CN111407811B - Application of total phenols extract of immature bitter orange in preparation of intestinal tract protection medicine - Google Patents
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Abstract
The invention belongs to the technical field of new application of medicines, and particularly relates to application of a total phenol extract of immature bitter oranges in preparation of an intestinal tract protection medicine, wherein the preparation method of the total phenol extract of immature bitter oranges comprises the following steps: crushing immature bitter oranges, soaking the crushed immature bitter oranges in 10 times of 50% ethanol for 30 minutes, then carrying out reflux extraction on the mixture for 1.5 hours, repeating the extraction for three times, sieving the mixture for 80 meshes, combining extracting solutions obtained in the three times, recovering the ethanol in a vacuum rotary evaporator, mixing and concentrating the water extract in the vacuum rotary evaporator, and drying the mixed water extract in a vacuum oven to obtain an immature bitter oranges total phenol extract; the intestinal protection is specifically as follows: up-regulating the growth of wart microbiaceae, lactobacillaceae, Prevoteriaceae, inhibiting the growth of pilospiraceae, Bacteroides, Porphyromonaceae and Enterobacteriaceae, and protecting the integrity of intestinal barrier. Compared with the prior art, the invention aims at specific diseases more accurately, so that patients can more clearly and quickly select the total phenol extract preparation of the immature bitter orange for treatment after the disease causes are detected.
Description
Technical Field
The invention belongs to the technical field of new application of medicines, and particularly relates to application of a total phenol extract of immature bitter oranges in preparation of an intestinal tract protection medicine.
Background
The intestinal flora is a complex, highly dynamic community of microorganisms. It is considered to be an indispensable "metabolic organ" involved in the nutritional processing and production of essential compounds such as short chain fatty acids and bile acids. In addition, the intestinal flora contributes to the maturation of the gastrointestinal system and the development of the immune system. The composition of the intestinal flora is constantly influenced by factors such as genetics, age, sex, diet, antibiotics, environment, etc. The most important of these is the antibiotic, which is widely used in the treatment and prevention of bacterial infections. However, there is increasing evidence that antibiotics have an adverse effect on the physiological function of the host, leading to a structural change in the intestinal flora, a condition known as "gut flora imbalance".
Broad spectrum antibiotics can affect the overall abundance of gut flora composition, resulting in a rapid decline in diversity, uniformity, and categorical richness. Several studies have shown that the use of broad spectrum antibiotics such as ampicillin, clindamycin and vancomycin can lead to a dysbacteriosis of the intestinal flora. The flora plays an important role in maintaining the integrity of the intestinal barrier, which is crucial for the homeostasis and function of intestinal and systemic diseases. The integrity of the intestinal barrier prevents the transfer of bacterial endotoxin from the intestine to the liver. Tight junctions, such as Zonula occludens 1(ZO-1) and Occludin, tightly join intestinal cells together and control cell permeability. Thus, claudin regulates the integrity of the intestinal barrier. In addition to altering intestinal barrier function, antibiotics also alter the function of the intestinal flora.
The fructus Aurantii Immaturus is dry young fruit of Citrus aurantium L. of Rutaceae and its cultivar or Citrus sinensis Osbeck. The fruits which fall from the tree in 5-6 months are collected, and the fruits are bitter, pungent and sour in taste, slightly cold in nature and enter spleen and stomach meridians. Has effects of relieving flatulence, resolving phlegm and relieving distension, and can be used for treating stagnation, distention and pain, dysentery, constipation, phlegm stagnation, qi stagnation, thoracic obstruction, and viscera prolapse. Immature bitter orange has abundant and widely distributed medicinal materials, and the producing areas mainly comprise Sichuan, Jiangxi, Hunan, Fujian and other places, and the sour orange produced in Jiangxi is used as a genuine medicinal material. The Total phenolic extract of immature bitter orange (TPE-CA) comprises flavan and glycosides thereof, polymethoxylated flavonoids, flavones (alcohols) and glycosides thereof, coumarins, limonoids, anthocyanidins and other polyphenols, and has important therapeutic effects of regulating balance, enhancing body homeostasis and the like on human health.
Although immature bitter orange is reported to have the effects of invigorating stomach and promoting digestion, the pharmacological actions related to invigorating stomach and promoting digestion are too extensive. For example, the drugs for treating gastric ulcers and gastric obstructions must not be used interchangeably, and immature bitter orange does not only act on gastric diseases. Therefore, the current research is not deep enough for the drugs requiring precise medical treatment. The stomach invigorating and digestion promoting effects of the total phenols extract of the immature bitter orange are analyzed, more accurate pharmacological effects are found, and the research and development of new drugs are facilitated.
Disclosure of Invention
In order to solve the technical problems, the invention provides application of total phenols extract of immature bitter orange in preparation of an intestinal tract protection medicament.
