Method for extracting leaf of psidium genus plant, leaf extract of psidium genus plant obtained thereby, and bioactive composition containing leaf extract of psidium genus plant

Abstract

The present invention provides a method for extracting an active ingredient from leaves of a Psidium plant. The method for extracting the leaves of the psidium plant comprises the following steps: (a) providing fragments or powder of the leaves of the psidium plant; (b) mixing the fragments or powder of the leaves of the guava plant with water to form a leaf fragment mixture or leaf powder suspension; (c) an enzyme preparation is added to the leaf fragment mixed solution or the leaf powder suspension to form a first mixture, an enzyme treatment procedure is carried out to obtain an enzyme treatment substance, and the weight ratio of the leaf powder of the psidium plant to the enzyme preparation is 1: 0.01-1; and (d) mixing the enzyme treatment material with an alcohol solvent to form a second mixture, and performing an extraction procedure to obtain an extract liquid which contains at least one active component.

Description

Technical Field

[0001] The present invention relates to a method for extracting plant materials, in particular to a method for extracting leaves of plants of the genus Psidium, a leaf extract of plants of the genus Psidium obtained thereby, and a bioactive composition containing the leaf extract of plants of the genus Psidium. Background Art

[0002] Plant tissues of agricultural by-products contain a large amount of cellulose, hemicellulose, proteins, etc. These components not only greatly limit the efficiency of extracting active substances in the pre-treatment and refining processes before recycling of agricultural by-products, but also result in a low residual concentration of the purified active substances. Moreover, the obtained solution containing active substances is prone to forming insoluble complexes due to the bonds formed with tannic catechins or gallic acid contained therein during storage, which is likely to cause precipitation and turbidity, thereby affecting the stability and activity of the active substances.

[0003] Guava (Psidium guajava) needs to be regularly pruned for growth and fruiting, and the waste such as branches and leaves produced during the pruning process is quite considerable. However, most of these discarded guava leaves are still disposed of by burying and burning, resulting in hidden carbon emissions. Currently, it is known that guava leaf extract contains a high concentration of tannin components, so it can be used as a target for effective recycling and utilization of agricultural by-products and / or agricultural waste.

[0004] Therefore, there is still an urgent need for a method that can more effectively recycle or extract agricultural by-products and / or agricultural waste. Summary of the Invention

[0005] The present invention provides a method for extracting active ingredients from the leaves of plants of the genus Psidium. The method for extracting the leaves of plants of the genus Psidium includes: (a) providing fragments or powder of the leaves of a plant of the genus Psidium; (b) mixing the fragments or powder of the leaves of the plant of the genus Psidium with water at a weight ratio of 1:7-21 to form a leaf fragment mixed solution or a leaf powder suspension; (c) adding an enzyme preparation to the leaf fragment mixed solution or the leaf powder suspension to form a first mixture, and performing an enzyme treatment process for 0.5-24 hours to obtain an enzyme-treated product, wherein the weight ratio of the fragments or powder of the leaves of the plant of the genus Psidium to the enzyme preparation is 1:0.01-1; and (d) mixing the enzyme-treated product with an alcohol solvent at a weight ratio of 1:0.1-2.5 to form a second mixture, and performing an extraction process to obtain an extract, wherein the extract contains at least one active ingredient. The formulation of the enzyme preparation includes one of the formulations listed in the following (i)-(iv): (i) Formulation (I), including an enzyme mixture, which includes: arabinase, cellulase, β-glucanase, hemicellulase and xylanase; (ii) Formulation (II), including the enzyme mixture of the above Formulation (I) and pectinase; (iii) Formulation (III), including tannase; and (iv) Formulation (IV), including the enzyme mixture of the above Formulation (I), pectinase and tannase.

[0006] The present invention also provides a bioactive ingredient obtained by the above method for extracting the leaves of plants of the genus Psidium.

[0007] Furthermore, the present invention also provides a bioactive composition including the above bioactive ingredient.

[0008] In order to make the above and other objects, features, and advantages of the present invention more obvious and understandable, the following preferred embodiments are specifically exemplified and described in detail in conjunction with the accompanying drawings as follows: Description of the Drawings

[0009] Figure 1 Showing the total polysaccharide contents of the extracts A to E of guava leaves and the commercially available guava leaf extract obtained by extraction methods A to E respectively.

[0010] Figure 2 Showing the ellagic acid contents of the extracts A to E of guava leaves and the commercially available guava leaf extract obtained by extraction methods A to E respectively.

[0011] Figure 3Shows the gallic acid contents of extracts A to E of guava leaves obtained by extraction methods A to E respectively, and a commercially available guava leaf extract.

[0012] Figure 4 Shows the quercetin contents of extracts A to E of guava leaves obtained by extraction methods A to E respectively, and a commercially available guava leaf extract. Detailed implementation manners

[0013] The present invention can provide a method for extracting the leaves of plants of the genus Psidium.

[0014] By the method for extracting the leaves of plants of the genus Psidium of the present invention, agricultural by-products and / or agricultural wastes obtained after harvesting the economic parts of plants of the genus Psidium can be effectively recycled and utilized, and thereby components with economic value, especially biologically active components, can be obtained, achieving the improvement of the overall economic value of crops and recycling.

[0015] The method for extracting the leaves of plants of the genus Psidium of the present invention can effectively increase the extraction amount of active components in the leaves of plants of the genus Psidium by combining specific enzyme treatment and solvent extraction, and / or can reduce ester bonds and depside bonds formed by high-concentration tannins in the extract, thereby improving the extraction efficiency and reducing precipitation, and enhancing stability.

[0016] Specifically, by using the method for extracting the leaves of plants of the genus Psidium of the present invention, the extraction efficiency of extracting active components from the leaves of plants of the genus Psidium can be improved, and agricultural by-products and / or agricultural wastes can be effectively utilized.

[0017] The above-mentioned active components may include, but are not limited to, polysaccharides, polyphenols, etc., or any combination thereof.

[0018] The above-mentioned polysaccharides may include glucan, fructan, xylan, glucomannan, etc., or any combination thereof, but are not limited thereto.

[0019] The above-mentioned polyphenols may include, but are not limited to, phenolic acids, flavonoids, stilbenoids, lignans, etc., or any combination thereof. Examples of the above-mentioned phenolic acids may include ellagic acid, gallic acid, quinic acid, etc., or any combination thereof, but are not limited thereto. Examples of the above-mentioned flavonoids may include, but are not limited to, quercetin, kaempferol, etc., or any combination thereof.

[0020] In the method for extracting the leaves of the Psidium plant of the present invention, the Psidium plant is not particularly limited as long as it is a Psidium plant. Examples of the Psidium plant may include, but are not limited to, Psidium guajava, Psidium littorale, Psidium acutangulum, etc. In one embodiment, the Psidium plant in the method for extracting the leaves of the Psidium plant of the present invention is Psidium guajava.

[0021] The above-mentioned method for extracting the leaves of the Psidium plant of the present invention may include, but is not limited to, the following steps.

