Preparation method and application of Centella asiatica extract for improving urease inhibition activity of yucca powder

Abstract

The invention discloses a preparation method and application of a Centella asiatica extract for improving the urease inhibition activity of yucca powder, and relates to the technical field of preparation of raw material components for pet food. Fresh Centella asiatica is treated with infrared radiation to passivate endogenous glycosidase and glycosyltransferase while drying the material, and then microwave-assisted acid extraction is performed to increase the content of active substances in the Centella asiatica extract. The Centella asiatica extract has the effect of improving the urease inhibition activity of yucca powder, and the extract is mixed with yucca powder in proportion and fed to dogs and cats, thereby reducing the amount of yucca powder used while ensuring the effect of alleviating the ammonia odor of dog and cat feces.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of plant active substances, and in particular to a method for preparing a Centella asiatica extract capable of improving the effect of yucca powder in inhibiting urease activity and application of the extract in dog and cat food. Background Art

[0002] Yucca is a perennial herb of the genus Yucca in the Asparagaceae family. It has certain medicinal value. It has a certain inhibitory effect on urease activity, thereby inhibiting the decomposition of urea in the excrement of dogs and cats to produce ammonia. However, yucca powder should not be used too much, as it can easily cause indigestion, nausea, vomiting and diarrhea in dogs and cats, and also interfere with the absorption of fat-soluble vitamins. The oxalate contained in yucca powder can cause renal failure if consumed excessively, so its use needs to be strictly controlled.

[0003] It has been reported that yucca powder is added to food or health products for dogs and cats to alleviate the problem of odor in dog and cat feces (a functional pet snack for reducing the odor of pet feces and a preparation method thereof, CN202410385913.3; a health food for regulating the intestinal function of pet dogs and cats and a preparation method thereof, CN201810121156.3; a powdered pet nutritional supplement for cats and dogs and a preparation method thereof, CN202211535782.X). In addition to adding yucca powder, these products also add proteases, probiotics, prebiotics or dietary fiber to assist in alleviating the odor of dog and cat feces. However, these components are mainly used to improve the digestibility and utilization of protein, reduce the amount of protein substrate remaining in the intestines and fermented by microorganisms, thereby reducing the fecal odor produced after protein fermentation.

[0004] Centella asiatica, also known as iron lantern, money tooth grass, copper coin grass, horse hoof grass and Gotu Kola, has the functions of clearing away heat and detoxifying, promoting dampness and swelling, and promoting wound healing. However, there is no report on the use of Centella asiatica to enhance the inhibitory effect of yucca powder on urease activity and reduce the amount of yucca powder used. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a Centella asiatica extract capable of improving the effect of yucca powder in inhibiting urease activity and the application of the Centella asiatica extract in dog and cat food.

[0006] In a first aspect of the present invention, there is provided a method for preparing a Centella asiatica extract capable of improving the inhibitory effect of yucca powder on urease activity, which is carried out according to the following steps:

[0007] (1) taking fresh Centella asiatica, treating it with infrared drying equipment, and then crushing and sieving it;

[0008] (2) placing the sieved material in an acid solution and performing a microwave-assisted acid extraction operation, adjusting the pH of the extract to 6.5-7.5 after cooling, filtering the filtrate, concentrating and freeze-drying it, and then crushing it to obtain a Centella asiatica extract.

[0009] In the above step (1), the infrared drying equipment has an infrared radiation surface temperature of 75°C to 90°C during treatment and reduces the moisture content of the treated material to 8% to 10%.

[0010] The pH of the acid solution in the above step (2) is 3.0 to 3.5.

[0011] In the microwave-assisted acid extraction in the above step (2), the microwave equipment used has a power of 500-2000W, a time of 10-30min, and a temperature of 40°C-60°C.

[0012] The second aspect of the present invention provides a method for using the Centella asiatica extract prepared by the above method to enhance the inhibitory effect of yucca powder on urease activity.