The application of the total phenols extract of immature bitter orange in the preparation of the intestinal tract protection medicine comprises the following steps: crushing immature bitter oranges, soaking the crushed immature bitter oranges in 10 times of 50% ethanol for 30 minutes, then carrying out reflux extraction on the mixture for 1.5 hours, repeating the extraction for three times, sieving the mixture for 80 meshes, combining extracting solutions obtained in the three times, recovering the ethanol in a vacuum rotary evaporator, mixing and concentrating the water extract in the vacuum rotary evaporator, and drying the mixed water extract in a vacuum oven to obtain an immature bitter oranges total phenol extract; the intestinal protection is specifically as follows: up-regulating the growth of wart microbiaceae, lactobacillaceae, Prevoteriaceae, inhibiting the growth of pilospiraceae, Bacteroides, Porphyromonaceae and Enterobacteriaceae, and protecting the integrity of intestinal barrier.
The application of the total phenols extract of immature bitter orange in the preparation of the intestinal tract protection medicament can correct the structural change of intestinal flora caused by antibiotics.
The application of the total phenols extract of immature bitter orange in the preparation of the intestinal tract protection medicine comprises the following components in a crude drug amount of 1 g/ml: neohesperidin 97244453.52ng/ml, naringin 61045773.9ng/ml, hesperidin 20603969.1ng/ml, narirutin 17752304.46ng/ml, hesperetin 3802648.92ng/ml, rhoifolin 2631472.98ng/ml, luteolin 2591288.1ng/ml, nobiletin 2291214.3ng/ml, eriocitrin 2166024.54ng/ml, rutin 852041.22ng/ml, diosmin 629279.22ng/ml, poncirin 520566.96ng/ml, hesperetin 340966.86ng/ml, naringenin 261388.26ng/ml, sinensetin 256528.62ng/ml, 5-nornobiletin 113856.18ng/ml, limonin 97171.56ng/ml, nomilin 50669.22ng/ml, diosgenin-7-O glucoside 34483.38ng/ml, apigenin 19076.58ng/ml, xanthotoxol 09ng/ml, kaempferitrinin 9062.52ng/ml, diosmin 5558.46ng/ml, diosmin 829 4ng/ml, 4218.9ng/ml eriodictyol, 4124.04ng/ml scoparone and 3898.14ng/ml isoanisic acid.
Compared with the prior art, the application of the total phenol extract of immature bitter orange in the preparation of the intestinal tract protection medicament aims at specific diseases more accurately, so that patients can more clearly and quickly select a total phenol extract preparation of immature bitter orange to treat the diseases after the disease causes of the patients are detected.
Drawings
FIG. 1: effect of TPE-CA on antibiotic-induced intestinal microflora composition. Relative abundance at phylum level of blank, antibiotic, TPE-CA and probiotic groups.
FIG. 2: effect of TPE-CA on antibiotic-induced intestinal microflora composition. Relative abundance at class level of blank, antibiotic, TPE-CA and probiotic groups.
FIG. 3: effect of TPE-CA on intestinal barrier integrity. A, the endotoxin expression levels of blank group, antibiotic group, TPE-CA group and probiotic group. B, expression levels of a blank group, an antibiotic group, a TPE-CA group and a probiotic group ZO-1. C, expression levels of placebo, antibiotics, TPE-CA, probiotic Ocplus. P <0.05, p <0.01, p <0.001, compared to the control group. Data are expressed as mean ± standard deviation, 10 animals per group.
Detailed Description
The following will further illustrate the application of the total phenols extract of citrus aurantium to the preparation of a drug for protecting the intestinal tract, but the scope of the present invention is not limited thereto.
Example 1
Experimental methods
Preparation of TPE-CA
400g of immature bitter orange is taken, crushed and soaked in 10 times of 50% ethanol for 30 minutes, and then the mixture is refluxed and extracted for 1.5 hours and repeated for three times. The three extracts were combined after sieving through a 80 mesh sieve, the ethanol was recovered in a vacuum rotary evaporator, the aqueous extracts were mixed and concentrated in a vacuum rotary evaporator, and dried in a vacuum oven to give 132 g of dry extract. The prepared immature bitter orange total phenol extract (TPE-CA) sample is stored in a refrigerator at the temperature of 20 ℃ below zero.
