TW201114433A - Loquat leaf cell extracts useful for hepatoprotection and/or reducing fat and preparation methods and uses of the same - Google Patents

Abstract

A loquat leaf cell extract and a pharmaceutical composition useful for hepatoprotection and/or reducing fat and comprising the compound of formula (1): are provided. Also provided are a method for the preparation of a loquat leaf cell extract and a use of the loquat leaf cell extract for the manufacture of a medicine.

Description

201114433 VI. Description of the Invention: [Technical Field] The present invention relates to an application of an extract of temporal lobe cells for protecting the liver and/or reducing fat, and a method for preparing the extract. [Prior Art] The scientific name of the scorpion is the name of the Chinese ancient name Lugan, also known as Jinmao, or the genus of the apple branch of the family Rosaceae, which is an evergreen small tree. The present study shows that the leaves have the effect of lowering body fat, anti-inflammatory, anti-tumor, blood sugar lowering and improving 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 (refer to 28 (10), 1995-1999 (2005)); and US patents US 6,9〇 8,632 B1, reveals that corosolic acid (a triterpenic acid) in the sputum extract 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 in addition to the batch, the two private acids (such as tomentic acid, hyptadienic acid, maslinic acid and strontium corosinate) may also be derived from other plants. _Get 'and have other biological activities. For example, in U.S. Patent No. 6,784,206 B2, it is taught that Corydalis in the leaves of B. sinensis (7) (10) μ • speck (10) or Banaba can be used for weight loss and regulating blood sugar; The acid component of Potentilla in the family can be used to slow down neurological or inflammatory pain (please refer to Ewrope (10) < 201114433 抑顾卿/吧,453 (2002), 2〇3_2〇8); in addition, behenic acid has The effect of lowering blood sugar (凊Review 0/· PWwm〇( 11 ) 2075-2078 ( 2007 )). The three IS acids can be extracted from different plants, but they are extremely low in natural plants. The right is to make triterpenic acid from natural plants. In order to meet market demand, plants must be planted. It also requires complicated purification steps and the use of a large amount of solvent, activated carbon and purified resin, thereby increasing the manufacturing cost. The industry has actively developed methods for the preparation of tri-salt, such as the use of tissue culture batches of leaf cells (clear reference corpse / ^ ioc / ze / m. kiss, 59, 315-323 (fine 2)), but its promotion The effect of tri-sodium acid content is limited. In WO 2007/004827 A1, a method for preparing corosolic acid is taught, which is a method of cultivating a high content of corosolic acid in a two-stage culture of eucalyptus or large flower leaf cells. However, this method must first screen cell lines with high productivity and add inducer to the culture medium, and it must undergo a two-stage culture step. 'Not only the process is quite complicated, but also the manufacturing cost is high, and only the corosolic acid can be raised. The content is not beneficial to the increase of other three (four) content. Therefore, a method for producing trisuccinic acid conveniently and in large quantities is urgently needed. The present invention is directed to the above-mentioned needs, and the inventors of the present invention can greatly increase the content of the three cells of the cultured loquat leaf cells by adding the leaf cell cell extract to the culture medium of the 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 ingredients of the potentic acid and the scented dilute acid in the extract can also be used for fat reduction. SUMMARY OF THE INVENTION A first object of the present invention is to provide a sample of a leaf cell for protecting a liver, which comprises a compound of the following formula: 201114433

Wherein, R and R2 are each independently hydrogen or a fluorenyl group; and the dentites 3 and I are each independently a gas sulfhydryl group or a hydroxy group, and the carbon atoms are bonded to the carbon atoms at positions indicated by No. 1 and No. 2 and form the following formula ( 2) The structure of the six-membered ring or the five-membered ring of the following formula (3):

(2) A second object of the present invention is to provide a method for preparing an extract of the above-mentioned batch of leaf cells. This "Month of the Month" is intended to provide a pharmaceutical composition & an effective amount of the above extract. A fourth object is to provide an application for the use of an extract of the above-mentioned temporal lobe cells for the manufacture of a medicament for protecting the liver. A fourth 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 formulas (4) and (5)^(5):

• A sixth object of the invention is to provide an extract for a fat-reducing 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. An eighth object of the present invention is to provide a 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. The ninth object of the present invention is to provide an agent for using a extract of eucalyptus cells to produce 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. [Embodiment] The inventors of the present invention found that the extract of the stalk leaf cells has the effect of protecting the liver after performing numerous in vitro cell experiments and animal loyalty tests in vivo, and the composition of the bitterness 201114433 is as follows (1) ) Compound:

Wherein I is independently hydrogen, and Ri and R2 are each independently hydrogen or a hydrazine; r3 and a methyl or hydroxy group; and R5 is bonded to a carbon atom at positions indicated by No. 1 and No. 2 and forms the following formula (2) The structure of the six-membered ring or the five-membered ring of the following formula (3):

HO

HO

Containing one or more of the following specific words, the extract of the temporal lobe cells of the present invention is a succinic acid compound: 201114433

Among them, the compounds of the formulae (4) to (7) are respectively a succinic 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). • As used herein, 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 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; b) extraction step a) cultured loquat leaf 201114433 wherein the loquat leaf cell line in step a) is obtained by: taking a leaf of a seedling with a wound and waiting for the wound to grow callus ( (10) cells; and 2) removing the callus cells and incubating them. In the foregoing step, the leaves of the seedlings of the seedlings can be wounded in any manner, for example, by cutting, tearing or shearing, obtaining the seedlings of the seedlings with the wound σ, preferably using the After that, 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 a suitable nitrogen source, carbon source, and plant growth regulator. , cell division regulating hormone (such as 6 · adenine (BA), phlegm (4) (10) (4), or zeatin (zeatin), or cell growth regulating hormone (such as α_naphthaleneacetic acid (naa), 2,4- Diphenoxypheneacetic acid (2,4_D) or (tetra)acetic acid (IAA)) #, cultured for about 2 months, see Avanzados for α/,p丨_(10)Cu-ep,