[0022] First, provide fragments or powder of the leaves of a Psidium plant. The method for obtaining the fragments or powder of the leaves of the above-mentioned Psidium plant is not particularly limited as long as fragments or powder of the leaves of a Psidium plant can be obtained. In one embodiment, the leaves or dried leaves can be chopped or ground into fragments or powder, but it is not limited thereto. Also, the size of the fragments or powder of the leaves of the above-mentioned Psidium plant is not particularly limited as long as it is helpful for the extraction of the active ingredients in the subsequent leaves. In one embodiment, the size of the fragments or powder of the leaves of the above-mentioned Psidium plant can be about 0.05 - 0.5 mm, such as about 0.1 - 0.5 mm, about 0.2 - 0.4 mm, about 0.1 mm, about 0.2 mm, about 0.25 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, etc., but it is not limited thereto. In a specific embodiment, the size of the fragments or powder of the leaves of the above-mentioned Psidium plant can be about 0.25 mm.

[0023] In the present invention, the so-called "about" refers to a range of ±10% to ±1% of the recited amount, such as a range of ±10%, a range of ±5%, a range of ±1%, etc., but it is not limited thereto.

[0024] Next, mix the fragments or powder of the leaves of the above-mentioned Psidium plant with water to form a leaf fragment mixed solution or a leaf powder suspension. The weight ratio of the fragments or powder of the leaves of the above-mentioned Psidium plant to water can be about 1:7 - 21, such as about 1:8 - 20, about 1:10 - 18, about 1:11 - 15, about 1:7 - 14, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, about 1:20, about 1:21, but it is not limited thereto. In one embodiment, the weight ratio of the fragments or powder of the leaves of the above-mentioned Psidium plant to water can be about 1:14.

[0025] Depending on the need, adjust the pH value of the above-mentioned leaf fragment mixed solution or leaf powder suspension. For example, the pH value of the above-mentioned leaf fragment mixed solution or leaf powder suspension can be adjusted to about 4.5 - 5.5, such as about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, etc., but not limited thereto. In one embodiment, the pH value of the above-mentioned leaf fragment mixed solution or leaf powder suspension can be adjusted to about 5.0.

[0026] In the method for extracting the leaves of the Psidium genus of the present invention, after mixing the fragments or powder of the leaves of the above-mentioned Psidium genus with water to form a leaf fragment mixed solution or a leaf powder suspension, an enzyme preparation can be added to the above-mentioned leaf fragment mixed solution or leaf powder suspension to form a first mixture, so as to perform an enzyme treatment process to obtain an enzyme-treated product.

[0027] The weight ratio of the fragments or powder of the leaves of the above-mentioned Psidium genus to the above-mentioned enzyme preparation can be about 1:0.01 - 1, such as about 1:0.02 - 0.9, about 1:0.05 - 0.8, about 1:0.1 - 0.7, about 1:0.1 - 0.3, about 1:0.2 - 0.6, about 1:0.3 - 0.5, about 1:0.01, about 1:0.05, about 1:0.1, about 1:0.2, about 1:0.3, about 1:0.4, about 1; 0.5, about 1:0.6, about 1:0.7, about 1:0.8, about 1:0.9, about 1:1, etc., but not limited thereto. In one embodiment, the weight ratio of the fragments or powder of the leaves of the above-mentioned Psidium genus to the above-mentioned enzyme preparation can be about 1:0.1. In another embodiment, the weight ratio of the fragments or powder of the leaves of the above-mentioned Psidium genus to the above-mentioned enzyme preparation can be about 1:0.2. In yet another embodiment, the weight ratio of the fragments or powder of the leaves of the above-mentioned Psidium genus to the above-mentioned enzyme preparation can be about 1:0.3.

[0028] And the formulation of the above-mentioned enzyme preparation can include, but is not limited to, one of the following formulations (I) to (IV).

[0029] Formulation (I), which can include an enzyme mixture. The above-mentioned enzyme mixture can include arabinase, cellulase, β - glucanase, hemicellulase, and xylanase. Examples of the above-mentioned enzyme mixture can include commercially available L, but not limited thereto.

[0030] Formulation (II) may include the enzyme mixture of the above-mentioned Formulation (I) and pectinase. In the above-mentioned Formulation (II), the weight ratio of the above-mentioned enzyme mixture to the above-mentioned pectinase may be about 1:0.5 - 2, such as about 1:0.5 - 1.8, about 1:0.6 - 1.7, about 1:0.5, about 1:0.6, about 1:0.7, about 1:0.8, about 1:0.9, about 1:1, about 1:1.1, about 1:1.2, about 1:1.3, about 1:1.4, about 1:1.5, about 1:1.6, about 1:1.7, about 1:1.8, about 1:1.9, about 1:2, etc., but not limited thereto. In one embodiment, in the above-mentioned Formulation (II), the weight ratio of the above-mentioned enzyme mixture to the above-mentioned pectinase may be about 1:1.

[0031] Formulation (III) may include tannase.

[0032] Formulation (IV) may include the enzyme mixture of the above-mentioned Formulation (I), pectinase and tannase. In the above-mentioned Formulation (IV), the weight ratio of the above-mentioned enzyme mixture, the above-mentioned pectinase and the above-mentioned tannase may be about 1:0.5 - 2:0.5 - 2, such as about 1:0.5 - 1.8:0.5 - 1.8, about 1:0.6 - 1.7:0.6 - 1.7, about 1:0.5:0.5, about 1:0.5:1, about 1:1:0.5, about 1:0.6:0.6, about 1:0.7:0.7, about 1:0.8:0.8, about 1:0.9:0.9, about 1:1:1, about 1:1.1:1.1, about 1:1.2:1.2, about 1:1.3:1.3, about 1:1.4:1.4, about 1:1.5:1.5, about 1:1:1.5, about 1:1.5:1, about 1:1.6:1.6, about 1:1.7:1.7, about 1:1.8:1.8, about 1:1.9:1.9, about 1:1:2, about 1:2:2, about 1:2:1, etc., but not limited thereto. In one embodiment, in the above-mentioned Formulation (IV), the weight ratio of the above-mentioned enzyme mixture, the above-mentioned pectinase and the above-mentioned tannase may be about 1:1:1.

[0033] In one embodiment, in the step of adding an enzyme preparation to the above-mentioned leaf fragment mixture or leaf powder suspension to form a first mixture for an enzyme treatment process to obtain an enzyme-treated product, the formulation of the above-mentioned enzyme preparation may be the above-mentioned Formulation (I), and the weight ratio of the fragments or powder of the leaves of the above-mentioned Psidium plants to the above-mentioned enzyme preparation may be about 1:0.1.

[0034] In another embodiment, in the step of adding an enzyme preparation to the above-mentioned leaf fragment mixture or leaf powder suspension to form a first mixture for an enzyme treatment process to obtain an enzyme-treated product, the formulation of the above-mentioned enzyme preparation can be the above-mentioned formulation (II), and the weight ratio of the fragments or powder of the leaves of the Psidium plant to the above-mentioned enzyme preparation can be about 1:0.2. And in the above-mentioned formulation (II), the weight ratio of the above-mentioned enzyme mixture to the above-mentioned pectinase can be about 1:1.

