A method for extracting sangpinoside A and oxyresveratrol from mulberry branches at one time

Abstract

This application relates to the technical field of one-time extraction of compounds from mulberry branches, and particularly relates to a method for one-time extraction of moracin A and oxyresveratrol from mulberry branches, which comprises the following steps: S1. Placing mulberry branches in ethanol with a concentration of 40% - 70% and heating for extraction at least once, and combining the extraction solutions; S2. Adsorbing the extraction solution with macroporous adsorption resin; S3. Eluting the macroporous adsorption resin after step S2 with ethanol with a concentration of 20% - 38% at a flow rate of 2.6 - 3.5 BV / h, and collecting the eluate; S4. Eluting the macroporous adsorption resin after step S3 with ethanol with a concentration of 70% - 90% at a flow rate of 3 - 4 BV / h, and collecting the eluate; S5. Concentrating and / or drying the eluates obtained in step S3 and step S4 to obtain moracin A and oxyresveratrol respectively. Two stilbene compounds are obtained by one-time extraction from mulberry branches using a relatively simple process. The preparation route is simple, and the costs of raw materials and auxiliary materials are relatively low, which is suitable for industrial production and is conducive to carrying out more complex applications in the future.

Description

Technical Field

[0001] The present application relates to the technical field of primary extraction of compounds from mulberry twigs, and particularly relates to a method for primary extraction of moracenin A and oxyresveratrol from mulberry twigs. Background Art

[0002] As a traditional tree species, mulberry trees are widely planted in China. The mulberry twigs of mulberry trees are included in the ancient Chinese pharmacopoeia due to their application in various diseases. Research shows that mulberry twigs are rich in secondary metabolites and have various biological activities. However, the development of mulberry twigs is seriously insufficient at present, and they are generally treated as agricultural waste or used as fuel, resulting in a great waste of resources.

[0003] Recently, the market has begun to study stilbenes, and mulberry twigs have received extensive attention due to their rich content of stilbenes. Oxyresveratrol is one of the main stilbene compounds in mulberry twigs and has antioxidant, anti-inflammatory, antiviral, and cytoprotective activities; Moracenin A is a glycosylated compound of oxyresveratrol, in which its 3'-OH and 4'-OH are substituted by glucose molecules, and it has various biological activities and pharmacological functions such as antioxidant, anti-inflammatory, antiviral, and anti-tyrosinase. In recent years, the reports on mulberry twigs mainly focus on total flavonoids, polysaccharides, alkaloids, etc., and there are few reports on stilbene compounds in mulberry twigs. At present, moracenin A and oxyresveratrol in mulberry twigs are often extracted separately, with complex operations. Moreover, for the separation and purification of oxyresveratrol, silica gel column chromatography is mostly used, and elution is carried out with a mixed solution of petroleum ether - ethyl acetate, which is not suitable for industrial production, or a biotransformation method is used, with complex operations. Summary of the Invention

[0004] The main purpose of the present application is to provide a method for primary extraction of moracenin A and oxyresveratrol from mulberry twigs, aiming to solve the technical problems in the prior art that moracenin A and oxyresveratrol in mulberry twigs need to be extracted separately, with complex processes and low extraction efficiency.

[0005] To achieve the above purpose, the method for primary extraction of moracenin A and oxyresveratrol from mulberry twigs proposed by the present application includes the following steps: S1. Place the mulberry twigs in ethanol with a concentration of 40% - 70% and heat and extract at least once, and combine the extraction solutions;

[0006] S2. Adsorb the extraction solution with macroporous adsorption resin;

[0007] S3. Elute the macroporous adsorption resin after step S2 with ethanol with a concentration of 20% - 38% at a flow rate of 2.6 - 3.5 BV / h, and collect the eluate;

[0008] S4. Elute the macroporous adsorption resin after step S3 with ethanol at a concentration of 70% - 90% at a flow rate of 3 - 4 BV / h, and collect the eluate.

[0009] S5. Concentrate and / or dry the eluates obtained in step S3 and step S4 to obtain sangpigenin A and oxyresveratrol respectively.