Methods in Μ-r Biobgy, vo丨, 318, p 59_7〇, 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 then the new shoots can be grown; if the cell suspension culture method is used By cultivating the callus cells, they can divide more new sputum leaf cells. See Ma Heart, Leaf ca||us mducti〇n _ sqing (10) i〇n culture establishment in lychee {Litchi chinensis Sonn.) Cv Huaizhi Acta Physio丨ogiae P丨antarum, v〇丨3丨, p 4〇丨·4〇5, 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. 10 201114433 The temporal lobe cell raffinate used in step a) of the method of the invention is obtained by the following steps: I) taking a batch of loquat leaf cells and extracting with a concentration of about 5% by volume to about 5% by volume of ethanol The loquat cell; U) the concentrated product of the step 丨) to obtain a first concentrate; III) dissolving the first concentrate using a concentration of from about 4% by volume to about 6% by volume of decyl alcohol Collecting a first undissolved portion; and IV) dissolving the first undissolved portion with a concentration of from about 70% by volume to about 95% by volume of methanol, collecting a first undissolved portion to obtain a temporal lobe cell raffinate Things. The temporal lobe cells in the above step 丨) can also be obtained by the following steps: ^) taking the leaves of the seedlings having the wound, and growing the callus cells after the wound, and 2) removing the callus cells And cultivated. The 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 9% by volume to about 5% by volume. The methanol concentration of the bovine sputum is preferably from about 45% by volume to about 55% by volume; Preferably, it is from about 80% by volume to about 9% by volume. In the method of the present invention

In the embodiment, when the concentration is about % by volume and about 85% by volume of methanol, respectively, in the step m) and the step IV), a particularly satisfactory concentration of the leaf fine residue can be obtained; in other words, The batch of leaf cell dwellings prepared by the steps of this particular partial aspect does not substantially contain about 5% by volume of methanol soluble partial volume / 〇 methanol soluble fraction. One to δ) In the total amount of the culture solution, the step a) allows the leaf cell to be depleted from 〇5 to about 约, preferably from about 1 to about 5 _, optimal = ::. If the content of leaf cell raffinate is lower than 〇_ a stimulus/induction:" The effect of self-sufficiency is limited; and if the content of sputum leaf cell raffinate is higher than: 201114433, it will inhibit the leaf blast cells. Growth, thereby affecting the yield of triterpenic acid. According to the method of the present invention for preparing a temporal lobe cell extract, after the completion of the culture procedure of step a), the extraction procedure of step b) is followed. Further, step b) is substantially the following steps: bl) extracting the temporal lobe cells cultured in step a) using ethanol having a concentration of about 80% by volume to about 1% by volume; b2) concentration step b 1) Extracting the product to obtain a second concentrate; b3) dissolving the second condensate using methanol having a concentration of about 4,000 vol% to about 60 vol%, collecting a third undissolved portion; The third undissolved portion is dissolved in about 70% by volume to about 95% by volume of methanol and filtered to collect the filtrate; and b5) the filtrate is concentrated as needed. In some embodiments of the method of the present invention, the above step is preferably an ethanol concentration of from about 90% by volume to about 98% by volume. The concentration of methanol is preferably from about μ% by volume to about 555% by volume; and the concentration of sterol in step b4) is preferably about 8 Torr. /〇 to about 90% by volume. Further, the extraction and/or dissolution steps in the plurality of steps can be repeated as needed, and the active ingredient in the temporal lobe cells can be separated from the ineffective components as much as possible, and all the active ingredients can be extracted as much as possible. In general, depending on the application form of the extract of the temporal lobe cells, the extract of the leaf cells obtained in the step b) can be completely dried as needed to facilitate transportation, storage or utilization. For example, a drying step (e.g., concentration under reduced pressure and/or gas introduction) may be utilized to remove the organic solvent remaining in the extract during the process. In the embodiment of the present invention, the extract was dried to obtain an extract of white powdery loquat cells. In an 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: extracting the cells of the cultivar 12 201114433 with about 95% by volume of ethanol, filtering with filter paper The filtrate was collected and concentrated under reduced pressure to obtain a concentrate. The concentrate was dissolved with 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 vol% of methanol to filter paper. Filtration, collection of the filtrate ► The filtrate was concentrated under reduced pressure to obtain an extract of the cells of the loquat leaf. The above operation is shown in Figure 1. The extract of the temporal lobe cells prepared by this embodiment does not substantially contain about 50% by volume of the sterol soluble component and about 85% by volume of the methanol insoluble component, and contains a particularly satisfactory amount of triterpenoids. Acid active ingredient. 4 , , , , , , , , , , , , , , , , , 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由. Compared with the prior art, the method of the present invention can be produced in a large amount economically and conveniently. In general, the content of the tri-acid component is at least about 6% by weight based on the dry weight of the extract of the temporal lobe cells. At least about 75% by weight, more preferably at least about 8% by weight, most preferably at least about 85% by weight. The invention also relates to a pharmaceutical composition for protecting the liver comprising an extract of an effective amount of eucalyptus cells. The extract 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 stalk leaf 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 by subcutaneous or intravenous administration. Further, the pharmaceutical composition of the present invention can be used alone or in combination with a pharmaceutical adjuvant, and can be practically used in veterinary medicine and human medicine. Furthermore, the present invention relates to the use of an extract of eucalyptus cells or a fine mosquito extract obtained from the method of the present invention, 13 201114433, which can be used to prepare a liver for protecting the liver in any suitable form. Pharmacy. For example, when an extract of eucalyptus cells is used for the manufacture of (4) for pour (four), usually about 25 to about 90 mg of compound of formula (4) and/or formula (5) per kilogram of body weight per day is used. Preferably, the compound of formula (4) and/or formula (5) is from about 3 to about 75 gram per kilogram of body weight. Wherein in the preparation of a medicament suitable for oral administration, the extract of the present invention: eucalyptus cells can be adapted to the project without adversely affecting the activity of the extract:

= agent, such as: solvent, oily solvent, thinner, An (four), absorption of soil Μ, Peng powder, emulsifier, adhesive 1 f 4, interslip agent, moisture absorbent and so on. For example: from water and ship liquid 'diluent can be selected from lactose, _ and _ 14 late agent can be selected from chitosan and glucosamine poly::: etc.: the agent can be selected from plant or animal oils, Such as 撖 sand oil, sunflower - two •: can: use ^ π W capsules, granules, suspensions, emulsions, cutting agents, etc. 4, solution, «agent,