[0035] In yet another embodiment, in the step of adding an enzyme preparation to the above-mentioned leaf fragment mixture or leaf powder suspension to form a first mixture for an enzyme treatment process to obtain an enzyme-treated product, the formulation of the above-mentioned enzyme preparation can be the above-mentioned formulation (III), and the weight ratio of the fragments or powder of the leaves of the Psidium plant to the above-mentioned enzyme preparation can be about 1:0.1.

[0036] Furthermore, in another embodiment, in the step of adding an enzyme preparation to the above-mentioned leaf fragment or leaf powder suspension to form a first mixture for an enzyme treatment process to obtain an enzyme-treated product, the formulation of the above-mentioned enzyme preparation can be the above-mentioned formulation (IV), and the weight ratio of the fragments or powder of the leaves of the Psidium plant to the above-mentioned enzyme preparation can be about 1:0.3. And in the above-mentioned formulation (IV), the weight ratio of the above-mentioned enzyme mixture, the above-mentioned pectinase and the above-mentioned tannase can be about 1:1:1.

[0037] In the step of adding an enzyme preparation to the above-mentioned leaf fragment mixture or leaf powder suspension to form a first mixture for an enzyme treatment process to obtain an enzyme-treated product, there is no special limitation on the temperature for carrying out the above-mentioned enzyme treatment process, as long as the above-mentioned enzyme preparation has the required enzyme activity at the adopted temperature. In one embodiment, the above-mentioned enzyme treatment process can be carried out at about 35-55 °C, such as about 36-54 °C, about 37-53 °C, about 38-50 °C, about 39-51 °C, about 40-50 °C, about 35 °C, about 36 °C, about 37 °C, about 37.5 °C, about 38 °C, about 39 °C, about 40 °C, about 41 °C, about 42 °C, about 43 °C, about 44 °C, about 45 °C, about 46 °C, about 47 °C, about 48 °C, about 49 °C, about 50 °C, about 51 °C, about 52 °C, about 53 °C, about 54 °C, about 55 °C, etc., but not limited thereto. In a specific embodiment, in the step of adding an enzyme preparation to the above-mentioned leaf fragment mixture or leaf powder suspension to form a first mixture for an enzyme treatment process to obtain an enzyme-treated product, the above-mentioned enzyme treatment process can be carried out at about 45 °C.

[0038] In the step of adding an enzyme preparation to the above-mentioned leaf fragment mixture or leaf powder suspension to form a first mixture, and performing an enzyme treatment process to obtain an enzyme-treated product, the time for performing the above-mentioned enzyme treatment process is not particularly limited, as long as the above-mentioned enzyme treatment process can be fully carried out. In one embodiment, the above-mentioned enzyme treatment process can be carried out for about 0.5 - 24 hours, such as about 0.5 - 21 hours, about 1 - 18 hours, about 2 - 18 hours, about 2.5 - 15 hours, about 3 - 15 hours, about 4 - 12 hours, about 5 - 10 hours, about 0.5 hours, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 14 hours, about 15 hours, about 16 hours, about 18 hours, about 20 hours, about 21 hours, about 22 hours, about 24 hours, but not limited thereto. In a specific embodiment, in the step of adding an enzyme preparation to the above-mentioned leaf fragment mixture or leaf powder suspension to form a first mixture, and performing an enzyme treatment process to obtain an enzyme-treated product, the above-mentioned enzyme treatment process can be carried out for about 1 hour.

[0039] Finally, after the step of adding an enzyme preparation to the above-mentioned leaf fragment mixture or leaf powder suspension to form a first mixture, and performing an enzyme treatment process to obtain an enzyme-treated product, the above-mentioned enzyme-treated product can be mixed with an alcohol solvent to form a second mixture, and an extraction process can be performed to obtain an extract as the leaf extract of the Psidium plant. The above-mentioned extract can contain at least one active ingredient.

[0040] In the step of mixing the above-mentioned enzyme-treated product with an alcohol solvent to form a second mixture, and performing an extraction process to obtain an extract as the leaf extract of the Psidium plant, the weight ratio of the above-mentioned enzyme-treated product to the above-mentioned alcohol solvent can be about 1:0.1 - 2.5, such as about 1:0.15 - 2.2, about 1:0.2 - 2.1, about 1:0.3 - 2, about 1:0.4 - 1.8, about 1:0.45 - 1.5, about 1:0.5 - 1, about 1:0.6 - 0.8, about 1:0.1, about 1:0.2, about 1:0.3, about 1:0.4, about 1:0.41, about 1:0.42, about 1:0.43, about 1:0.44, about 1:0.45, about 1:0.5, about 1:0.6, about 1:0.7, about 1:0.8, about 1:0.9, about 1:1, about 1:1.5, about 1:2, about 1:2.5, etc., but not limited thereto. In one embodiment, the weight ratio of the above-mentioned enzyme-treated product to the above-mentioned alcohol solvent can be about 1:0.43. In another embodiment, the weight ratio of the above-mentioned enzyme-treated product to the above-mentioned alcohol solvent can be about 1:0.42.

[0041] Furthermore, in the step of mixing the enzyme-treated material with an alcohol solvent to form a second mixture for performing an extraction process to obtain an extract which is a leaf extract of plants of the genus Psidium, in the above-mentioned second mixture, the final concentration of the alcohol solvent may be about 20-50%, such as about 20-48%, about 22-47%, about 25-46%, about 30-45%, about 35-40%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, etc., but not limited thereto. In one embodiment, in the step of mixing the enzyme-treated material with an alcohol solvent to form a second mixture for performing an extraction process to obtain an extract which is a leaf extract of plants of the genus Psidium, in the above-mentioned second mixture, the final concentration of the alcohol solvent may be about 30%.

[0042] Moreover, in the step of mixing the enzyme-treated material with an alcohol solvent to form a second mixture for performing an extraction process to obtain an extract which is a leaf extract of plants of the genus Psidium, examples of the alcohol solvent may include, but are not limited to, methanol, ethanol, ethylene glycol, propanol, isopropanol, etc., or any combination thereof. In one embodiment, in the step of mixing the enzyme-treated material with an alcohol solvent to form a second mixture for performing an extraction process to obtain an extract which is a leaf extract of plants of the genus Psidium, the alcohol solvent may be ethanol.

[0043] In the step of mixing the enzyme-treated material with an alcohol solvent to form a second mixture for performing an extraction process to obtain an extract which is a leaf extract of plants of the genus Psidium, the temperature for performing the above-mentioned extraction process is not particularly limited as long as the active ingredients can be extracted. In one embodiment, the above-mentioned extraction process may be carried out at about 50-70°C, such as about 52-68°C, about 53-67°C, about 54-66°C, about 55-65°C, about 56-64°C, about 57-63°C, about 50°C, about 51°C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, etc., but not limited thereto. In a specific embodiment, in the step of mixing the enzyme-treated material with an alcohol solvent to form a second mixture for performing an extraction process to obtain an extract which is a leaf extract of plants of the genus Psidium, the above-mentioned extraction process may be carried out at about 60°C.