[0013] Furthermore, the Centella asiatica extract prepared by the above method is added to yucca powder in proportion and mixed for use. Specifically, by weight, the Centella asiatica extract and yucca powder total 100 parts, of which the Centella asiatica extract is 10 to 30 parts and the rest is yucca powder.

[0014] The third aspect of the present invention provides the use of the Centella asiatica extract prepared by the above method in dog and cat food.

[0015] Furthermore, the above-mentioned composite plant extract can also be applied to other pets such as foxes and hamsters.

[0016] Furthermore, the application method is to feed the Centella asiatica extract prepared by the above method to dogs and cats by directly feeding or mixing it into dog and cat food (or snacks).

[0017] The present invention has the following beneficial effects:

[0018] Active substances in fresh Centella asiatica, such as asiatica glycoside, are easily destroyed by endogenous glycosidases and glycosyltransferases. In the present invention, infrared pretreatment can effectively passivate these enzymes and reduce the degradation degree of asiatica glycoside. During the infrared pretreatment, the water can be quickly dried and removed to reduce the influence of water on the ethanol concentration during alcohol extraction. Microwave action can destroy the dense cell wall structure of Centella asiatica, promote the effective release of active substances therein, and thus improve the extraction rate of active substances such as asiatica glycoside during alcohol extraction.

[0019] The Centella asiatica extract prepared by the method of the present invention has the effect of synergizing with yucca powder to inhibit the activity of urease. The IC 50 The inhibitory effect of Centella asiatica extract on urease activity was not as good as that of Yucca powder (IC50 100.84 mg / mL), after the yucca powder and Centella asiatica extract were mixed in equal mass ratio, the IC 50 It dropped to 14.25-16.66 mg / mL, which was 40.82%-49.38% lower than yucca powder and 83.48%-85.87% lower than Centella asiatica extract. When the mixture of yucca powder and Centella asiatica extract was mixed in equal mass ratios and fed to dogs and cats with severe fecal ammonia odor problems, it was found that compared with feeding yucca powder alone, the urease activity, odor score, volatile basic nitrogen content and ammonia content in cat feces decreased by more than 24%, and the urease activity, odor score, volatile basic nitrogen content and ammonia content in dog feces decreased by more than 28%; compared with feeding Centella asiatica extract alone, the urease activity, odor score, volatile basic nitrogen content and ammonia content in cat feces decreased by more than 41%, and the urease activity, odor score, volatile basic nitrogen content and ammonia content in dog feces decreased by more than 43%. DETAILED DESCRIPTION

[0020] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be further described in detail below in conjunction with specific embodiments and with reference to data. It should be understood that these embodiments are only further explanations of the present invention and cannot be construed as limiting the scope of protection of the present invention. Technical engineers in this field may make some non-essential improvements and adjustments to the present invention based on the content of the above invention.

[0021] In the following examples, various processes and methods not described in detail are conventional methods known in the art. The sources, trade names and components of the reagents used are indicated when they first appear, and the same reagents used thereafter are the same as those indicated for the first time unless otherwise specified.

[0022] In this embodiment and the comparative example, the fresh Centella asiatica and yucca powder are commercially available.

[0023] The total glycoside content of the samples in the present embodiment and the reference examples is determined as follows:

[0024] Take 1 g of the sample and dissolve it in 80% ethanol solution. After diluting it to a suitable multiple, determine the total glycoside content in the sample. The specific determination steps refer to the method of Lu Xingyi (UV determination of the content of total glycosides of Centella asiatica, 2006).

[0025] The half inhibition concentration (IC 50 ) determination method:

[0026] Prepare urease solution, and at the same time, centrifuge the aqueous solution of the test sample and retain the supernatant. Mix the supernatant and urease solution in equal volumes and let stand at 37°C for 10 minutes. Determine the urease activity according to the method of Zhou Dongkai (Study on the Extraction of Soybean Urease and Its Influencing Factors, 2008). The control group used distilled water instead of supernatant for enzyme activity determination, and other operations remained the same. The enzyme activity inhibition rate was calculated according to the formula: Enzyme activity inhibition rate (%) = [(enzyme activity of the control group - enzyme activity of the test group) / enzyme activity of the control group] × 100%. Use the concentration of the test sample aqueous solution as the horizontal axis and the enzyme activity inhibition rate as the vertical axis to draw a curve and calculate its IC 50 .