As for the subcutaneous or static _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ pulse infusion, a solvent that can be used, for example: liquid injection and the like. . κ physiological saline solution 'alcohol alcohol, glycerol, etc.), sugar solution (~ 贞 (for example: ethanol, a combination of C. (d) sugar or mannitol solution), or as described above, except for the above In addition to the adjuvant component of the Xile preparation, an additive such as a tempering agent, a toner, a coloring agent, and the like may be added to improve the mouthfeel and visual feeling of the obtained medicament; and a reasonable amount of preservative and antiseptic may be added. An antibacterial agent, an antifungal agent, or the like, for improving the storage property of the obtained drug. Further, the pharmaceutical composition (or agent) of the present invention may be further contained in the pharmaceutical composition (or agent) of the present invention to further enhance the pharmaceutical composition of the present invention. The efficacy of the substance (or pharmaceutical agent) or the flexibility and degree of formulation of the formulation of the preparation, 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 in the following examples The inventor of the present invention unexpectedly found that the extract of the leaf cells of the eucalyptus leaf has the following formula (4) and the potentilla compound of the formula (5) has a fat-reducing and/or The effect of protecting the liver, such as reducing body fat accumulation or saltiness, blood, and glycerol, is particularly useful for the treatment of hypertriglyceridemia or weight loss, including weight loss, weight control, obesity, etc. Without being bound by theory, Xianxinshan aromanic acid or potentic acid can achieve the effect of reducing fat by inhibiting the formation of fat cells. In this paper, the word "fat reducing" covers reducing tissues. The body fat and/or blood fat in the blood. Therefore, the present invention also relates to a pharmaceutical composition for reducing fat and/or protecting the liver, which comprises an effective amount of the following formula (4) and/or formula ( 5) Compound:

15 201114433 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 the natural loquat leaves, the content of the mountain succinic acid (i.e., the compound of the formula (4)) is extremely low, and even cannot be measured, and the amount which can be improved in the prior art is also limited. 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 temporal lobe 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 2 ounces by weight. /. Preferably, it is about 30% by weight, more preferably at least about 4% by weight of 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 selenic acid or potentic acid, for example, by liquid phase partitioning or column chromatography, etc. The morphology was purified by high performance liquid chromatography to obtain the standard compounds of sulphuric 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) or the formula (5) is not absolutely critical as long as the fat-reducing and/or liver-protecting benefits are provided. can. Further, the present invention relates to the use of a compound of the formula (4) and/or the formula (5) or a pharmaceutically acceptable salt thereof or the use of the formula (4) and/or the formula (5). The extract of the leaf cell is used in the manufacture of the medicament, and the medicament is used in the system; the fat is and/or the liver is protected. 16 201114433 Similar to the above-mentioned agents (or pharmaceutical compositions) for protecting the liver, the agent for reducing fat can also be prepared in any suitable form, and can be used without adverse effects (4) (ie, scented ene The adjuvant or other active ingredient of the acid (and acid) and/or the compound of the formula (5) (ie, potentic acid) is mixed to increase the applicability of the agent. 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. To about 180 mg of the compound of formula (4) and/or formula (5), preferably from about 90 to about 15 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 by way of illustration only and are not intended to limit the scope of the invention. [Example 1 [Example 1 丨 Preparation of extract of eucalyptus cells (1) Establishment of sterile seedlings from the harvesting of the mature 枇杷 (―, 』P ^ Lmd1·) seeds in the orchard of the head of the pit in Taichung County Rinse for 30 minutes. Soak the seeds with 70% ethanol for j 1 knife, then immerse them in a solution containing 0 〇 1% Tween 2〇 次% sodium sulphate: and place them in an ultrasonic absorber for surface disinfection for about 15 minutes before moving To the aseptic console. The seeds were washed three to four times with sterile water, and the seeds were cultured in MS medium (broken (8) § _ coffee) containing 3 gram grams per liter of sucrose. After two to three 4 'the seeds began to germinate', the leaves of the seedlings two months after the germination were collected for use as test materials. 17 201114433 (2) Callus tissue The leaves of the seedlings are cut into about 0.3 cm and transferred to solid medium (MS basic formula contains 2.5 ppm of 6-benzyl adenine (BA, 6_benzyladenine), Cultured in 1 ppm alpha-naphthaleneacetic acid (NAA), 3% (30 g/L) sucrose, and 0.3% (3 g/L) crystal geir (geirite). One month later, a pale yellow callus grew from the cut wound. (3) cultivating eucalyptus cells to take about 60 g of the above-mentioned pale yellow callus (which can be differentiated into new scutellaria sylvestris) and sieved through a 590 micron sieve to disperse the callus cells into a certain size. After that, transfer it to a bioreactor containing 4,0 (8) ml of culture medium (2.5 ppm BA in MS basic formula, 1 ppm NAA & 3% (3 gram gram / liter) sucrose) (BioFlol 1 〇 Bioreactor'New Cultured in Brunswick Scientific, USA) (culture conditions: aeration volume of 0.3 vv m (gas volume flow per unit of liquid volume per minute per minute), stirring speed of 40 rpm, The temperature is 24 to 26 «>C) (J is subcultured every 10 days, and each subculture, leaving 5 ml of the old suspension cell solution (ie, cultured liquid), and then added 4,500 ml of new culture medium. Transfer another 4,5 ml of old suspension cell solution (containing about 32 g of fresh cell weight) into 25.5 liters of culture medium (MS basic formula contains 2.5 ppm BA, 1 ppm) NAA and 3% (30 g/L) cane) A bioreactor (3 liter liter stainless steel bottle) was added with 1 ppm of eucalyptus cell raffinate to the culture medium to continue to culture the differentiated temporal lobe cells. After 18 days, the culture was terminated. 4) Preparation of eucalyptus cell raffinate 18 201114433; In a separate culture batch, cultured eucalyptus cells are obtained and dried (dry Μ 5, gram gram 'about 32 () gram after drying), and After reflux extraction three times with % by volume of ethanol, the mixture was filtered through a filter paper, and the mash was collected and concentrated under reduced pressure. The concentrate was dissolved in 50% by volume of decyl alcohol, dissolved twice, and then transferred to the paper to obtain undissolved. After 0f5 knives, '85 vol% sterol was used to dissolve the undissolved part, dissolved three times to take care of, and the undissolved part was collected to obtain the eucalyptus cell raffinate. (5) Preparation of eucalyptus leaves Cell extract Φ In a separate culture batch, cultured loquat cells were obtained and dried at 60 ° C (about 5,400 g before drying, about 32 g after drying), and 95 liters in ι 5% by volume of ethanol for reflux extraction After that, the paper was passed through a paper, and the liquid was collected and concentrated under reduced pressure. After dissolving the condensate in 5 liters of 5 vol% of methanol, the condensate was dissolved twice, and then filtered through a filter paper to obtain an undissolved portion. Thereafter, 1 再 was used. The liters of 85 vol% of methanol dissolved the undissolved portion, dissolved three times, filtered through a filter paper, collected mash, and concentrated under reduced pressure to obtain about 53 g of a white powdery eucalyptus cell extract. The eucalyptus cell extract obtained by the above procedure does not substantially contain 5% by volume of the sterol-soluble component and the 5% by volume of the sterol-insoluble component. (6) Purification of triterpenic acid compound The extract of 1 g of white powdered eucalyptus cells in a spoon was placed on a lycopene (L丨Chroprep RP-18, E. Merck, 40 to 63 μm) with a sterol pair. The water ratio is 8:2 to 10:0, and the preparative high-performance liquid chromatography (HPLC 'Jipu: Shimadzu LC-8A (Japan, Kyoto); mobile phase: 85 vol. 0/〇) Methanol; flow rate: 3 ml / min; column · YMC, factory Sphere series ODS-H80 column (inner diameter 丨〇 mm, length 25 〇 mm, particle size is $ 19 201114433 μm) purification, can get two A triterpenic acid compound. The purified compound was analyzed by mass spectrometry (Jeol GCmate 'Japan, Tokyo) and NMR (丨Η, 丨3C, DEPT, COSY 'HMQC, HMBC, Jeol 500 MHz, 曰本, Tokyo) The main component was confirmed to be hyptadienic acid (a compound of the formula (4)) and tonicic acid (a compound of the formula (5)).