[0044] In the step of mixing the above enzyme-treated product with an alcohol solvent to form a second mixture for performing an extraction process to obtain an extract which is a leaf extract of the Psidium plant, there is no special limitation on the time for performing the above extraction process, as long as the above extraction process can be fully carried out. In one embodiment, the above extraction process can be carried out for about 0.5 - 24 hours, such as about 0.5 - 21 hours, about 1 - 18 hours, about 2 - 18 hours, about 2.5 - 15 hours, about 3 - 15 hours, about 4 - 12 hours, about 5 - 10 hours, about 0.5 hours, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 14 hours, about 15 hours, about 16 hours, about 18 hours, about 20 hours, about 21 hours, about 22 hours, about 24 hours, but not limited thereto. In a specific embodiment, in the step of mixing the above enzyme-treated product with an alcohol solvent to form a second mixture for performing an extraction process to obtain an extract which is a leaf extract of the Psidium plant, the above extraction process can be carried out for about 1 hour.

[0045] In the step of mixing the above enzyme-treated product with an alcohol solvent to form a second mixture for performing an extraction process to obtain an extract which is a leaf extract of the Psidium plant, at least one active ingredient contained in the obtained extract may include, but is not limited to, polysaccharides, polyphenols, etc., or any combination thereof.

[0046] The above polysaccharides may include glucan, fructan, xylan, glucomannan, etc., or any combination thereof, but not limited thereto.

[0047] The above polyphenols may include, but are not limited to, phenolic acids, flavonoids, stilbenoids, lignans, etc., or any combination thereof. Examples of the above phenolic acids may include gallic acid, ellagic acid, quinic acid, etc., or any combination thereof, but not limited thereto. Examples of the above flavonoids may include, but are not limited to, quercetin, psidoflavin, etc., or any combination thereof.

[0048] In one embodiment, at least one active ingredient contained in the extract obtained in the step of mixing the above enzyme-treated product with an alcohol solvent to form a second mixture for performing an extraction process to obtain an extract which is a leaf extract of the Psidium plant may include polysaccharides, gallic acid, ellagic acid and quercetin.

[0049] The above method for extracting the leaves of the Psidium plant of the present invention may include, but is not limited to, the following steps.

[0050] In addition, in one embodiment of the method for extracting leaves of the Psidium plant of the present invention, in addition to the above steps, before the step of providing fragments or powder of the leaves of the Psidium plant, a leaf treatment procedure may also be included to obtain the fragments or powder of the leaves of the Psidium plant, but is not limited thereto.

[0051] The above leaf treatment procedure may include, but is not limited to, the following steps.

[0052] First, the leaves of a Psidium plant are subjected to a drying procedure to obtain dried leaves.

[0053] There is no special limitation on the way the above drying procedure is carried out, and it can be adjusted according to the requirements and / or the environmental conditions at that time, as long as the leaves can be dried and there is no negative impact on the active ingredients in the leaves. In one embodiment, the above drying procedure is carried out by drying.

[0054] There is no special limitation on the temperature for carrying out the above drying procedure, and it can be adjusted according to the requirements and / or the environmental conditions at that time, as long as the leaves can be dried and there is no negative impact on the active ingredients in the leaves. In one embodiment, the above drying procedure can be carried out at about 40 - 60 °C, such as about 42 - 58 °C, about 43 - 57 °C, about 44 - 56 °C, about 45 - 55 °C, about 46 - 54 °C, about 47 - 53 °C, about 40 °C, about 41 °C, about 42 °C, about 43 °C, about 44 °C, about 45 °C, about 46 °C, about 47 °C, about 48 °C, about 49 °C, about 50 °C, about 51 °C, about 52 °C, about 53 °C, about 54 °C, about 55 °C, about 56 °C, about 57 °C, about 58 °C, about 59 °C, about 60 °C, etc. for drying, but is not limited thereto. In a specific embodiment, the above drying procedure can be carried out at about 50 °C.

[0055] There is also no special limitation on the time for carrying out the above drying procedure, and it can be adjusted according to the requirements and / or the environmental conditions at that time, as long as the leaves can be dried and there is no negative impact on the active ingredients in the leaves.

[0056] In one embodiment, the above drying procedure is carried out on the leaves of the Psidium plant at 50 °C until the leaves are dry.

[0057] Furthermore, in the above leaf treatment procedure, after the step of subjecting the leaves of a Psidium plant to a drying procedure to obtain dried leaves, the above dried leaves are subjected to a chopping or grinding procedure to obtain the fragments or powder of the leaves of the Psidium plant.

[0058] There is no special limitation on the way the above chopping or grinding procedure is carried out, and it can be adjusted according to the requirements and / or the environmental conditions at that time, as long as the dried leaves can be made into fragments or powder.

[0059] Furthermore, in one embodiment of the above-mentioned leaf treatment process, before the step of drying the leaves of a Psidium plant to obtain dried leaves, a pretreatment process may further be included.

[0060] The above-mentioned pretreatment process may include, but is not limited to, the following steps.

[0061] First, select intact leaves without withering or rot from the branches and leaves of guava, then separate them from the branches and remove the petioles to obtain the selected leaves. Then, wash the above-mentioned selected leaves to obtain the washed leaves. The above-mentioned washed leaves can be used as the leaves of the Psidium plant in the above-mentioned leaf treatment process.

[0062] Also, in another embodiment, in the method for extracting the leaves of a Psidium plant of the present invention, in addition to the above steps, after the step of mixing the enzyme-treated product with an alcohol solvent to form a second mixture for an extraction process to obtain an extract which is the leaf extract of the Psidium plant, it may further include subjecting the above extract to at least one filtration process to obtain a filtrate, and the above filtrate may contain the above at least one active ingredient, but is not limited thereto. The manner of the above at least one filtration process is not particularly limited as long as the solids in the above extract can be removed. Also, the number of times of performing the above filtration process is not particularly limited and can be determined according to the requirements and the situation at that time. If the filtration process is more than once, the manner of each filtration process can be the same or different. For example, filter papers with different pore sizes can be used to perform the above at least one filtration process on the above extract.

[0063] Also, in this embodiment, after the step of subjecting the above extract to at least one filtration process to obtain a filtrate, it may further include subjecting the above filtrate to a centrifugation process to obtain a supernatant, and the above supernatant may contain the above at least one active ingredient.

[0064] Furthermore, in this embodiment, after the step of subjecting the above filtrate to a centrifugation process to obtain a supernatant, it may further include subjecting the above supernatant to a drying process to form an extract powder, and the above extract powder may contain the above at least one active ingredient. The manner of performing the above drying process is not particularly limited and can be adjusted according to the requirements and / or the environmental conditions at that time as long as the supernatant can be dried into an extract powder without negative effects on the active ingredients therein. In one embodiment, freeze-drying can be used to perform the above drying process.

[0065] In addition, the present invention can also provide a leaf extract of a Psidium plant. The leaf extract of the Psidium plant of the present invention described above may contain at least one bioactive ingredient, and it can be obtained by any of the methods for extracting the leaves of the Psidium plant of the present invention described above.

[0066] Furthermore, the present invention can also provide a bioactive composition. The bioactive composition of the present invention described above may include, but is not limited to, any of the leaf extracts of the Psidium plant of the present invention described above.