[0010] In the extract after heating and reflux extraction with ethanol at a concentration of 40% - 70%, there are both sangpigenin A and oxyresveratrol. Then, taking advantage of the polarity and molecular weight differences between sangpigenin A and oxyresveratrol, sangpigenin A has a larger polarity and molecular weight, and in this solution, ethanol at a concentration of 20% - 38% is used for elution. While oxyresveratrol has a smaller polarity and molecular weight and stronger adsorption on the resin, and ethanol at a concentration of 70% - 90% is used to elute it. Finally, the purpose of separating both sangpigenin A and oxyresveratrol is achieved. The eluates at two concentration gradients are collected separately, concentrated or dried respectively to obtain two refined components. The purity of sangpigenin A obtained through the above extraction process can reach 65 - 80%, and the purity of oxyresveratrol can reach 60 - 85%.

[0011] The porous structure of the macroporous adsorption resin has a screening effect on substances with different molecular sizes, and the macromolecular substances are eluted first. At the same time, the higher the concentration of ethanol, the stronger the elution effect. In the present invention, the separated sangpigenin A is a glycoside compound of oxyresveratrol, in which the 3'-OH and 4'-OH are substituted by glucose molecules, and its molecular weight is larger than that of oxyresveratrol. During the process of free dispersion of the extract in the resin, low-concentration ethanol can elute sangpigenin A alone, and high-concentration ethanol can elute both sangpigenin A and oxyresveratrol. By eluting step by step in the order of first eluting sangpigenin A with low-concentration ethanol and then eluting oxyresveratrol with high-concentration ethanol, sangpigenin A and oxyresveratrol can be obtained successively. On the contrary, if high-concentration ethanol is used to elute oxyresveratrol first, then sangpigenin A and oxyresveratrol will be eluted simultaneously, and the effect of obtaining both sangpigenin A and oxyresveratrol cannot be achieved.

[0012] After eluting with ethanol at a concentration of 20% - 38% in the present invention, under the same resin conditions, ethanol at a concentration of 40% - 80% is continuously used for elution. The two groups of eluates are collected separately to obtain the refined products of sangpigenin A and oxyresveratrol respectively. The steps are simple. Only one extraction with macroporous adsorption resin can obtain two components, the extraction cycle is shortened, the utilization degree of the macroporous adsorption resin is improved, and it is more economical.

[0013] In this solution, macroporous adsorption resin is used to extract morusin A and oxyresveratrol. The macroporous adsorption resin mainly uses styrene and propionate as monomers, adds ethylbenzene as a cross-linking agent, and toluene and xylene as pore-forming agents, and cross-links and polymerizes with each other to form a porous skeleton structure. The macroporous adsorption resin selectively adsorbs morusin A and oxyresveratrol from the extract by physical adsorption, so as to achieve the purpose of extraction. After the extraction is completed, this solution will also regenerate the macroporous adsorption resin for recycling.

[0014] Optionally, in step S3, ethanol with a concentration of 27% - 35% is used to elute the macroporous adsorption resin after step S2 at a flow rate of 2.6 - 3.5 BV / h, and the eluate is collected.

[0015] In step S4, ethanol with a concentration of 75% - 82% is used to elute the macroporous adsorption resin after step S3 at a flow rate of 3 - 4 BV / h, and the eluate is collected. Within the above preferred range, the purity of the extracted morusin A and oxyresveratrol is higher.

[0016] Optionally, in step S1, the heating temperature > 95°C. Similarly, this solution raises the heating temperature to above 95°C, and the extraction effect is better, which can increase the simultaneous extraction amount of morusin A and oxyresveratrol components in mulberry twigs.

[0017] Optionally, in step S2, the macroporous adsorption resin is a non-polar or weakly polar macroporous resin. The above two types of macroporous adsorption resins have good extraction effects on morusin A and oxyresveratrol. The macroporous adsorption resin can specifically be AB-8 macroporous resin (styrene-type weakly polar macroporous resin), etc.

[0018] Optionally, the macroporous adsorption resin is spherical particles, and the specific surface area ≥ 500m 2 / g.

