit the expression of CD14. Therefore, the extract of temporal lobe cells can inhibit the activation of the cells or inhibit the liver. Inflammatory response.

From the results of Tables 3 to 5 and 2, 3, and 4, it can be seen that the extract of the temporal lobe cells inhibits inflammation of the liver and inhibits liver fibrosis.

[Example 3] Extract of temporal lobe cells, potentic acid and squalic acid inhibit activation of liver bank cells

Since nitric oxide is produced after activation of the cells, the concentration of nitric oxide can be measured to observe the activation of the cells or the degree of inflammation of the liver.

The liver bank of the rat (purchased from Leko Biotech Co., Ltd.) was isolated according to the following method: First, the rat was anesthetized, and then 0.05% by weight of the fourth type dissolved in HBSS (Hanks' balanced salt solution) was used. Collagenolytic enzyme (type IV collagenase) was perfused at a flow rate of 20 ml per minute (200 ml total). Thereafter, the rat liver was taken out and cut, and then digested with 0.5 g/liter of type IV collagen degrading enzyme for 10 minutes. Next, the liver samples were filtered with a sterile nylon guze, and the filtrate was washed and centrifuged at a gradient of 50%/25% by volume percoll solution. After centrifugation, 25% percoll and 50% percoll of cell fluid were collected, centrifuged again, and the isolated cells were cultured in DMEM containing medium (Dulbecco's modified Eagle's medium, Hyclone, USA, containing 10% fetal bovine serum, 100 The active unit/liter of penicillin, 100 μg/ml of streptomycin, and 2 mmol/L of L-glutamic acid in 96-well plates (5×10 ⁵ cells/well). After 15 minutes of culture, the culture supernatant was removed, and then DMEM medium was added. After 24 hours of culture, the medium was changed, and different concentrations of leaf cell extracts (10, 50 or 100 μg/ml) and potentic acid were added. 5, 25 or 50 μg/ml), or succinic acid (10, 25 or 50 μg/ml), for another 60 minutes. Thereafter, a lipopolysaccharide of 0.1 micromolar concentration was added to the medium, and after 24 hours, the supernatant of the culture medium was collected, and the content of nitric oxide was measured using Griess reagent (purchased from Sigma). MTS (3-(4,5-di-methylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, available from Promega, Madison, Wisconsin, USA) Cell viability was measured. Among them, the principle of action of MTS is: since living cells have the activity of dehydrogenase, MTS can be reduced to a reddish purple water-soluble product, and its maximum absorbance at a wavelength of 490 nm, therefore, the absorbance value The ratio can be used to determine the survival rate of the cells, and the cell survival rate is calculated by the following formula. The results of the above tests are summarized in Table 6.

Survival rate = absorbance of the experimental group / absorbance of the control group × 100%

It can be seen from Table 6 that the lipopolysaccharide at a concentration of 0.1 micromolar does not cause the cell to die. Under this condition, the cell extract of the loquat leaf, the acid of the potentic acid and the seletaric acid can be inhibited by the lipopolysaccharide. One of the nitrogen oxides is induced and has a concentration dependence. Therefore, the above results demonstrate that the extract of the eucalyptus cells, the potentic acid and the seletaric acid can achieve the effect of inhibiting liver fibrosis by inhibiting the activation of the cells or the inflammatory reaction of the liver.

[Example 4] Extract of temporal lobe cells inhibits high fat diet induced mouse obesity Experiment A, extract of temporal lobe cells inhibited body weight and fat weight in mice

Experiments were performed on male C57BL/6 mice (purchased from Taipei Leko Biotech Co., Ltd.). 24 mice were divided into four groups, one group was fed normal feed (control group), and the other three groups were fed high fat feed 58Y1 (purchased from TestDiet) and 0.5% by weight of carboxymethyl cellulose solvent, 200 mg/ Extracts of eucalyptus cells in kilograms or 400 mg/kg, and recorded weekly average food intake and body weight changes for each mouse for a period of four weeks. The results are shown in Tables 7 and 8 below.

After the mice were subjected to the above-mentioned four weeks of experimentation with carbon dioxide, blood was collected and the fat on both sides of the sputum was taken out, and after weighing, a part of the fat was soaked in 10% by volume of neutral fumarin.

As shown in Table 7, the food intake of the mice fed the high-fat diet was lower than that of the mice fed the normal feed, and in the mice administered with the extract of the temporal lobe cells, the dose was 400 mg/kg. The rats' food intake decreased only in the first week, while the second to four-week food intake did not significantly affect.