[0067] In one embodiment, the bioactive composition of the present invention described above may be a pharmaceutical composition, and it may further include, but is not limited to, a pharmaceutically acceptable carrier or salts. In this embodiment, in the bioactive composition of the present invention described above, the content of the leaf extract of the Psidium plant of the present invention described above may be about 10 - 99.5 wt%, such as about 10 - 50 wt%, about 50 - 99.5 wt%, etc., but is not limited thereto.

[0068] The pharmaceutically acceptable carrier described above may include, but is not limited to, solvents, dispersion media, coatings, antibacterial and antifungal agents, and isotonic and absorption delaying agents, etc., which are compatible with pharmaceutical administration. For different administration routes, the pharmaceutical composition can be formulated into a dosage form by general methods.

[0069] The pharmaceutically acceptable salts described above may include, but are not limited to, salts including inorganic cations, such as alkali metal salts, such as sodium, potassium or amine salts, alkaline earth metal salts, such as magnesium, calcium salts, salts containing divalent or tetravalent cations, such as zinc, aluminum or zirconium salts. In addition, they can also be organic salts, such as dicyclohexylamine salts, methyl - D - glucamine, amino acid salts, such as arginine, lysine, histidine, glutamine amide.

[0070] The above-mentioned pharmaceutical composition in the present invention can be administered in a non-oral, oral, via inhalation spray, or through an implanted reservoir manner. Non-oral administration can include applying to any part or the desired part of the skin, subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional injection, and perfusion techniques.

[0071] In one embodiment, the pharmaceutical composition in the present invention can be an oral dosage form, and the above-mentioned oral dosage form can include, but is not limited to, tablets, granules, powders, pellet capsules, capsules, coated tablets, emulsions, solutions, aqueous suspensions, dispersions, instant beverages, etc.

[0072] In another embodiment, the pharmaceutical composition in the present invention can be a topically applied dosage form by rubbing, and the above-mentioned topically applied dosage form by rubbing can include ointments, emulsions, liquids, gels, mousses, etc., but is not limited thereto.

[0073] Furthermore, in one embodiment, the above-mentioned bioactive composition of the present invention can be a health food composition, and may further include, but is not limited to, a food-acceptable additive. In this embodiment, in the above-mentioned bioactive composition of the present invention, the content of the leaf extract of the Psidium plant of the present invention can be about 10 - 99.5 wt%, such as about 10 - 50 wt%, about 50 - 99.5 wt%, etc., but is not limited thereto.

[0074] The above-mentioned food-acceptable additives can include, but are not limited to, preservatives, bactericides, antioxidants, bleaching agents, color retention agents, swelling agents, colorants, flavoring agents, sweeteners, thickeners (pastes), binders, emulsifiers, carriers, etc., or any combination thereof.

[0075] The health food composition in the present invention can be in an oral form, and the above-mentioned oral form can include, but is not limited to, tablets, granules, powders, pellet capsules, capsules, coated tablets, emulsions, solutions, aqueous suspensions, dispersions, instant beverages, etc.

[0076] Furthermore, in one embodiment, the bioactive composition of the present invention may be a skin care composition, and may further include, but not limited to, a pharmaceutically acceptable carrier. For the description of the pharmaceutically acceptable carrier, reference may be made to the relevant description above, and thus it will not be elaborated herein. In this embodiment, in the bioactive composition of the present invention, the content of the leaf extract of the Psidium plant of the present invention may be about 10-99.5 wt%, such as about 10-50 wt%, about 50-99.5 wt%, etc., but not limited thereto.

[0077] The skin care composition described in the present invention may be in a form for topical application by rubbing, and the above-mentioned form for topical application by rubbing may include ointments, emulsions, liquids, gels, mousses, etc., but not limited thereto.

[0078] In addition, the administration target of the bioactive composition of the present invention may include, but not limited to, a vertebrate. Furthermore, the aforementioned vertebrate may include fish, amphibians, reptiles, birds or mammals, but not limited thereto. Examples of mammals may include, but not limited to, humans, orangutans, monkeys, horses, donkeys, dogs, cats, rabbits, guinea pigs, rats and mice. In one embodiment, the administration target of the bioactive composition of the present invention may be humans.

[0079] Examples

[0080] A. Experimental methods

[0081] A-1. Extraction method of guava leaves

[0082] 1. Extraction method A (only extraction with solvent)

[0083] (1) Select intact leaves without withering or rotting from the branches and leaves of guava, then separate them from the branches and remove the petioles to obtain the selected leaves. Then wash the above-mentioned selected leaves.

[0084] (2) Dry the washed leaves at a constant temperature of 50 °C, and then grind the dried leaves into powder, and the size (particle size) of the powder is about 0.25 mm.

[0085] (3) Mix 1 g of the powder of the above-obtained guava leaves with 14 g of RO water to form a leaf powder suspension, and adjust the pH value of the leaf powder suspension to 5.0 with citric acid.

[0086] (4) Add 6.5 g of ethanol to the above leaf powder suspension, and ultrasonically vibrate it at 60 °C for 1 hour to carry out the extraction process to obtain the extract.

[0087] (5) First filter the obtained extract with a filter paper with a pore size of 6 μm, and then filter it with a filter paper with a pore size of 1 μm to obtain the filtrate.

[0088] (6) Centrifuge the obtained filtrate at 25 °C at 5000 rpm for 15 minutes.

[0089] (7) After centrifugation, take the supernatant and freeze-dry it into a powder.

[0090] 2. Extraction method B (stir for 1 hour + extract with solvent)

[0091] (1) Screen the leaves from the branches and leaves of guava to select complete leaves without withering or rot. Then separate them from the branches and remove the petioles to obtain the screened leaves. Then wash the above-mentioned screened leaves.

[0092] (2) Dry the washed leaves at a constant temperature of 50 °C, and then grind the dried leaves into a powder, and the size (particle size) of the powder is about 0.25 mm.

[0093] (3) Mix 1 g of the powder of the previously obtained guava leaves with 14 g of RO water to form a leaf powder suspension, and adjust the pH value of the leaf powder suspension to 5.0 with citric acid.

[0094] (4) Stir the above-mentioned leaf powder suspension at 45 °C for 1 hour.

[0095] (5) Then, add 6.5 g of ethanol to the above-mentioned leaf powder suspension, and ultrasonically oscillate it at 60 °C for 1 hour to carry out the extraction process to obtain an extract.

[0096] (6) First filter the obtained extract with a filter paper with a pore size of 6 μm, and then filter it with a filter paper with a pore size of 1 μm to obtain a filtrate.

[0097] (7) Centrifuge the obtained filtrate at 25 °C at 5000 rpm for 15 minutes.

[0098] (8) After centrifugation, take the supernatant and freeze-dry it into a powder.

[0099] 3. Extraction method C (using enzyme formula α)

[0100] (1) Screen the leaves from the branches and leaves of guava to select complete leaves without withering or rot. Then separate them from the branches and remove the petioles to obtain the screened leaves. Then wash the above-mentioned screened leaves.

[0101] (2) Dry the washed leaves at a constant temperature of 50 °C, and then grind the dried leaves into a powder, and the size (particle size) of the powder is about 0.25 mm.

[0102] (3) Mix 1 g of the powder of the guava leaves obtained above with 14 g of RO water to form a leaf powder suspension, and adjust the pH value of the leaf powder suspension to 5.0 with citric acid.