[0019] Optionally, the particle size range of the macroporous adsorption resin is that 0.315 - 0.5 mm accounts for 10 - 30%, 0.5 - 1 mm accounts for 50% - 70%, and above 1 mm accounts for 20 - 30%. Controlling the macroporous adsorption resin within the above particle size range has good extraction effects on both morusin A and oxyresveratrol, and the obtained morusin A and oxyresveratrol have higher purity.

[0020] Optionally, in steps S3 and S4, the volume of the ethanol is 2 BV - 4 BV.

[0021] Optionally, in step S1, the mass ratio of ethanol to mulberry twigs is (6 - 12):1. Controlling the mass ratio of ethanol to mulberry twigs within the above range can better extract the morusin A and oxyresveratrol components in mulberry twigs.

[0022] Optionally, in the step S1, the single extraction time is 0.5 - 2 h.

[0023] Optionally, in the step S1, the number of extractions is 1 - 4 times.

[0024] The method for extracting sangpinoside A and resveratrol oxide from mulberry twigs in one step in this application has the following beneficial effects: The main preparation method includes selecting mulberry twigs, extracting with an ethanol - water mixed solvent, refining the extract with macroporous resin, eluting with an ethanol - water mixed solvent, and concentrating and / or drying the eluate. Finally, high - purity sangpinoside A and resveratrol oxide can be obtained simultaneously. Two stilbene compounds are extracted from mulberry twigs at one time using a relatively simple process. Its preparation route is simple, the extraction efficiency is high, the costs of raw materials and auxiliary materials are low, it is suitable for industrial production, and it is conducive to carrying out more complex applications in the future. Description of the Drawings

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present application. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0026] Figure 1 It is the chromatogram when the present invention extracts mulberry twigs with 50% ethanol;

[0027] Figure 2 It is the chromatogram of the product obtained after eluting successively with 50% ethanol and 40% ethanol in the present invention;

[0028] Figure 3 It is the chromatogram of the product obtained after eluting successively with 38% ethanol and 80% ethanol in the present invention;

[0029] Figure 4 It is the chromatogram when the present invention extracts mulberry twigs with 60% ethanol;

[0030] Figure 5 It is the chromatogram of the product obtained after eluting successively with 60% ethanol and 20% ethanol in the present invention;

[0031] Figure 6 It is the chromatogram of the product obtained after eluting successively with 20% ethanol and 80% ethanol in the present invention;

[0032] Figure 7 It is the chromatogram when the present invention extracts mulberry twigs with 70% ethanol;

[0033] Figure 8It is the chromatogram of the product obtained after eluting successively with ethanol at a concentration of 70% and ethanol at a concentration of 30% in the present invention;

[0034] Figure 9 It is the chromatogram of the product obtained after eluting successively with ethanol at a concentration of 30% and ethanol at a concentration of 70% in the present invention;

[0035] Figure 10 It is a schematic diagram showing the influence of different eluents on the desorption effect in the present invention.

[0036] The realization of the purpose of this application, functional features and advantages will be further described in conjunction with the embodiments with reference to the accompanying drawings. Detailed Embodiments

[0037] To make the purposes, technical solutions and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0038] In the embodiments of this application, a method for extracting moracin A and oxyresveratrol from mulberry branches at one time includes the following steps:

[0039] S1. Place the mulberry branches in ethanol with a concentration of 40% - 70% and heat to >95°C for extraction 1 - 4 times, with the single extraction time being 0.5 - 2 h. Combine the extraction solutions, and the mass ratio of ethanol to mulberry branches is (6 - 12):1;

[0040] The multiple extractions in step S1 are mainly for more complete extraction. Considering the need to soak the medicinal materials during the first extraction, more ethanol will be used than in subsequent extractions. And during the first extraction, most of the components have been extracted. The subsequent extractions are to extract the remaining components as much as possible. Therefore, the corresponding extraction times are different, and the first extraction time is longer.

[0041] S2. Adsorb the extraction solution with macroporous adsorption resin. The macroporous adsorption resin is non-polar or weakly polar macroporous resin. The macroporous adsorption resin is spherical particles, with a specific surface area ≥500m 2 / g, and the particle size range of the macroporous adsorption resin is that 10 - 30% is 0.315 - 0.5 mm, 50% - 70% is 0.5 - 1 mm, and 20 - 30% is above 1 mm;

[0042] Before step S3, generally water is used for elution first to remove the polysaccharide components in the extraction solution.