RRLC-QqQ-MSnThe TPE-CA component of the immature bitter orange extract is analyzed, and the specific process is shown in the article for administration. The crude drug amount of 1g/ml comprises the following components in percentage by weight: 97244453.52ng/ml neohesperidin, 61045773.9ng/ml naringin, 20603969.1ng/ml hesperidin, 17752304.46ng/ml rutin naringin, 3802648.92ng/ml hesperetin, 2631472.98ng/ml rhoifolin, 2591288.1ng/ml luteolin, 2291214.3ng/ml nobiletin, 2166024.54ng/ml eriocitrin, 852041.22ng/ml rutin, 629279.22ng/ml diosmin, 520566.96ng/ml poncirin, 340966.86ng/ml hesperetin, naringin, 17752304.46ng/ml rutin261388.26ng/ml, sinensetin 256528.62ng/ml, 5-nornobiletin 113856.18ng/ml, limonin 97171.56ng/ml, nomilin 50669.22ng/ml, diosmetin-7-O-glucoside 34483.38ng/ml, apigenin 19076.58ng/ml, xanthotoxol 15009ng/ml, kaempferide 9062.52ng/ml, diosmetin 5558.46ng/ml, eriodictyol 4218.9ng/ml, scoparone 4124.04ng/ml, and isoanisidine 3898.14 ng/ml.
Grouping animals
Healthy male C57BL6 mice (weight 20. + -.2 g) were purchased from Experimental animals technology Inc. of Wei Tong Hua, Beijing. Animals are raised in a temperature control room (22-25 ℃), and the light and shade alternating cycle is automatically controlled for 12 hours (6.30 in the morning and 6.30 in the afternoon). These mice were given standard laboratory food and water. According to the guidelines for the care and use of laboratory animals, specific nonpathogenic (SPF) class laboratories have been developed. One week after adaptive feeding, mice were randomized into normal, antibiotic, TPE-CA, probiotic groups of 10 per group. The normal group and the antibiotic group were given normal food and water before the end of the experiment; the TPE-CA group was administered by gavage with 8.7g/kg concentration of TPE-CA extract (26g/kg technical drug) once a day for four weeks. The probiotic group was administered at a concentration of 109Lactic acid bacteria 100uL for 4 weeks. Throughout the experiment, body weight of each mouse was recorded weekly and water was changed every 3 days during the experiment. Three other groups, in addition to the normal group, were molded. The molding method comprises the following steps: streptomycin, clindamycin and ampicillin were mixed into sterile drinking water (1 mg/mL) and water containing antibiotics was drunk for two weeks. During the experiment, water was replaced and/or replenished every 3 days. Antibiotic-induced dysbiosis was assessed by changes in microbiota abundance and intestinal permeability.
Obtaining animal materials
Fasting is carried out on the day after the last administration, water is not forbidden, feces are collected, the feces are weighed on the 2 nd day, then, 10% chloral hydrate is injected into the abdominal cavity for anesthesia, blood is taken from the abdominal aorta, the abdominal aorta is collected in a centrifugal tube, centrifugation is carried out at 10000rpm and 4 ℃ for 15min, and serum is taken and stored at minus 80 ℃. The liver, ileum and caecum contents of the mouse are immediately frozen in liquid nitrogen and then stored in a refrigerator at the temperature of-80 ℃.
Intestinal flora community analysis
The cecal contents (100mg) were subjected to DNA extraction using the Power Soil DNA isolation kit (MoBio Laboratories, Carlsbad, Calif.) according to the instructions and the purity and quality of the genomic DNA was checked with 0.8% agarose gel. Deep sequencing was performed on the Miseq platform of oweison gene, beijing. After run, image analysis and error estimation were performed using Illumina analysis Pipeline Version 2.6 software.
Serum endotoxin detection
Adding 100uL of water for bacterial endotoxin test, endotoxin standard solution, or test sample into pyrogen-free test tube. Then 100uL limulus reagent solution was added, mixed well, covered with aluminum foil paper, and incubated at 37 ℃ for 30 min. After the incubation, 100uL of the chromogenic substrate solution was added, mixed well and incubated at 37 ℃ for 6 min. After the incubation, 500uL of the azotization reagent 2 solution is added and mixed evenly, 500uL of the azotization reagent 3 solution is added and mixed evenly, the mixture is kept still for 5min, and the absorbance value is read at the 545nm wavelength.
Analysis results
Modulation of abundance composition of intestinal flora by TPE-CA
Changes in the abundance composition of the gut flora were detected using high-throughput sequencing. The abundance of intestinal flora is expressed in taxonomic units (OTUs). At the phylum level, nearly 95% of the bacteria belong to 10 phyla, the rest being assigned to other unclassified bacteria. The results show that the antibiotic groups bacteroidetes and proteobacteria are significantly increased and the firmicutes and actinomycetes are significantly decreased compared to the control group (FIG. 1). However, the treatment with TPE-CA and probiotics was completely opposite to the antibiotic group. At the family level, the TPE-CA treated group significantly upregulated the wart family, the lactic acid family, the prevotella family, significantly inhibited the growth of the pilospiraceae family, the bacteroidaceae family, the porphyromonadaceae family and the enterobacteriaceae family compared to the antibiotic group (fig. 2).