Shandienic acid (7.2 mg): iH-NMR (pyridine-d5): (5 0.98, 1.08, 1.1 5, 1. Π, 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, 5, H-18), 4.44, 4.56 (each 1H, d, J = 14.9 Hz, Hl), 5.60 · ( 1H,i, / = 3.4 Hz, H-12). 13C-NMR (pyridine-d5): 316.7 (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-ll), 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-I4), 42.4 (C-9), 43.6 ( C-20), 48.2 (C-17), 50.9 (C-10), 54.7 (C-18), 60.8 (Cl), 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). 20 201114433 Potentillanic acid (25.3 mg): 'H-NMRp ratio pyridine-d5) J 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), δ 1.65 (3H, s, H-27), 1.74 (1H, t, 7 = 12.7 Hz, Hl ), 1.90 ( 1H, dd, /= 12.0, 3.8 Hz, Hl ), ό 2.34 (1H, td, J= 13.2. 4.1Hz, H-15), δ 3.05 (3H, s, H-18), δ 3.14 (1H, td, /= 13.1, 4.1 Hz, H-l6 ), <5 3.77 (3H, s, H-3 ), δ 4.31 (1H, dt, ·/= 10, 2.6 Hz, H-2 ), (5 5.59 (1H, s, H-12). I3C-NMR (mouth-d5): <517.1 (C- 30), 17.2 (C-25), 19.1 (C-6), 22.8 (C-24), 17.7 (C-26), 24.5 (C-ll), 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 (Cl), 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 triterpenic acid content of extract of eucalyptus cells The triterpenic acid content of eucalyptus cell extract was determined by HPLC. The conditions used for hplC are as follows: the pump system Shimadzu LC-lOATvp; the refractive index detector Shimadzu RID-10A; the column column HyPLiRiTY C-18 column (the inner diameter is 4.6 mm, the length is 250 mm, and the particle size is 5 μm). The solvent system was methanol/〇丨5 vol% aqueous acetic acid 曱 sterol: 0.15 vol% aqueous acetic acid = 85:15 volume ratio, flow rate 〇5 ml/min, temperature 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. · 21 201114433 Table 1 Culture batch of dry weight of alfalfa leaf cells (g) Triterpenic acid content (mg/cell dry weight) Extract dry weight of alfalfa leaf cells (g) Eucalyptus dilute acid content of alfalfa leaf cell extract ( %) Potentillanic acid content in loquat leaf cell extract (%) Total triterpenic acid content in loquat leaf cell extract (%) Control group (no added loquat cell cell raffinate) 269.9 66.8 29.35 2.52 18.08 66.9 1 323.4 192.6 53.7 5.46 39.02 80.8 2 346.1 162.86 55.3 4.19 40.57 83.21 3 310.5 165.24 41.9 5.56 37.73 75.57 Table 2 \Source ingredient eucalyptus (mg/g) Tissue culture Phytochemistry, 59, 315-323 (2002) (mg/g) Two-stage culture W02007/004827A1 (mg/g) Eucalyptus cells prepared by the method of the present invention (mg/g) Potentillanic acid 0 20 - 73.7 Shandienic acid 0 1.2 - 10.1 Behenic acid 0.8 5.6 - 28.0 Corosolic acid 4.6 6.3 110.95 62.1 Total 5.4 33.1 110.95 173.9

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 WO2007/004827A1, 枇 22 201114433 杷 leaf 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 36 ICR mice (purchased from Lesco Biotechnology) The company was divided into four groups, one group was the control group, and the other three groups were induced by the four-gasification carbon to induce sputum damage and liver fibrosis in mice. The mice were divided into 5% by weight of carboxymethylcellulose (CMC) vehicle or eucalyptus cell extract (70 or 200 mg/kg body weight). On the other hand, induction of liver fibrosis in mice was performed. ICR mice were administered twice a week with four carbonized carbon (10 vol% 'dissolved in eucalyptus oil (〇 1 ml / 1 〇 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 immersed in 10 volumes of 〇/〇 neutral formalin for pathological sectioning. One of the wounds was dried at a temperature of 丨00 〇C for liver fibrosis & ampere measurement' and the other portions were stored at _8 〇〇C for determination of glutathione. In addition, 'after taking the blood of the mouse', it was centrifuged at 4,700 rpm for 15 knives' and used an automatic biochemical analyzer (Cobas Mira, Roche, Rotkreuz, Switzerland) and a commercially available reagent (Hoche Diagnostics, Mannheim, Germany). The activity of alanine transaminase (ALT) and glutamic acid 23 201114433 aspartate transaminase (AST) was measured in mouse blood. Among them, the index of transaminase aminotransferase and glutamic acid oxalic acid transaminase 'enzyme liver inflammation, the results are shown in Table 3. The determination of protein in liver tissue was carried out according to the method of Lowry, and the bovine plasma protein was used as a standard (this can be found in Lowry cz/. Protein measurement with the folin phenol reagent. ·/. 5ζ·ο/_ CAem. 1951; 193 : 262-275, the contents of which is incorporated herein by reference. The determination of the amino-based sulfur of the liver tissue t is based on the method of Hissin, which can be found in the Hessin et al. A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal. Biochem. 1976; 74: 214-226, the document The contents are hereby incorporated by reference. Among them, weigh 0.5 gram of liver tissue 'and add 5 ml of 1 · 15% by weight potassium chloride solution, homogenize with homogenizer, take 1 ml of homogenate' and add 1 ml of 1 〇 wt% three Gaseous acetic acid, after mixing well, centrifuged at 3000 g for 15 minutes. Next, take 1 ml of the supernatant, and add 0.18 ml of phosphate-EDTA buffer and 〇.1 ml of σ-o-phthalaldehyde (σ-phthaldehyde ' 1 mg/ml sterol) solution was mixed and detected with a fluorescence of 420 nm and an excitation wavelength of 350 nm. The content of the surface amine sulfur is expressed 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. The amount of hydroxyproline can be used to estimate the extent of liver fibrosis. The determination of collagen (or lysine) in liver tissue by 24 201114433 is based on the method of Neuman, see Neuman ei a/. The determination of hydroxyproline. J. Biol. Chem. 1950; 184: 299- 306, the disclosure of which is incorporated herein by reference. Among them, the liver tissue was hydrolyzed, and the absorbance was measured at a wavelength of 540 nm by hydrogen peroxide oxidation and coloring with p-dimethylaminobenzoa丨dehyde. 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. Liver fibrosis was measured 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. table 3