[0103] (4) Add the enzyme preparation of Formula α (0.1 g of enzyme mixture ( L, which contains arabinase, cellulase, β-glucanase, hemicellulase and xylanase)) and 0.1 g of pectinase) to the above leaf powder suspension, and stir at 45 °C for 1 hour to carry out an enzymatic depolymerization reaction to obtain an enzyme-treated product.

[0104] (5) Add 6.5 g of ethanol to the above enzyme-treated product, and perform ultrasonic oscillation at 60 °C for 1 hour to carry out an extraction process to obtain an extract.

[0105] (6) First filter the obtained extract with a filter paper having a pore size of 6 μm, and then filter it with a filter paper having a pore size of 1 μm to obtain a filtrate.

[0106] (7) Centrifuge the obtained filtrate at 25 °C at 5000 rpm for 15 minutes.

[0107] (8) After centrifugation, take the supernatant and freeze-dry it into a powder.

[0108] 4. Extraction method D (using enzyme formula β)

[0109] (1) Select intact leaves without withering or rotting from the branches and leaves of guava, then separate them from the branches and remove the petioles to obtain the selected leaves. Then wash the above-selected leaves.

[0110] (2) Dry the washed leaves at a constant temperature of 50 °C, and then grind the dried leaves into powder, and the size (particle size) of the powder is about 0.25 mm.

[0111] (3) Mix 1 g of the powder of the guava leaves obtained above with 14 g of RO water to form a leaf powder suspension, and adjust the pH value of the leaf powder suspension to 5.0 with citric acid.

[0112] (4) Add the enzyme preparation of Formula β (0.1 g of tannase) to the above leaf powder suspension, and stir at 45 °C for 1 hour to carry out an enzymatic depolymerization reaction to obtain an enzyme-treated product.

[0113] (5) Add 6.5 g of ethanol to the above enzyme-treated material and ultrasonically oscillate it at 60 °C for 1 hour to perform an extraction procedure to obtain an extract.

[0114] (6) First filter the obtained extract through a filter paper with a pore size of 6 μm, and then filter it through a filter paper with a pore size of 1 μm to obtain a filtrate.

[0115] (7) Centrifuge the obtained filtrate at 25 °C at 5000 rpm for 15 minutes.

[0116] (8) After centrifugation, take the supernatant and freeze-dry it into a powder.

[0117] 5. Extraction method E (using enzyme formulation γ)

[0118] (1) Select intact leaves without withering or rot from the branches and leaves of guava, then separate them from the branches and remove the petioles to obtain the selected leaves. Then wash the above-selected leaves.

[0119] (2) Dry the washed leaves at a constant temperature of 50 °C, and then grind the dried leaves into a powder, and the size (particle diameter) of the powder is about 0.25 mm.

[0120] (3) Mix 1 g of the powder of the above-obtained guava leaves with 14 g of RO water to form a leaf powder suspension, and adjust the pH value of the leaf powder suspension to 5.0 with citric acid.

[0121] (4) Add the enzyme preparation of formulation γ (0.1 g of enzyme mixture ( L, which contains arabinase, cellulase, β-glucanase, hemicellulase and xylanase), 0.1 g of pectinase and 0.1 g of tannase) to the above leaf powder suspension, and stir at 45 °C for 1 hour to perform an enzymatic depolymerization reaction to obtain an enzyme-treated material.

[0122] (5) Add 6.5 g of ethanol to the above enzyme-treated material and ultrasonically oscillate it at 60 °C for 1 hour to perform an extraction procedure to obtain an extract.

[0123] (6) First filter the obtained extract through a filter paper with a pore size of 6 μm, and then filter it through a filter paper with a pore size of 1 μm to obtain a filtrate.

[0124] (7) Centrifuge the obtained filtrate at 25 °C at 5000 rpm for 15 minutes.

[0125] (8) After centrifugation, take the supernatant and freeze-dry it into a powder.

[0126] A-2. Method for determining the content of active ingredients

[0127] 1. Method for determining total polysaccharide content

[0128] The total polysaccharide content in the extract of guava leaves was determined by the 3,5-dinitrosalicylic acid (DNS) method

[0129] After 3,5-dinitrosalicylic acid reacts with reducing sugar by heating, it will be reduced to a brownish-red amino compound, and the absorbance value of this brownish-red amino compound can be measured at a wavelength of 540 nm using a spectrophotometer. Within a certain range, the color depth of this brownish-red amino compound is proportional to the concentration of reducing sugar. If there is reducing sugar present, the color will change from yellow to brownish-red. Therefore, the concentration of reducing sugar in a sample can be determined by colorimetry, and thus the total polysaccharide content in a sample can also be determined

[0130] (1) Preparation of glucose calibration curve

[0131] 1 mL of glucose solutions with different concentrations (0, 100, 200, 400, 800 μg / mL) were respectively mixed evenly with 1 mL of DNS reagent to form separate reaction solutions. The above reaction solutions were heated at 100 °C for 10 minutes and cooled in an ice box after cooling. Then, 3 mL of distilled water was added to the reaction solutions, and their absorbance values were measured at a wavelength of 540 nm. The glucose concentration calibration curve was plotted with the glucose solutions of each concentration corresponding to their absorbance values, and the equation corresponding to the calibration curve was obtained

[0132] (2) Determination of total polysaccharide content in the extract of guava leaves

[0133] The powder of the extract of guava leaves was prepared into a sample solution with a concentration of 10000 ppm with water

[0134] 1 mL of the sample solution was mixed evenly with 1 mL of DNS reagent to form a separate reaction solution. The above reaction solution was heated at 100 °C for 10 minutes and cooled in an ice box after cooling. Then, 3 mL of distilled water was added to the reaction solution, and its absorbance value was measured at a wavelength of 540 nm. The absorbance value of the sample solution was compared with the equation corresponding to the calibration curve to calculate its corresponding total polysaccharide content

[0135] 2. Method for determining the contents of ellagic acid, gallic acid and quercetin

[0136] (1) Preparation of calibration curves for substances to be measured

[0137] Prepare standard samples of the substance to be measured (ellagic acid standard, gallic acid standard or quercetin standard) with different concentrations (100 ppm, 50 ppm, 25 ppm, 12.5 ppm, 6.25 ppm) in ethanol.

[0138] Use a C18 chromatographic column and measure the above standard samples with different concentrations by high performance liquid chromatography (HPLC). The conditions of high performance liquid chromatography are as follows:

[0139] Detection wavelength: 270 nm;

[0140] Mobile phase A: 0.1% acetic acid + water;

[0141] Mobile phase B: 0.1% acetic acid + acetonitrile;

[0142] Flow rate: 1 mL / min;

[0143] Injection volume: 10 μL;

[0144] Analysis gradient: 0 - 5 minutes, mobile phase A 90%, mobile phase B 10%; 5 - 30 minutes, mobile phase A from 90% to 0%, mobile phase B from 10% to 100%; 30 - 35 minutes, mobile phase A 0%, mobile phase B 100%; 35 - 35.5 minutes, mobile phase A from 0% to 90%, mobile phase B from 100% to 10%; 35.5 - 40 minutes, mobile phase A 90%, mobile phase B 10%.