[0043] S3. Elute the macroporous adsorption resin after step S2 with ethanol at a concentration of 20% - 38% at a flow rate of 2.6 - 3.5 BV / h, and collect the eluate. The volume of the ethanol is 3 BV - 4 BV;

[0044] S4. Elute the macroporous adsorption resin after step S3 with ethanol at a concentration of 70% - 90% at a flow rate of 3 - 4 BV / h, and collect the eluate. The volume of the ethanol is 3 BV - 4 BV;

[0045] S5. Concentrate and / or dry the eluates obtained in step S3 and step S4 to obtain sangpigenin A and oxyresveratrol respectively.

[0046] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0047] Example 1

[0048] A method for extracting sangpigenin A and oxyresveratrol from mulberry branches at one time, comprising the following steps:

[0049] S1. Place 500 g of mulberry branches in ethanol with a mass of 8 times, 6 times, and 6 times and a concentration of 60% respectively, and heat and extract 3 times. The extraction times are 2 h, 1 h, and 1 h respectively, and the extraction temperature is > 95°C. Combine the extraction liquids and recover the ethanol until there is no alcohol smell;

[0050] S2. Adsorb the extraction liquid with macroporous adsorption resin. The macroporous adsorption resin used is 1.4 Kg of AB-8 resin, the column volume is 1.8 L, and the specific surface area is 500 m 2 / g;

[0051] Elute the macroporous adsorption resin after step S2 with 3 times the column volume (3 BV) of water, and collect the eluate;

[0052] S3. Elute the above macroporous adsorption resin with ethanol at a concentration of 20% and a volume of 3 BV at a flow rate of 2.6 BV / h to obtain another group of eluates;

[0053] S4. Elute the above macroporous adsorption resin with ethanol at a concentration of 80% and a volume of 3 BV at a flow rate of 4 BV / h to obtain another group of eluates;

[0054] S5. Concentrate the eluates obtained in step S3 and step S4 and then freeze-dry them to obtain refined products of sangpigenin A and oxyresveratrol respectively. Among them, the purity of sangpigenin A is 71.9%, and the purity of oxyresveratrol is 61.2% (confirmed by detection and analysis).

[0055] Example 2

[0056] A method for extracting sangpicoside A and oxyresveratrol from mulberry branches at one time, comprising the following steps:

[0057] S1. Place 1500 g of mulberry branches in ethanol with a mass 10 times, 8 times, and 6 times and a concentration of 70% respectively, and heat and extract 3 times. The extraction times are 1 h, 0.5 h, and 0.5 h respectively, and the extraction temperature is >95°C. Combine the extraction solutions and recover ethanol until there is no alcohol smell;

[0058] S2. Adsorb the extraction solution with macroporous adsorption resin. The macroporous adsorption resin used is 2.4 Kg of D101 resin, the column volume is 1.8 L, and the specific surface area is 500 m 2 / g;

[0059] Elute the macroporous adsorption resin after step S2 with 4 BV of water, and collect the eluate;

[0060] S3. Elute the above macroporous adsorption resin with ethanol with a concentration of 30% and a volume of 4 BV at a flow rate of 3 BV / h to obtain another group of eluates;

[0061] S4. Elute the above macroporous adsorption resin with ethanol with a concentration of 70% and a volume of 4 BV at a flow rate of 3.5 BV / h to obtain another group of eluates;

[0062] S5. Concentrate the eluates obtained in step S3 and step S4 and dry them in an oven to obtain refined products of sangpicoside A and oxyresveratrol respectively. Among them, the purity of sangpicoside A is 76.6%, and the purity of oxyresveratrol is 75% (confirmed by detection and analysis).