' Wi ALT AST _ (mg / kg) (activity unit / liter) (activity unit / liter) $ group ~ 32.2 ± 7.6 62.4 ± 15.7 ~ ~ ~ four gasification carbon + CMC - 2833.1 ± 815.9_ 2415.5 ± 602.8_ Four carbonized carbon extracts 70 2107.9 + 631.5 1743.8 ±615.0 gasified carbon + extract __200__1851.41538 0* 1383.1 ± 669.1** The values of the stones are average ± standard error sampling number = 9). The phase is controlled by ^ρ<〇〇〇1 ;- compared to the four gasified carbon plus CMC group, *P<〇.〇5, **p< o.oi. 25 201114433 Table 4 Dose protein (mg/kg) (mg/g tissue) Control group four gasified carbon + CMC four gasified carbon + extract four gasified carbon + extract via proline (microgram / gram tissue) 鞑Amino sulphur (micromole / gram of tissue) 176.5 soil 11,4 1 Β · 4 ± 14.9 _ 131.3 ± 6.2 * 221.5 soil 33.9 363.8 ± 51.6 ^ — 298.8 soil 53.7 * compared to the four gasification carbon plus CMC group, Control group,, <0·001; 70 200 32.2 ±5.4 19.2 Soil 6.0 foot 26.413.3* 28.6+4.3** ***P< 0.001

Him-~ Four gasified carbon + CMC. H±〇·6 fine Four gasified carbon + extract 7. ^±17 J gasification carbon + extract 200 21ΪΠ7ΙΙ ***P< 0.001

#户^Values are the average number of samples of the corpse<ο·οοι; compared to the four gasified carbon plus CMC group' (1)I- As can be seen from Table 3, the extract of the temporal lobe cells can be inhibited by the four gasified carbon The increase in plasma ALT and AST activity in mice> This result indicates that the extract of leaf cells can alleviate the inflammation of the liver caused by four-carbonized carbon. In addition, as can be seen from Table 4, the liver damage caused by the gasification of the gasification of the four gasifications will cause a decrease in the protein content in the liver, and the protein content in the liver is decreased due to its ability to inhibit the synthesis of proteins in the liver. The oxidative stress or damage caused by the four gas (four) consumes and reduces the content of the amino-based sulfur in the liver, and Table 4 shows that the extract of the leaf cells of the present invention can increase the content of the protein and the face amine base, indicating that Can reduce liver damage. Furthermore, as in the second _*, the pathological W (four) shows that the stroke of the temporal lobe cells can improve the severe necrosis of the hepatocytes caused by the four gasified carbon. The results in Table 4 also show that the extract of the leaf cells of the mites can reduce the growth of the liver towel _(4) containing the four gasified carbons. Therefore, the extract of the leaf cells can inhibit liver fibrosis 26 201114433. 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, the extract of the temporal lobe cells inhibits the performance of the cell surface receptor CD丨4

Extracts from temporal lobe cells may reduce liver fibrosis by inhibiting inflammation. For this mechanism, see Salminea ei d·, Terpenoids: natural inhibitors of NF-kB signaling with anti-inflammatory and anticancer potential. Ce" A/o/. Li /e 2008; 65: 2979-2999, the disclosure of which is incorporated herein by reference. The four-gasification carbon will cause the increase of lipopolysaacharide 'LPS in the blood. After the lipopolysaccharide enters the liver, it can interact with the surface receptor CD14 of the macrophage (Kupffercell) to activate Libupolysaccharides in liver injury: molecular mechanisms of Kupffer cell activation, dm. J. 283, G256-G265, the mechanism of cytokine production and the promotion of cytokine production, causing inflammatory reactions, which may be referred to in Su, 2002. Lipopolysaccharides in liver injury: molecular mechanisms of Kupffer cell activation, dm. J. 283, G256-G265 The contents are hereby incorporated by reference. Therefore, the immunostaining of the surface receptor CD14 was carried out by observing the expression of CD 14 to understand whether the temporal lobe cell extract reduced liver fibrosis by inhibiting inflammatory action. 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. Subsequently, the cD丨4 antibody was added, and after incubation at room temperature for 2 hours, it was washed with a phosphate buffer solution (pBS), and a secondary antibody was added thereto, followed by incubation at room temperature for 30 minutes. Using an immunodetection kit (purchased from BioGenex, San Ramon, Canada) and diaminobenzidine 27 201114433 (diaminobenzidme, DAB) for coloration, followed by heparin staining, dehydration and sealing, the results are as follows Figure 4 shows. 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 Figs. 2, 3, and 4, it is understood that the extract of the eucalyptus cells can inhibit liver inflammation and inhibit liver fibrosis. [Example 3] Extract of temporal lobe cells, potentic acid and selenate inhibit the activation of liver bank cells. Since nitric oxide is produced after activation of cells, the concentration of nitric oxide can be determined by measuring the concentration of nitric oxide. 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 reused in a 0.05% by weight type 4 dissolved in HBSS (Hanks, balanced salt solution). Collagen degrading enzyme (type ιν 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/L of the fourth type collagenase to reach a minute. Next, the liver sample was passed through 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 Active unit/liter of penicillin, 100 μg/201114433 ml of streptomycin, and 2 mmol/L of L-glutamic acid in 96-well plates (5χ105 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 eucalyptus cell extract (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, 0.1 μmol of lipopolysaccharide was added to the medium, and after 24 hours, the supernatant of the medium was collected, and the content of the gas was determined using Griess reagent (purchased from Sigma). MTS(3-(4,5-di-methylthiazol -2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliu m, purchased from Promega, Madison, Wisconsin , USA) measures cell viability. 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 X 100%