Effect of TPE-CA on gut barrier integrity
Intestinal permeability is used as an indicator of altered intestinal integrity. It has been shown that ZO-1 and occludin play an important role in intestinal permeability and endotoxin is also one of the markers. In order to further research the intestinal permeability, the content of endotoxin in serum is detected by biochemical indexes, and the content of related ZO-1 and occludin proteins is detected by biological indexes. The significantly higher serum endotoxin levels in the antibiotic group compared to the blank group indicated an increased intestinal permeability following antibiotic treatment (p <0.01, fig. 3A). The reduced enzyme activity of ZO-1 and occludin of the antibiotic group (p <0.01, fig. 3B, fig. 3C) indicates compromised mucosal integrity following antibiotic exposure. After treatment for 4 weeks, the TPE-CA or probiotic group can obviously improve the contents of ZO-1 and occludin and obviously reduce the content of endotoxin. These results indicate that TPE-CA has a strong protective effect on the integrity of intestinal barriers of mice.
Claims (3)
1. An application of total phenols extract of immature bitter orange in preparation of an intestinal tract protection medicament is characterized in that the preparation method of the total phenols extract of immature bitter orange comprises the following steps: crushing immature bitter oranges, soaking the crushed immature bitter oranges in 10 times of 50% ethanol for 30 minutes, then carrying out reflux extraction on the mixture for 1.5 hours, repeating the extraction for three times, sieving the mixture for 80 meshes, combining extracting solutions obtained in the three times, recovering the ethanol in a vacuum rotary evaporator, mixing and concentrating the extracting solutions in the vacuum rotary evaporator, and drying the extracting solutions in a vacuum oven to obtain an immature bitter oranges total phenol extract; the intestinal protection is specifically as follows: up-regulating the growth of wart microbiaceae, lactobacillaceae, Prevoteriaceae, inhibiting the growth of pilospiraceae, Bacteroides, Porphyromonaceae and Enterobacteriaceae, and protecting the integrity of intestinal barrier.
2. The use of total phenols from citrus aurantium as claimed in claim 1 in the preparation of a medicament for the protection of the intestinal tract, wherein the protection is a correction of the structural changes of the intestinal flora caused by antibiotics.
3. The application of the total phenols extract of immature bitter orange in the preparation of an intestinal tract protection medicament as claimed in claim 1, wherein the total phenols extract of immature bitter orange comprises the following components in a crude drug amount of 1 g/ml: 97244453.52ng/ml neohesperidin, 61045773.9ng/ml naringin, 20603969.1ng/ml hesperidin, 17752304.46ng/ml rutin naringin, 3802648.92ng/ml hesperetin, 2631472.98ng/ml rhoifolin, 2591288.1ng/ml luteolin, 2291214.3ng/ml nobiletin, 2166024.54ng/ml eriocitrin, 852041.22ng/ml rutin, 629279.22ng/ml diosmin, 520566.96ng/ml poncirin, 340966.86ng/ml hesperetin, 261388.26ng/ml naringenin, 256528.62ng/ml sweet orange flavone, 113856.18ng/ml 5-nornobiletin, 97171.56ng/ml limonin, 50669.22ng/ml nomilin, 7-O glucoside 34483.38ng/ml diosmin, 19076.58ng/ml apigenin, 15009ng/ml xanthotoxol, 15032 ng/ml xanthotoxol, Kaempferide 9062.52ng/ml, diosmetin 5558.46ng/ml, eriodictyol 4218.9ng/ml, scoparone 4124.04ng/ml, and isoanisic lactone 3898.14 ng/ml.
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| CN105995326A (en) * | 2016-05-20 | 2016-10-12 | 西南大学柑桔研究所 | Method for acquiring pectin, tangerine peel fiber and tangerine peel fermented beverage by classifying citrus peels |
| CN107028962A (en) * | 2017-04-14 | 2017-08-11 | 杭州禹正医药科技有限责任公司 | Application of the neohesperidin in preventing and treating tumour medicine is prepared |
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| CN105995326A (en) * | 2016-05-20 | 2016-10-12 | 西南大学柑桔研究所 | Method for acquiring pectin, tangerine peel fiber and tangerine peel fermented beverage by classifying citrus peels |
| CN107028962A (en) * | 2017-04-14 | 2017-08-11 | 杭州禹正医药科技有限责任公司 | Application of the neohesperidin in preventing and treating tumour medicine is prepared |
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| Naturally Occurring TPE-CAMaintains Gut Microbiota and Bile Acids Homeostasis via FXR Signaling Modulation of the Liver–Gut Axis;Linlin Liu. et al.;《Frontiers in Pharmacology》;20200206;第11卷;第1-16页 * |
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