[0063] Example 3

[0064] A method for extracting sangpicoside A and oxyresveratrol from mulberry branches at one time, comprising the following steps:

[0065] S1. Place 800 g of mulberry branches in ethanol with a mass 10 times, 8 times, and 8 times and a concentration of 50% respectively, and heat and extract 3 times. The extraction times are 2 h, 0.5 h, and 0.5 h respectively, and the extraction temperature is >95°C. Combine the extraction solutions and recover ethanol until there is no alcohol smell;

[0066] S2. Adsorb the extraction solution with macroporous adsorption resin. The macroporous adsorption resin used is 1.3 Kg of HPD300 resin, the column volume is 1.8 L, and the specific surface area is 500 m 2 / g;

[0067] Elute the macroporous adsorption resin after step S2 with 2 BV of water, and collect the eluate;

[0068] S3. Elute the above macroporous adsorption resin with ethanol at a concentration of 38% and a volume of 2BV at a flow rate of 3.5BV / h to obtain another group of eluates;

[0069] S4. Elute the above macroporous adsorption resin with ethanol at a concentration of 80% and a volume of 2BV at a flow rate of 3BV / h to obtain another group of eluates;

[0070] S5. Concentrate the eluates obtained in step S3 and step S4 and then dry them under vacuum at low temperature to obtain refined products of morusin A and resveratrol oxide respectively. Among them, the purity of morusin A is 66.2%, and the purity of resveratrol oxide is 65.6% (confirmed by detection and analysis).

[0071] Example 4

[0072] All conditions in this example are the same as those in Example 1, except that: steps S3 and S4 are adjusted to the preferred parameters. Specifically, in step S3, elute the macroporous adsorption resin after step S2 with ethanol at a concentration of 30% at a flow rate of 3BV / h and collect the eluate; in step S4, elute the macroporous adsorption resin after step S3 with ethanol at a concentration of 80% at a flow rate of 3.5BV / h and collect the eluate. Among them, the purity of the obtained morusin A is 72%, and the purity of resveratrol oxide is 81% (confirmed by detection and analysis).

[0073] After further optimizing the particle size range of the macroporous adsorption resin to be 10 - 30% of 0.315 - 0.5mm, 50% - 70% of 0.5 - 1mm, and 20% - 30% of above 1mm, the purity of the obtained morusin A can reach about 80%, and the purity of resveratrol oxide can reach about 85%.

[0074] Comparative Example 1

[0075] Take three macroporous resins AB - 8 (styrene - type weakly polar macroporous resin), D101 (styrene - type non - polar macroporous resin), and HPD300 (medium - polar macroporous adsorption resin) for conventional industrial resin treatment. Weigh 2g and add them to the adsorption column, suck 10ml of the sample solution, pass through the above macroporous resins at a flow rate of 1ml / min, and elute them with 10ml of water and absolute ethanol respectively. Collect the eluates, water eluates, and alcohol eluates, calculate the content of the active ingredients, and calculate the adsorption rate and desorption rate. The results are shown in the following table:

[0076]

[0077] Adsorption rate calculation formula (%) = [(C0V0 - C1V1) / C0V0] × 100%; C0: Initial concentration of mulberroside A and resveratrol oxide in mg / mL; V0: Sample loading volume (mL); C1: Concentration of the liquid passing through the column and the water washing liquid (mg / mL); V1: Total volume of the liquid passing through the column and the water washing liquid (mL).

[0078] Desorption rate (%) = Resolution rate (%) = [C2V2 / (C0V0 - C1V1)] × 100%; C0: Initial concentration of mulberroside A and resveratrol oxide in mg / mL; V0: Sample loading volume (mL); C1: Concentration of the liquid passing through the column and the water washing liquid (mg / mL); V1: Total volume of the liquid passing through the column and the water washing liquid (mL); C2: Concentration of mulberroside A and resveratrol oxide in the eluent (mg / mL); V2: Volume of the eluent (mL).

[0079] From the test results in the above table, it can be concluded that when using weakly polar macroporous resins such as AB-8, the adsorption effect on mulberroside A and resveratrol oxide is the best, with the adsorption rate of mulberroside A being 86.55% and the adsorption rate of resveratrol oxide being 100%. When using non-polar macroporous adsorption resins such as D101, the adsorption effect on mulberroside A and resveratrol oxide slightly decreases. When using medium-polar macroporous adsorption resins such as HPD300, the adsorption effect on mulberroside A and resveratrol oxide decreases to a greater extent.