Table 6 Concentration survival rate (%) Nitric oxide inhibition rate (%) Control group 100 - Lipopolysaccharide + dimethyl sulfoxide 119_9 ± 14.1 0 (DMSO) Lipopolysaccharide + extract 10 μg / ml 101.7 ± 16.6 5.0 ± 5_6 Lipopolysaccharide + extract 50 μg / ml 98.5 ± 7.3 25.6 ± 5.9 (4) Lipopolysaccharide + extract 100 μg / ml 95_3 ± 8.2 37.7 ± 3.9 (4) Control group 100 - Lipopolysaccharide + dimethyl sulfoxide - 113.0 ± 0.1 0 Lipopolysaccharide + Potentillanic Acid 5 μg / ml 114.3 ± 2.6 1.3 Soil 1.4 Lipopolysaccharide + Potentillanic Acid 25 μg / ml 102.3 ± 1.6*** 47.8 ± 3.9 *** Lipopolysaccharide + Potentillax 50 μg / ML 104.5 ± 2.6 ** 87.1 ± 1.2 control group 100 - 29 201114433 Lipopolysaccharide + dimercapto sulphate - 102.0 ± 24.7 〇 lipopolysaccharide + succinic acid 10 μg / ml 103.8 ± 27.9 29.1 soil 10.4 * lipopolysaccharide + Shandienic acid 25 μg/ml 94.9± 14.8 43.1 ± 10.4** Lipopolysaccharide + succinic acid 50 μg/ml 113.6 ± 35.8_75.9 ±2.5** All values are average soil standard error (sample number = 3). Compared with the control group, _ household <0.001; compared to lipopolysaccharide plus dimethyl sub-group, ^ (0.05, ** P < 0.01. As can be seen from Table 6, 0.1 micro-mole concentration of fat The polysaccharide does not cause the cell to die. Under these conditions, the eucalyptus cell extract, the potentic acid and the sulphuric acid can inhibit the production of one of the nitrogen oxides induced by the lipopolysaccharide, and have a concentration dependence. Therefore, the above results demonstrate that the extract of the loquat leaf 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 liver inflammatory reaction. [Example 4] The extract of temporal lobe cells inhibits obesity induced by high-fat diet. Experiment A. Extracts of temporal lobe cells inhibited body weight and fat weight in mice. Male C57BL/6 mice (purchased from Taipei Lesco Biotech Co., Ltd.) were used for experiments. 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 respectively. / kg or 400 mg / kg of loquat leaves The extracts of the cells were recorded, and the weekly average food intake and body weight change of each mouse were recorded for a period of four weeks. The results are shown in Tables 7 and 8 below. After the mice were subjected to carbon dioxide anesthesia for four weeks after the experiment, blood was collected. 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 normal feeding. In the mice fed the feed, and the mice fed the extract of the temporal lobe cells, the dose of 30 201114433 for the mice of 400 mg/kg decreased only for the first week, while the second to four weeks of food intake There was no significant effect. 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 the extract of the temporal lobe cells was administered compared to the mice administered with CMC. The final body weight of the mice, the absolute weight of the scorpion fat and the relative weight (as a percentage of body weight) were lower.

Dosage __ (mg/kg) 1 ^ Food intake (g) Week 2 Week 3 Week 4 3. 4 r 〇. β 3· 4 ± 〇· 3 3. 3 ± 0. 3 3· 6 ± 0. 2 2,1 ± 2. 5±0.521 Control Group"" South fat feed + CMC ~ ^IIWW 200 2.3 ± 0.2 2.5 ± 0.2 2. 5 ± 0. 2 2.5 ± 0. South fat feed + extraction物400_1. ± 0.3 木土 2 4 + 0 2 ? 5 + 0 All values are mean soil standard error (sample number compared to control group, . 1 compared to high fat feed plus CMC group, 叩 < 0.05.,·' Table 8 2. 2 ± 〇. 2. 5 ± 〇. 4ί: 0· 5"

Dosage (mg/kg) Precursor t (g) Dipper fat Final weight absolute weight Relative weight control group 24.9 ± 1.7 27. 1 ± 1.3 0. 5 ± 0· 1 1-7 ± 0.2 High fat feed + CMC 29.3 ± 34.9. Ten 1.9 soil 0·5:: ί 5. 3 ± 〇. 9fai 1.6*·* 2. 6*'1** Pulp feed + extract 200 28.7 ± 1.7 .33. 2 ± 2.3 1.6 ± 0.4 U ± 1.0 400 28.7 ± 1.9 30. 8 ± 4. 1* 1. 3 ± 0.4* 4. 2 ± 〇. 7* All values are the mean ± standard error (samples = 6). Compared to the control group, Na p < 〇.〇〇1 ·, compared to the high fat feed plus CMC group ' * P < 0.05. 31 201114433 Experiment B, extract of temporal lobe cells inhibits the concentration of total cholesterol and triglyceride in mouse plasma using an automatic biochemical analyzer (Cobas Mira, Roche, Rotkreuz, Switzerland) and commercially available reagents (Roche Diagnostics, Mannheim, Germany) The concentration of total cholesterol and triglyceride in mouse plasma was determined, and the results are shown in Table 9. Table 9 'Deterage Total Cholesterol Triglyceride