[0027] Method for determining urease activity in dog and cat feces in this embodiment and control examples:

[0028] Fresh dog and cat feces samples were collected and mixed with physiological saline in a mass ratio of 1:1, and then the tissue was crushed and centrifuged (10000r / min, 10min) to keep the supernatant. The urease activity was determined according to the method of Zhou Dongkai (Study on the Extraction of Soybean Urease and Its Influencing Factors, 2008).

[0029] Manual evaluation method of dog and cat feces odor in this embodiment and reference example:

[0030] 30g of fresh dog or cat feces was placed in a 50mL stoppered test tube, and the tester conducted a sensory evaluation of the odor of the dog or cat feces at a distance of 10cm from the tube mouth. The higher the odor, the higher the score, with a faint odor of 1 point, a perceptible odor of 2 points, a tolerable odor of 3 points, a strong odor that is difficult to tolerate of 4 points, and an extremely strong odor that is unbearable of 5 points.

[0031] The method for determining the volatile basic nitrogen content in dog and cat feces in this embodiment and the reference example is as follows:

[0032] Weigh 5g of fresh feces sample from dogs and cats into a conical flask, add 10mL trichloroacetic acid solution (150g / L) and 40mL distilled water, and stir and extract for 30min. Take 30mL of the extract and centrifuge it (3500r / min, 10min), then take 10mL of the supernatant into the digestive tube, add 10mL of sodium hydroxide solution (40g / L) and immediately distill it on the machine. Refer to "GB / T 32141-2015 Determination of Volatile Basic Nitrogen in Feed" to determine the volatile basic nitrogen content of the supernatant.

[0033] Method for determining ammonia content in dog and cat feces in this embodiment and reference examples:

[0034] Weigh 10g of fresh feces sample from dogs and cats and put it into a ziplock bag. Insert the probe of the portable gas detector into the bag and ensure that the insertion length and angle are consistent. Record the reading after it stabilizes.

[0035] The yucca powder, Centella asiatica extract, and the mixture of Centella asiatica extract and yucca powder in the control example and the embodiment were directly mixed into the daily diet of dogs and cats at a mass ratio of 0.1% for supplementary feeding, and no supplementary feeding was performed in the control example 1. The feces of the fed dogs and cats had a serious odor (the odor score exceeded 4 points). In the cat feeding test, 18 Maine Coons, Oriental Shorthair cats, Tabby cats, and Silver Gradient cats were randomly divided into 6 groups, each group containing 3 cats of the above 4 breeds; in the dog feeding test, 18 Teddy dogs, Golden Retrievers, Beagles, and Chihuahuas were randomly divided into 6 groups, each group containing 3 dogs of the above 4 breeds. After 3 days of supplementary feeding with mixed grain, the feces were taken for fecal urease activity determination, odor degree evaluation, volatile basic nitrogen content, and ammonia content determination.

[0036] Comparative Example 1

[0037] Dogs and cats with severe fecal ammonia odor problems were fed a normal diet without adding extracts for supplementary feeding. After the feeding test, the fecal urease activity of cats was 1.88U / g, the odor score was 4.4 points, the volatile basic nitrogen content was 146.5mg / 100g, and the ammonia content was 1.08ppm / g; the fecal urease activity of dogs was 1.96U / g, the odor score was 4.5 points, the volatile basic nitrogen content was 139.4mg / 100g, and the ammonia content was 1.17ppm / g.

[0038] Comparative Example 2

[0039] Yucca powder is subjected to physical, chemical and animal testing.

[0040] The results of physical and chemical tests showed that the IC value of yucca powder on urease activity was 50 It is 28.15mg / mL.