[0080] Comparative Example 2

[0081] Optimization of the eluent concentration: Take the pretreated AB-8 macroporous resin, weigh 6 g, add it to the adsorption column, draw 100 ml of the sample loading solution and pass it through the resin at a flow rate of 1 ml / min, and elute with 100 ml of water, 100 ml of 20% ethanol, 40% ethanol, 60% ethanol, and 80% ethanol respectively. Collect the eluent, water washing eluent, and alcohol eluents with different concentrations, calculate the content of the active ingredients, and make a curve graph of the eluent concentration and desorption rate, as Figure 10 shown. From the test results, it can be concluded that 20%-80% ethanol has little effect on the desorption rate of mulberroside A, while for resveratrol oxide, as the ethanol concentration increases, the desorption rate gradually approaches 100%. Mulberroside A is eluted with 20%-38% ethanol, and resveratrol oxide is eluted with 70%-80% ethanol.

[0082] Comparative Example 3

[0083] All conditions in this comparative example are the same as those in Example 1, except that: the heating extraction temperature is the temperature in the following table, and the purity test results of mulberroside A and resveratrol oxide are shown in the following table:

[0084]

[0085] From the test results in the above table, it can be concluded that when eluting with 2BV ethanol, sangpigenin A and oxyresveratrol were not completely eluted, and the purity was relatively low. However, when eluting with 3BV - 4BV ethanol, the purity increased. Finally, it was determined to use 3BV - 4BV ethanol for elution.

[0086] The above are only the preferred embodiments of the present application, and do not limit the patent scope of the present application. Any equivalent structural transformation made under the inventive concept of the present application by using the content of the specification and drawings of the present application, or directly / indirectly applied in other related technical fields, is included in the patent protection scope of the present application.

Claims

1. A method for extracting sangpinoside A and oxyresveratrol from mulberry branches at one time, characterized in that, It includes the following steps: S1. Place mulberry twigs in ethanol with a concentration of 40% - 70% and heat for extraction at least once, then combine the extracts; S2. Adsorb the extract with macroporous adsorption resin; the macroporous adsorption resin is non-polar or weakly polar macroporous resin; the macroporous adsorption resin is spherical particles with a specific surface area ≥ 500 m 2 / g; S3. Elute the macroporous adsorption resin after step S2 with ethanol with a concentration of 20% - 38% at a flow rate of 2.6 - 3.5 BV / h, and collect the eluate; S4. Elute the macroporous adsorption resin after step S3 with ethanol with a concentration of 70% - 90% at a flow rate of 3 - 4 BV / h, and collect the eluate; S5. Concentrate and / or dry the eluates obtained in step S3 and step S4 to obtain sangpigenin A and resveratrol oxide respectively.

2. The method for extracting moracin A and resveratrol oxide from mulberry branches at one time according to claim 1, characterized in that, In step S3, elute the macroporous adsorption resin after step S2 with ethanol with a concentration of 27% - 35%, and collect the eluate; In step S4, elute the macroporous adsorption resin after step S3 with ethanol with a concentration of 75% - 82%, and collect the eluate.

3. The method for extracting sangpinoside A and oxyresveratrol from mulberry branches at one time as described in claim 1, characterized in that, In step S1, the heating temperature > 95°C.

4. The method for extracting moracin A and resveratrol oxide from mulberry branches at one time as claimed in claim 1, wherein The particle size range of the macroporous adsorption resin is that 10 - 30% is 0.315 - 0.5 mm, 50% - 70% is 0.5 - 1 mm, and 20 - 30% is above 1 mm.

5. The method for extracting sangpinoside A and resveratrol oxide from mulberry twigs at one time as described in claim 1, characterized in that, In step S3 and step S4, the volume of the ethanol is 2 BV - 4 BV.

6. The method for extracting sangpigenin A and oxyresveratrol from mulberry branches at one time according to claim 1, characterized in that In step S1, the mass ratio of ethanol to mulberry twigs is (6 - 12):

1.

7. The method for extracting moracin A and resveratrol oxide from mulberry branches at one time as claimed in claim 1, wherein In step S1, the single extraction time is 0.5 - 2 h.

8. The method for extracting sangpicoside A and resveratrol oxide from mulberry branches at one time according to claim 1, wherein In step S1, the number of extraction times is 1 - 4 times.

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