__(mg/kg) (mg/min liter)_(mg/min liter) 5 纽 - 85.8 ± 10.6 67.2 ± 5. 4 High fat feed + CMC High fat feed + extract 200 400 130.4 ± 8.5ts < 118.4 ± 12.6 110.5 ± 11.4 101.6 ± 9. 96. 0 ± 23.0 80. 0 ± 4. All values are average soil standard error (sampling number = 6). Compared to the control group, _Ρ<0·001; compared to the high fat feed plus CMC group, 叩 < 0.05. 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 diameter of the fat cells was analyzed by an image analysis system. The results are shown in Table 5 and Table 5. The figure shows. Table 10 Table 10 Dose (mg/kg) Adipocytes straight (micron) Control group 65.8 ± 2.4 for fat feed + QIC 15.3. 8 ± 8. 8# pull fat material + extract 200 157. 9 ± 8.9 400 100. 3 ± 11. 7 wood* All values are the mean standard error (samples = 6). Compared with the control group, ρ·〇·〇〇1 ; compared with the CMC group, ***ρ<〇.〇ι 〇32 201114433 The results of the fat cell analysis shown in Table 10 and Figure 5 show The mice that received 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] Extract of eucalyptus cells, oleic acid and succinic acid inhibited adipocyte differentiation In an incubator having a saturated humidity of 5% carbon dioxide and a temperature of 37 ° C, 10 volumes were contained. In a DMEM medium (Hyclone, USA) of abortive bovine serum (Hyclone 'USA), 3T3-L1 pre-fat fines were cultured at a density of lx10 4 cells/cm 2 . 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, 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), oleic acid, or sulphuric acid, and 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, the 3T3-L1 cells changed from a spindle shape to a 201114433 sphere and increased, and oil droplets distributed in the cytoplasm were found. At this time, 3T3-L1 cells have differentiated into fat cells (at this time). Adipocyte ). The fat cells were fixed with 10% by volume of neutral formalin for one hour. After staining with Oil red 0 (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, the film 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 the differentiation of the adipocytes by the extract of the eucalyptus cells, the potentic acid, or the sulphuric acid was calculated. The inhibition rate of cell differentiation is calculated by the following formula. Inhibition rate of differentiation (%) = [(differentiator + dimethyl sulfoxide) absorbance - control group absorbance - (differentiator + test substance) absorbance] / [(differentiator + dimethyl sulfoxide) absorbance - Control group absorbance value] X 100% In addition, in the presence of a differentiation agent, MTS observation of test substances (ie, extracts of loquat leaf cells, potentic acid and selenate) on the survival rate of 3T3-L1 cells The results are shown in Table 11. Survival rate = absorbance of the experimental group / absorbance of the control group X 100% Table 11. · Concentration survival rate differentiation inhibition rate (%) (%) Control group 100 Differentiator + dimethyl sulfoxide 85.5 ± 6.4 - 0 Differentiation Agent + extract 0.01 μg / ml 80.4 ± 1.8 31.4 ± 6.1 *** Differentiation agent + extract 0.1 μg / ml 82.3 ± 3.0 47.3 ± 5.2 *** Differentiation agent + extract 1.0 μg / ml 78.3 ± 2.8 55.8 ± 2.4 *" Control group 100 Differentiating agent + dimethyl sulfoxide - 84.0 Soil 0.5s 0 Differentiating agent + Potentillanic acid 0.05 μg / ml 81.3 ± 7.6 26.412.8 + Differentiation agent + Potentilla acid 0.5 μg / ml 77.8 ±7.8 57.7 ± 7.9 (4) Differentiation agent + Potentillanic acid 5.0 μg/ml 72.5 ± 11.1 72·7 ± 16.1*** 34 201114433 Controlled componentizing agent + dimethyl sulfoxide differentiation agent + seledionic acid differentiation agent + Santodiene acid differentiation agent + seledionate 0.05 μg/ml 0.5 μg/ml 5.0 μg/ml 100 71.3 6.7 画 67.9 ± 6.0 67.7 士 3.1 58.7 ±6.0* 0 27_9±9'7** 42.8 ± 12.7* ** 50.5 ±2.5*** All values are mean ± standard error (number of samples = 3). Compared to the control group, #Ρ<0.05,_Ρ<0·001; compared to the differentiation agent plus dimethyl sulfoxide group, *Ρ<0.05, **Ρ<0.01, ***Ρ<0.001. 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 adipocytes. Therefore, in this example, it was first demonstrated that potentillanic acid and seletaric acid have the effect of reducing fat. The above-described 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 appended claims. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a flow chart showing the preparation of an extract of temporal lobe cells in an embodiment of the present invention; and Fig. 2 is a view showing the extraction of an extract of temporal lobe cells by four gasification. Η.Ε. staining map of liver tissue cells after carbon-induced liver fibrosis in mice; Figure 3 shows the extract of temporal lobe cells after administration of mice induced by four-carbonized carbon to induce liver fibrosis The Sirius Red staining map of the liver tissue cells; Figure 4 shows the immunostaining map of CD14 of the cells of the cells after the extract of the temporal lobe cells was administered to mice induced by carbon tetrachloride-induced liver fibrosis. ; 201114433 Figure 5 shows a micrograph of the fat cells after the extract of the temporal lobe cells is administered to the mouse; and Figure 6 shows the extract of the temporal lobe cells, the potentilla, or the mountain A staining pattern of the olefinic acid which is differentiated into adipocytes after administration of 3T3-L1 preadipocytes. [Main component symbol description] (none).

36

Claims (1)