[0145] According to the UV absorption spectrum at 270 nm, calculate the integral area of the characteristic peak of standard samples with different concentrations, and plot the calibration curve of concentration against the integral area of the characteristic peak and obtain the equation corresponding to the calibration curve.

[0146] (2) Determination of ellagic acid content in the extract of guava leaves

[0147] Prepare a sample solution with a concentration of 10000 ppm from the powder of the extract of guava leaves in ethanol.

[0148] Obtain the UV absorption spectrum at 270 nm of the sample solution by high performance liquid chromatography under the same conditions as above, and calculate the integral area of its characteristic peak. Compare the calculated integral area of the characteristic peak of the sample solution with the equation corresponding to the calibration curve to convert the content of the substance to be measured corresponding to it.

[0149] B. Experimental results

[0150] B-1. Obtaining the extract of guava leaves

[0151] According to extraction methods A to E described in the "Determination Method of the Content of Active Ingredients" in the aforementioned "A. Experimental Methods", extracts A to E of guava leaves were obtained respectively.

[0152] Subsequently, the contents of active ingredients in extracts A to E of guava leaves and commercially available guava leaf extract were determined.

[0153] B-2. Determination of the Contents of Active Ingredients in Extracts A to E of Guava Leaves and Commercially Available Guava Leaf Extract

[0154] 1. Determination of the Total Polysaccharide Content in Extracts A to E and Commercially Available Guava Leaf Extract

[0155] According to the "1. Determination Method of Total Polysaccharide Content" described in the "A-2. Extraction Method of Guava Leaves" in the aforementioned "A. Experimental Methods", the total polysaccharide contents of extracts A to E of guava leaves and commercially available guava leaf extract (GuavaLeaf Extract Powder, Japan, Bizen Kasei Co., Ltd.) were determined respectively.

[0156] The results are as Figure 1 shown.

[0157] Figure 1 It shows that compared with extract A of guava leaves obtained by extraction method A with only solvent extraction, extract B of guava leaves obtained by extraction method B with stirring for 1 hour plus solvent extraction, and commercially available guava leaf extract, extract C of guava leaves obtained by extraction method C with enzyme formula α treatment plus solvent extraction, extract D of guava leaves obtained by extraction method D with enzyme formula β treatment plus solvent extraction, and extract E of guava leaves obtained by extraction method E with enzyme formula γ treatment plus solvent extraction all have higher total polysaccharide contents.

[0158] Compared with extraction method B with stirring for 1 hour plus solvent extraction, extraction method E with enzyme formula γ treatment plus solvent extraction can increase the total polysaccharide extraction amount by 137.8%. Moreover, compared with commercially available guava leaf extract, extraction method E with enzyme formula γ treatment plus solvent extraction can further increase the total polysaccharide extraction amount by 147.2%.

[0159] 2. Determination of the Ellagic Acid Content in Extracts A to E and Commercially Available Guava Leaf Extract

[0160] According to the "2. Determination method of ellagic acid, gallic acid and quercetin content" described in "A-2. Extraction method of guava leaves" in the previous "A. Experimental method", the ellagic acid contents of extracts A to E of guava leaves and the commercially available guava leaf extract (Guava Leaf Extract Powder, Japan, Bizen Kasei Co., Ltd.) were determined respectively.

[0161] The results are as Figure 2 shown.

[0162] Figure 2 shown that, compared with extract B of guava leaves obtained by extraction method B of stirring for 1 hour plus solvent extraction, extracts A of guava leaves obtained by extraction method A of only solvent extraction, extract D of guava leaves obtained by extraction method D of treatment with enzyme formula β plus solvent extraction, and extract E of guava leaves obtained by extraction method E of treatment with enzyme formula γ plus solvent extraction all have higher ellagic acid contents.

[0163] Compared with extraction method B of stirring for 1 hour plus solvent extraction, extraction method D of treatment with enzyme formula β plus solvent extraction can increase the ellagic acid extraction amount by 47.9%.

[0164] Also, compared with extraction method B of stirring for 1 hour plus solvent extraction, extraction method E of treatment with enzyme formula γ plus solvent extraction can increase the ellagic acid extraction amount by 32.7%.

[0165] 3. Determination of gallic acid contents of extracts A to E and the commercially available guava leaf extract

[0166] According to the "2. Determination method of ellagic acid, gallic acid and quercetin content" described in "A-2. Extraction method of guava leaves" in the previous "A. Experimental method", the gallic acid contents of extracts A to E of guava leaves and the commercially available guava leaf extract (Guava Leaf Extract Powder, Japan, Bizen Kasei Co., Ltd.) were determined respectively.

[0167] The results are as Figure 3 shown.

[0168] Figure 3It is shown that, compared with the extract A of guava leaves obtained by extraction method A using only solvent extraction, the extract B of guava leaves obtained by extraction method B with 1-hour stirring plus solvent extraction, and the commercially available guava leaf extract, the extract D of guava leaves obtained by extraction method D with enzyme formula β treatment plus solvent extraction and the extract E of guava leaves obtained by extraction method E with enzyme formula γ treatment plus solvent extraction all have higher gallic acid contents.

[0169] Moreover, compared with extraction method B with 1-hour stirring plus solvent extraction, extraction method D with enzyme formula β treatment plus solvent extraction can increase the extraction amount of gallic acid by 557.0%. Also, similarly, compared with the commercially available guava leaf extract, extraction method D with enzyme formula β treatment plus solvent extraction can also increase the extraction amount of gallic acid by 557.0%.

[0170] In addition, compared with extraction method B with 1-hour stirring plus solvent extraction, extraction method E with enzyme formula γ treatment plus solvent extraction can increase the extraction amount of gallic acid by 528.4%. Also, similarly, compared with the commercially available guava leaf extract, extraction method E with enzyme formula γ treatment plus solvent extraction can also increase the extraction amount of gallic acid by 528.4%.

[0171] 4. Determination of the quercetin contents of extracts A to E and the commercially available guava leaf extract

[0172] According to the "2. Determination method of ellagic acid, gallic acid and quercetin contents" described in "A-2. Extraction method of guava leaves" in the previous "A. Experimental method", the quercetin contents of extracts A to E of guava leaves and the commercially available guava leaf extract (Guava Leaf Extract Powder, Japan, Bizen Kasei Co., Ltd.) were determined respectively.

[0173] The results are as Figure 4 shown.

[0174] Figure 4 It is shown that, compared with the extract A of guava leaves obtained by extraction method A using only solvent extraction and the extract B of guava leaves obtained by extraction method B with 1-hour stirring plus solvent extraction, the extract C of guava leaves obtained by extraction method C with enzyme formula α treatment plus solvent extraction, the extract D of guava leaves obtained by extraction method D with enzyme formula β treatment plus solvent extraction, and the extract E of guava leaves obtained by extraction method E with enzyme formula γ treatment plus solvent extraction all have higher quercetin contents.

[0175] Also,Figure 4 It is also shown that compared with the commercially available guava leaf extract, the extract C of guava leaves obtained by extraction method C using enzyme formula α treatment plus solvent extraction and the extract E of guava leaves obtained by extraction method E using enzyme formula γ treatment plus solvent extraction both have higher quercetin contents.