As shown in Table 8, after four weeks, the weight of the mice fed with the high fat diet was significantly higher than that of the control group, and compared with the mice administered with CMC, the mice administered with the extract of the temporal lobe cells. The final weight, the absolute weight of the by-line fat and the relative weight (as a percentage of body weight) are lower.

Experiment B, extract of temporal lobe cells inhibited the concentration of total cholesterol and triglyceride in mouse plasma

The concentrations of total cholesterol and triglyceride in mouse plasma were measured using an automatic biochemical analyzer (Cobas Mira, Roche, Rotkreuz, Switzerland) and a commercially available reagent (Roche Diagnostics, Mannheim, Germany). The results are shown in Table 9.

As shown in Table 9, the final plasma total cholesterol and triglyceride concentrations of the mice administered with the extract of the temporal lobe cells were lower than those of the mice administered with CMC.

Experiment C, extract of temporal lobe cells inhibited the growth of mouse adipocytes

The fat sections obtained in Experiment A were subjected to H.E. staining, and the diameters of the fat cells were analyzed by an image analysis system. The results are shown in Tables 10 and 5.

The results of the fat cell analysis of Tables 10 and 5 showed that the mice which were subjected to the high dose of the extract of the temporal lobe cells had smaller adipocyte diameters than the mice administered with CMC. Therefore, the extract of the temporal lobe cells of the present invention has an effect of inhibiting the growth of fat cells.

Tables 7 to 10 and Fig. 5 clearly show that the extract of the temporal lobe cells of the present invention can effectively reduce the formation and content of body fat, and also has the effect of lowering blood fat.

[Example 5] Extracts of temporal lobe cells, potentic acid and squalic acid inhibited adipocyte differentiation

In an incubator with a saturated humidity of 5% carbon dioxide and a temperature of 37 ° C, in a DMEM medium (Hyclone, USA) containing 10% by volume of fetal bovine serum (Hyclone, USA), 1 × 10 ⁴ cells 3T3-L1 preadipocytes were cultured at a density of / square centimeter.

The 3T3-L1 cells were cultured in a 24-well plate for two days, and the cells were subjected to monolayer confluents and subjected to contact inhibition, and a differentiation agent (including 5 μg/ml insulin, 0.5 mmol/min) was administered. Liters of IBMX (3-isobutyl-1-methylxanthine) and 1 micromolar/liter DEX (dexamethason), and Dimethyl sulfoxide (DMSO) or Extracts of eucalyptus cells at different concentrations (0.5, 5 or 50 μg/ml), potentic acid, or seletaric acid were cultured. Two days later, the culture medium (DMEM medium containing 5 μg/ml insulin and 10% by volume of FBS) was renewed, and the culture solution was changed every two days until the eighth day after the addition of the differentiation agent.

During the culture, under the observation of the microscope, 3T3-L1 cells became spherical and enlarged from the spindle shape, and oil droplets distributed in the cytoplasm were found. At this time, 3T3-L1 cells had differentiated into adipocytes (adipocyte). ). The fat cells were fixed with 10% by volume of neutral formalin for one hour. After staining with Oil red O (0.1 mg/ml) for two hours at room temperature, the oil droplets were stained red, and the cells were washed three times with deionized water. Thereafter, it was observed under a microscope and photographed, followed by dissolution of the oil droplets with a quantitative amount of isopropyl alcohol. Finally, the absorbance was measured at a wavelength of 492 nm, and the inhibition rate of adipocyte differentiation by extracts of eucalyptus cells, oleic acid, or sulphuric acid was calculated. The inhibition rate of cell differentiation was calculated by the following formula.

Inhibition rate of differentiation (%) = [(differentiation agent + dimethyl hydrazine) absorbance - control group absorbance - (differentiation agent + test substance) absorbance] / [(differentiation agent + dimethyl hydrazine) absorbance - Control group absorbance value] × 100%

Further, in the presence of a differentiation agent, the effect of the test substance (i.e., extract of eucalyptus cells, oleic acid and selenate) on the survival rate of 3T3-L1 cells was observed by MTS, and the results are shown in Table 11.

Survival rate = absorbance of the experimental group / absorbance of the control group × 100%

It can be clearly seen from Fig. 6 and Table 11 that the extract of the temporal lobe cells, the potentic acid and the salicylic acid can inhibit the differentiation of the fat cells. Therefore, in this example, it was first proved that potentillanic acid and seleric acid have the effect of reducing fat.

The above embodiments are merely illustrative of the principles and effects of the invention and are not intended to limit the invention. Modifications and variations of the above-described embodiments can be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention should be as set forth in the scope of the patent application described hereinafter.