201114433 VII. Patent application scope: 1. An extract for protecting the liver's temporal lobe cells, which comprises the following formula (compound: 'The heart and the R2 are each independently hydrogen or methyl; the heart and the & are each formed as hydrogen, methyl or silk; the r5 is linked to the &quot;Tiger and No. 2 = and forms the following formula (7) The five-membered ring of the following formula (3) = . An extract of the sputum, which contains - or a plurality of the following compounds: 37 201114433 ch3 ?η3 3. The extract of claim 1 which comprises the following compounds: An extract according to claim 1, which is for protecting liver damage caused by a chemical substance or inhibiting liver fibrosis. 38 4, 201114433 5. The extract of claim 4, wherein the chemical is carbon tetrachloride, ethanol or thiacetacetamide. 6. The extract of claim 1 which comprises at least about 60% by weight of a compound of formula (I), based on the dry weight of the extract. 7. The extract of claim 1 which comprises at least about 75% by weight of a compound of formula ,, based on the dry weight of the extract. 8. The extract according to claim ,, which comprises at least about 80% by weight of a compound of formula (I), based on the dry weight of the extract. 9. The extract of claim 1 which comprises at least about 85% by weight of a compound of formula (j), based on the dry weight of the extract. 10. A method of preparing an extract of a temporal lobe cell according to any one of claims 1 to 9, comprising: a) cultivating a leaf cell in the culture medium, wherein the culture medium comprises a leaf cell extract Residues and the temporal lobe cells; and b) extraction of the temporal lobe cells cultured in step a). The method of claim 10, 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 the callus cell from the wound; and 2) The callus cells were removed and incubated. 12. The method of claim 1 wherein, in the total amount of the culture solution, the amount of the leaf cell residue in the step is about 5 叩 to about Μ叩. 13. 4 The method of the long term 1 wherein the amount of the loquat cell cell raffinate in step a) is from about 丨ppm to about 5 ppm. The method of any one of claims 1 to 3, wherein the batch of leaf cell raffinate in step a) is obtained by the following steps: I) taking a leaf cell and using the concentration Extracting the eucalyptus leaves from about 8 vol% to about 10,000 vol% ethanol; π) concentrating the extracted product of step I) to obtain a first concentrate. ΠΙ) using a concentration of about 40 vol % to about 60% by volume of methanol dissolves the first pores, collecting a first undissolved portion; and IV) dissolving the first undissolved portion 'collection' using a concentration of from about 70% by volume to about 95% by volume of methanol The second undissolved portion is used to prepare the batch of leaf cell raffinate. 15. The method of claim 4, wherein the leaf cell line in the step is obtained by the following steps: picking up the leaves of the seedlings with the sigma sigma and waiting for the wound to grow out of the callus cells; and 2) taking out The callus cells are incubated. 16· As in May, the method of item 14, which step! The concentration of ethanol is from about 90% by volume to about 98% by volume, the concentration of methanol in the step (IV) I) is from about 45% by volume to about 55% by volume, and the concentration of the alcohol in the step iv) is from about 5% by volume to about 90% by volume. 17. If the request item 丨〇 to 丨3 _ 顼 ^ _ , method, /, step b) comprises the following steps: 1 / called using a concentration of about 8 〇 vol% to about 1 (9) vol% of ethanol extraction step a) the cultured temporal lobe cells; 呦concentrating the extracted product of step bl) to obtain a second concentrate; 201114433 b3) dissolving the second using a concentration of from about 40% by volume to about 6% by volume of methanol Concentrate, collecting a third undissolved portion; b4) dissolving the third undissolved portion using a concentration of from about 7 G% by volume to about 95% by volume of methanol and collecting the liquid; and b5) concentrating as needed filtrate. 18. 19. 21. 22. 23. 24. 25. 26. The method of claim </ RTI> wherein the concentration of ethanol is from about 卯 vol% to about 98 vol%, and the concentration of methanol in step b3) is from about Μ vol% to about The concentration of methanol of 55 vol% 'and this step b4) is from about (9) vol% to about 90 vol. /" ° A kind of medicinal herb for the protection of the liver - a job, a composition containing - an effective amount of the extract of any one of claims 1 to 9. '月求丨丨9 The pharmaceutical composition 'is used for protecting liver damage caused by chemical substances or inhibiting liver fibrosis. Amine composition 'where the chemical_four gas... - a pharmaceutical composition for protecting the liver, comprising an effective amount The extract obtained by the method of any one of the items of Item No. 8. The pharmaceutical composition of Moonlight Item 22 is used for protecting liver damage or inhibiting liver fibrosis by chemical substances. Item 23 of the Japanese medicine &amp; ^ ^ &&lt;&gt; Emirates composition, wherein the chemical substance is a tetra-glycolated alcohol or a sulphur-ethylamine. The use of the extract of any of the items 1 to 9 is manufactured. The Pharmacy Gallery is used to protect the liver. % 士 聋 聋 j j jp ) 5 , - ^ application, which is used to protect liver damage or inhibit liver fibrosis caused by chemical substances 41 201114433. 27 28. 29. 30. 31. • The application of claim 26, wherein the chemical is tetra-carbonized carbon, ethanol or thioacetamide. An application of the extract obtained by the method of any one of claims 10 to 18 for the manufacture of a medicament, wherein the medicament is for protecting the liver. The application of claim 28 wherein the agent is used to protect against liver damage caused by chemicals or to inhibit liver fibrosis. The application of claim 29 wherein the chemical is carbon tetrachloride, ethanol or thioacetamide. A medical compound for reducing fat and/or protecting the liver, comprising an effective amount of a compound of the following formula (4) and/or formula (5): 32. 33. Or a pharmaceutically acceptable salt or ester thereof. The pharmaceutical composition of claim 31 is derived from eucalyptus leaves. The pharmaceutical composition of the compound of the formula (4) and the formula (5), which is used for weight loss, treatment =, protection of liver damage caused by chemical substances, 42 201114433 includes the following formulas (4) and 35. An extract of a cell for reducing fat, or a compound of formula (5): ch3 36. An extract according to claim 35, which is for use in the treatment of weight loss or in the treatment of hypertriglyceridemia. 37. The extract of claim 35, which comprises at least about 3 weights of a compound of formula 4 / or greater, based on the dry weight of the extract. 38. The extract of claim 35, which comprises a compound of formula (4) of at least about 4 deniers per ounce of lanthanum, based on the dry weight of the extract. 39. The extract of claim 35, which comprises at least about 5 servants, j) a compound of the formula (4P compound, based on the dry weight of the extract). 40. The extract of claim 35, which comprises at least about 2% by weight of a compound of formula ο), based on the dry weight of the extract. 41. The extract of claim 35, which comprises at least a compound of the formula (5), based on the dry weight of the extract. VIIIC. The extract of claim 35, which comprises at least about 4 〇. The weight of the compound, based on the dry weight of the extract. 43. - A method for preparing a formula containing the following formula (4) and/or formula (1), which is a method for the treatment of Litian Method of clothing - 杬杷叶乡43 201114433 Compound of the extract of the cell: 44. 45. and carry out tenderness (4) and / or formula (5?h3 The method is as described in claim 43. Wherein the purification step comprises performing - high performance liquid ==r(5)— 46. 47. The agent is used to reduce fat and/or protect the liver. The use of claim 45, wherein the agent is for use in the treatment of weight loss, treatment of high triglyceride 'hemorrhagic acid caused by chemical substances, or inhibition of liver disease. For example, in the application of the month 45, the chemical is carbon tetrachloride, ethanol or sulfur. 44 201114433 Acetamide. 48. Use of an extract of eucalyptus cells for the manufacture of a medicament, wherein the extract comprises a compound of the following formula (4) and/or formula (5): ch3 ch3 And the agent is used to reduce fat. 49. The use of claim 48, wherein the agent is for weight loss or for treating hypertriglyceridemia.