[0176] Moreover, compared with extraction method B which involves stirring for 1 hour plus solvent extraction, extraction method E using enzyme formula γ treatment plus solvent extraction can increase the quercetin extraction amount by 411.9%. Additionally, compared with the commercially available guava leaf extract, extraction method E using enzyme formula γ treatment plus solvent extraction can further increase the quercetin extraction amount by 79.2%.

[0177] According to the above experimental results, it can be known that by using the above enzyme formula in combination with solvent extraction, the extraction amounts of active ingredients such as polysaccharides and polyphenols in guava leaves can be more effectively increased.

[0178] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in this art can make some modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the appended claims.

Claims

1. A method for extracting the leaves of the genus Psidium, comprising: (a) providing fragments or powder of the leaves of a Psidium plant; (b) mixing the fragments or powder of the leaves of the Psidium plant with water at a weight ratio of 1:7 - 21 to form a leaf fragment mixture or a leaf powder suspension; (c) adding an enzyme preparation to the leaf fragment mixture or the leaf powder suspension to form a first mixture, and performing an enzyme treatment process for 0.5 - 24 hours to obtain an enzyme-treated product, wherein the weight ratio of the fragments or powder of the leaves of the Psidium plant to the enzyme preparation is 1:0.01 - 1, and wherein the formulation of the enzyme preparation includes one of the formulations listed in the following (i) - (iv): (i) Formulation (I), which includes an enzyme mixture, and the enzyme mixture includes: arabinase, cellulase, β - glucanase, hemicellulase, and xylanase; (ii) Formulation (II), which includes the enzyme mixture and pectinase; (iii) Formulation (III), which includes tannase; and (iv) Formulation (IV), which includes the enzyme mixture, the pectinase, and the tannase; and (d) mixing the enzyme-treated product with an alcohol solvent at a weight ratio of 1:0.1 - 2.5 to form a second mixture, and performing an extraction process to obtain an extract, which is an extract of the leaves of a Psidium plant, wherein the extract contains at least one active ingredient.

2. The method for extracting the leaves of a Psidium plant according to claim 1, wherein the Psidium plant includes Psidium guajava, Psidium littorale, or Psidium acutangulum.

3. The method for extracting the leaves of a Psidium plant according to claim 1, wherein the Psidium plant is Psidium guajava.

4. The method for extracting the leaves of a Psidium plant according to claim 1, wherein the at least one active ingredient includes at least one of the following ingredients: polysaccharides and polyphenols.

5. The method for extracting the leaves of a Psidium plant according to claim 4, wherein the polysaccharides include at least one of the following polysaccharides: glucan, fructan, xylan, and glucomannan.

6. The method for extracting the leaves of a Psidium plant according to claim 4, wherein the polyphenols include at least one of the following polyphenols: phenolic acids, flavonoids, stilbenoids, and lignans.

7. The method for extracting the leaves of a Psidium plant according to claim 6, wherein the phenolic acids include at least one of the following phenolic acids: ellagic acid, gallic acid, and quinic acid, and wherein the flavonoids include at least one of the following flavonoids: quercetin and kaempferol.

8. The method for extracting leaves of the Psidium plant according to claim 1, wherein the size of the fragments or powder of the leaves of the Psidium plant is 0.05 - 0.5 mm.

9. The method for extracting leaves of the Psidium plant according to claim 1, wherein in step (b), the pH value of the leaf fragment mixture or leaf powder suspension is adjusted to 4.5 - 5.

5.

10. The method for extracting leaves of the Psidium plant according to claim 1, wherein in step (c), the enzymatic treatment process is carried out at 35 - 55 °C.

11. The method for extracting leaves of the Psidium plant according to claim 1, wherein in step (c), the weight ratio of the fragments or powder of the leaves of the Psidium plant to the enzyme preparation is 1:0.1 - 0.

3.

12. The method for extracting leaves of the Psidium plant according to claim 1, wherein in formula (II), the weight ratio of the enzyme mixture to the pectinase is 1:0.5 - 2.

13. The method for extracting leaves of the Psidium plant according to claim 1, wherein in formula (IV), the weight ratio of the enzyme mixture, the pectinase, and the tannase is 1:0.5 - 2:0.5 - 2.

14. The method for extracting leaves of the Psidium plant according to claim 1, wherein in step (c), the formula of the enzyme preparation is formula (I), and the weight ratio of the fragments or powder of the leaves of the Psidium plant to the enzyme preparation is 1:0.

1.

15. The method for extracting leaves of the Psidium plant according to claim 1, wherein in step (c), the formula of the enzyme preparation is formula (II), and the weight ratio of the fragments or powder of the leaves of the Psidium plant to the enzyme preparation is 1:0.2, and in formula (II), the weight ratio of the enzyme mixture to the pectinase is 1:

1.

16. The method for extracting leaves of the Psidium plant according to claim 1, wherein in step (c), the formula of the enzyme preparation is formula (III), and the weight ratio of the fragments or powder of the leaves of the Psidium plant to the enzyme preparation is 1:0.

1.

17. The method for extracting leaves of the Psidium plant according to claim 1, wherein in step (c), the formula of the enzyme preparation is formula (IV), and the weight ratio of the fragments or powder of the leaves of the Psidium plant to the enzyme preparation is 1:0.3, and in formula (IV), the weight ratio of the enzyme mixture, the pectinase, and the tannase is 1:1:

1.

18. The method for extracting the leaves of the Psidium plant according to claim 1, wherein in step (d), in the second mixture, the final concentration of the alcohol solvent is 20 - 50%.

19. The method for extracting the leaves of the Psidium plant according to claim 1, wherein in step (d), the alcohol solvent includes methanol, ethanol, ethylene glycol, propanol or isopropanol.

20. The method for extracting the leaves of the Psidium plant according to claim 1, wherein in step (d), the extraction procedure is carried out at 50 - 70 °C for 0.5 - 24 hours.

21. The method for extracting the leaves of the Psidium plant according to claim 1, after step (d), further comprising: (e) subjecting the extract to a filtration procedure to obtain a filtrate, wherein the filtrate contains the at least one active ingredient.

22. The method for extracting the leaves of the Psidium plant according to claim 21, after step (e), further comprising: (f) subjecting the filtrate to a centrifugation procedure to obtain a supernatant, wherein the supernatant contains the at least one active ingredient.

23. The method for extracting the leaves of the Psidium plant according to claim 22, after step (f), further comprising: (g) subjecting the supernatant to a drying procedure to form an extract powder, wherein the extract powder contains the at least one active ingredient.

24. A leaf extract of the Psidium plant, which contains at least one bioactive ingredient and is obtained by the method for extracting the leaves of the Psidium plant according to any one of claims 1 to 23.

25. A bioactive composition comprising the leaf extract of the Psidium plant according to claim 24.

26. The bioactive composition according to claim 25, which is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier or salt.

27. The bioactive composition according to claim 25, which is a health food composition and further comprises a food acceptable additive.

28. The bioactive composition according to claim 25, which is a skin care composition and further comprises a pharmaceutically acceptable carrier.

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