[0041] The results of animal tests showed that after the feeding test, the urease activity of cat feces was 1.26U / g, the odor score was 3.3 points, the volatile basic nitrogen content was 109.1mg / 100g, and the ammonia content was 0.79ppm / g; the urease activity of dog feces was 1.41U / g, the odor score was 3.1 points, the volatile basic nitrogen content was 105.4mg / 100g, and the ammonia content was 0.83ppm / g.

[0042] Comparative Example 3

[0043] Fresh Centella asiatica was treated with infrared drying equipment, the infrared radiation surface temperature was 90°C, and the moisture content of the treated material was reduced to 8%. The treated Centella asiatica was crushed and passed through a 40-mesh sieve, 50 g of the sieved material was weighed and placed in 1L of hydrochloric acid solution (pH 3.0), and microwave-assisted acid extraction (2000W, 10min, 60°C) was performed. After the extraction, the pH of the extract was adjusted to 7.5 with a NaOH solution, and the extract was filtered after cooling. The filtrate was concentrated under reduced pressure, freeze-dried, and then ground into powder to obtain a Centella asiatica extract.

[0044] The results of physical and chemical tests showed that the IC value of this Centella asiatica extract on urease activity was 50 It is 100.84mg / mL.

[0045] The results of animal tests showed that after the feeding test, the urease activity of cat feces was 1.73U / g, the odor score was 4.2 points, the volatile basic nitrogen content was 139.9mg / 100g, and the ammonia content was 1.02ppm / g; the urease activity of dog feces was 1.68U / g, the odor score was 4.1 points, the volatile basic nitrogen content was 132.6mg / 100g, and the ammonia content was 1.05ppm / g.

[0046] Example 1

[0047] Fresh Centella asiatica was treated with infrared drying equipment, the infrared radiation surface temperature was 90°C, and the moisture content of the treated material was reduced to 8%. The treated Centella asiatica was crushed and passed through a 40-mesh sieve, 50 g of the sieved material was weighed and placed in 1L of hydrochloric acid solution (pH 3.0), and microwave-assisted acid extraction (2000W, 10min, 60°C) was performed. After the extraction, the pH of the extract was adjusted to 7.5 with a NaOH solution, and the extract was filtered after cooling. The filtrate was concentrated under reduced pressure, freeze-dried, and then ground into powder to obtain a Centella asiatica extract.

[0048] The Centella asiatica extract was mixed with yucca powder in a mass ratio of 1:9, and the mixture was tested.

[0049] The results of physical and chemical tests showed that the IC value of this mixture on urease activity was 50 Dropped to 16.66mg / mL.

[0050] The results of animal tests showed that after the feeding test, the urease activity of cat feces dropped to 0.81U / g, the odor score dropped to 1.9 points, the volatile basic nitrogen content dropped to 77.7mg / 100g, and the ammonia content dropped to 0.45ppm / g; the urease activity of dog feces was 0.66U / g, the odor score was 1.7 points, the volatile basic nitrogen content was 71.5mg / 100g, and the ammonia content was 0.37ppm / g.

[0051] Example 2

[0052] Fresh Centella asiatica was treated with infrared drying equipment, the infrared radiation surface temperature was 75°C, and the moisture content of the treated material was reduced to 10%. The treated Centella asiatica was crushed and passed through a 40-mesh sieve, 50 g of the sieved material was weighed and placed in 1L of hydrochloric acid solution (pH 3.5), and microwave-assisted acid extraction (500W, 30min, 40°C) was performed. After the extraction, the pH of the extract was adjusted to 6.5 with a NaOH solution, and the extract was filtered after cooling. The filtrate was concentrated under reduced pressure, freeze-dried, and then ground to obtain a Centella asiatica extract.

[0053] The Centella asiatica extract was mixed with yucca powder in a mass ratio of 3:7, and the mixture was tested.