Figure 1 is a flow chart showing the preparation of extracts of temporal lobe cells in an embodiment of the present invention;

Figure 2 is a diagram showing the H.E. staining of liver tissue cells after the extract of the temporal lobe cells was administered to mice which were induced by carbon tetrachloride to induce liver fibrosis;

Figure 3 is a Sirius Red staining diagram of liver tissue cells after administration of extracts of temporal lobe cells to mice subjected to liver fibrosis induced by carbon tetrachloride;

Figure 4 is a diagram showing the immunostaining of CD14 of the cells of the cells after administration of the extract of the temporal lobe cells to mice which have been induced by carbon tetrachloride in liver fibrosis;

Figure 5 is a micrograph of the fat cells of the extract of the temporal lobe cells after administration to the mouse;

Fig. 6 is a staining diagram showing differentiation of a temporal lobe cell extract, a potentic acid, or a salicylic acid into a fat cell after administration of 3T3-L1 preadipocytes.

Claims (15)

A method for preparing an extract of a temporal lobe cell, comprising: a) cultivating a loquat leaf cell in a culture solution, wherein the culture solution comprises a loquat leaf cell raffinate and the loquat leaf cell, and the loquat leaf cell raffinate The system is obtained by the following steps: I) taking a leaf cell and extracting the leaf cells with a concentration of about 80% by volume to about 100% by volume of ethanol; II) concentrating the extracted product of step I) to Obtaining a first concentrate; III) dissolving the first concentrate using a concentration of from about 40% by volume to about 60% by volume of methanol, collecting a first undissolved portion; and IV) using a concentration of about 70% by volume to about 95% by volume of methanol dissolves the first undissolved portion, and a second undissolved portion is collected to prepare the temporal lobe cell raffinate, and the temporal lobe cell line is obtained by the following steps: 1) taking a wound The leaves of the seedlings are planted, and the callus cells are grown from the wound; and 2) the callus cells are removed and cultured; and b) the temporal lobe cells cultured in step a) are extracted. The method of claim 1, wherein the amount of the loquat cell cell raffinate in step a) is from about 0.5 ppm to about 20 ppm based on the total amount of the culture solution. The method of claim 1, wherein the amount of the loquat cell cell raffinate in step a) is from about 1 ppm to about 5 ppm based on the total amount of the culture solution. The method of claim 1, wherein the temporal lobe cell line in step I) is obtained by the following steps: 1) taking a leaf of a seedling with a wound, and growing the callus cell after the wound; and 2) taking out The callus cells are incubated. The method of claim 1, wherein the concentration of the ethanol in the step I) is from about 90% by volume to about 98% by volume, and the concentration of the methanol in the step III) is from about 45% by volume to about 55% by volume, and the step IV The concentration of methanol is from about 80% by volume to about 90% by volume. The method of any one of claims 1 to 3, wherein the step b) comprises the step of: b1) extracting the temporal lobe cells cultured in the step a) using an ethanol having a concentration of from about 80% by volume to about 100% by volume; b2) Concentrating the extracted product of step b1) to obtain a second concentrate; b3) dissolving the second concentrate using a concentration of from about 40% by volume to about 60% by volume of methanol to collect a third undissolved portion; b4 The third undissolved portion is dissolved using methanol having a concentration of from about 70% by volume to about 95% by volume and filtered to collect the filtrate; and b5) the filtrate is concentrated as needed. The method of claim 6, wherein the concentration of the ethanol in the step b1) is about From 90% by volume to about 98% by volume, the concentration of methanol in the step b3) is from about 45% by volume to about 55% by volume, and the concentration of methanol in the step b4) is from about 80% by volume to about 90% by volume. A pharmaceutical composition for protecting the liver, comprising an effective amount of the extract obtained by the method of any one of claims 1 to 7. A pharmaceutical composition according to claim 8 which is for protecting liver damage caused by a chemical substance or inhibiting liver fibrosis. The pharmaceutical composition of claim 9, wherein the chemical is carbon tetrachloride, ethanol or thioacetamide. An application of the extract obtained by the method of any one of claims 1 to 7 for the manufacture of a medicament, wherein the medicament is for protecting the liver. The use of claim 11, wherein the agent is for protecting liver damage caused by a chemical substance or inhibiting liver fibrosis. The use of claim 12, wherein the chemical is carbon tetrachloride, ethanol or thioacetamide. A method for producing a compound of the following formula (4) and/or formula (5), which comprises the method of preparing a loquat cell extract by the method of any one of claims 1 to 7, and performing a purification Steps to obtain a compound of formula (4) and/or formula (5): The method of claim 14, wherein the purifying step comprises performing a high performance liquid chromatography.