[0054] The results of physical and chemical tests showed that the IC value of this mixture on urease activity was 50 Dropped to 14.83mg / mL.

[0055] The results of animal tests showed that after the feeding test, the urease activity of cat feces dropped to 0.76U / g, the odor score dropped to 2.1 points, the volatile basic nitrogen content dropped to 82.4mg / 100g, and the ammonia content dropped to 0.49ppm / g; the urease activity of dog feces was 0.73U / g, the odor score was 1.8 points, the volatile basic nitrogen content was 73.9mg / 100g, and the ammonia content was 0.43ppm / g.

[0056] Example 3

[0057] Fresh Centella asiatica was treated with infrared drying equipment, the infrared radiation surface temperature was 80°C, and the moisture content of the treated material was reduced to 9%. The treated Centella asiatica was crushed and passed through a 40-mesh sieve, 50 g of the sieved material was weighed and placed in 1L of hydrochloric acid solution (pH 3.3), and microwave-assisted acid extraction (1200W, 20min, 5°C) was performed. After the extraction, the pH of the extract was adjusted to 7.0 with a NaOH solution, and the extract was filtered after cooling. The filtrate was concentrated under reduced pressure, freeze-dried, and then ground to obtain a Centella asiatica extract.

[0058] The Centella asiatica extract was mixed with yucca powder in a mass ratio of 2:8, and the mixture was tested.

[0059] The results of physical and chemical tests showed that the IC value of this mixture on urease activity was 50 Dropped to 14.83mg / mL.

[0060] The results of animal tests showed that after the feeding test, the urease activity of cat feces dropped to 0.76U / g, the odor score dropped to 2.1 points, the volatile basic nitrogen content dropped to 82.4mg / 100g, and the ammonia content dropped to 0.49ppm / g; the urease activity of dog feces was 0.73U / g, the odor score was 1.8 points, the volatile basic nitrogen content was 73.9mg / 100g, and the ammonia content was 0.43ppm / g.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a Centella asiatica extract capable of improving the inhibitory effect of yucca powder on urease activity, characterized in that Follow these steps: (1) taking fresh Centella asiatica, treating it with infrared drying equipment, and then crushing and sieving it; (2) placing the sieved material in an acid solution and performing a microwave-assisted acid extraction operation, adjusting the pH of the extract to 6.5-7.5 after cooling, filtering the filtrate, concentrating and freeze-drying it, and then crushing it to obtain a Centella asiatica extract.

2. The method for preparing a Centella asiatica extract capable of improving the inhibitory effect of yucca powder on urease activity according to claim 1, characterized in that In the above step (1), the infrared drying equipment has an infrared radiation surface temperature of 75°C to 90°C during treatment and reduces the moisture content of the treated material to 8% to 10%.

3. The method for preparing a Centella asiatica extract capable of improving the inhibitory effect of yucca powder on urease activity according to claim 1, characterized in that The pH of the acid solution in the above step (2) is 3.0 to 3.

5.

4. The method for preparing a Centella asiatica extract capable of improving the inhibitory effect of yucca powder on urease activity according to claim 1, characterized in that In the microwave-assisted acid extraction in the above step (2), the microwave equipment used has a power of 500-2000W, a time of 10-30min, and a temperature of 40°C-60°C.

5. A method for using the Centella asiatica extract prepared according to the method of claim 1 to enhance the inhibitory effect of yucca powder on urease activity, comprising adding the Centella asiatica extract prepared above to yucca powder in proportion and mixing them for use, wherein the Centella asiatica extract and yucca powder total 100 parts by weight, of which the Centella asiatica extract accounts for 10 to 30 parts and the rest is yucca powder.

6. Use of the Centella asiatica extract prepared according to the method of claim 1 in dog and cat food.

7. The Centella asiatica extract prepared by the method according to claim 1 is used in other pets such as foxes and hamsters.

8. The Centella asiatica extract prepared according to the method of claim 1 is fed to dogs and cats by direct feeding or mixing with dog and cat food (or snacks).