Use of Quzhou Aurantii Fructus and extract thereof in prevention and treatment of renal injury

Abstract

Provided is the use of Quzhou Aurantii Fructus (QAF) and a QAF extract in the prevention and treatment of renal injury, specifically for preventing and treating acute renal injury induced by a chemotherapy drug such as cisplatin. The Quzhou Aurantii Fructus (QAF) and QAF extract may improve cisplatin-induced renal injury by inhibiting cisplatin-induced oxidative stress and apoptosis of renal tissue through upregulation of peroxidase 3 and BCL-2 expression. A method for the preparation of the QAF extract is also provided which comprises conducting reflux extraction and filtration in sequence to obtain a filtrate which may be evaporated to obtain the QAF extract. A preparation process of QAF extract has a simple route and low production cost and can be used for preparing a drug for inhibiting cisplatin-induced oxidative stress and apoptosis of renal tissue as well as improving a cisplatin-induced renal injury.

Description

[0001] The present disclosure belongs to the technical field of natural medicine and specifically relates to the use of Quzhou Aurantii Fructus (QAF) and a QAF extract in the prevention and treatment of renal injury. BACKGROUND

[0002] Cisplatin, as a widely used anti-tumor drug in clinical practice, is widely used in the treatment of solid organ cancers such as ovarian cancer, breast cancer, lung cancer, and testicular cancer. However, during clinical use, it is found that the side effects of cisplatin, such as nephrotoxicity, ototoxicity, and neurotoxicity, have greatly limited the clinical use of cisplatin. In particular, nephrotoxicity is the most common and serious side effect and directly affects the efficacy and prognosis of chemotherapy. Cisplatin is used in about 10% to 20% of cancer patients, of which 30% may suffer from acute renal injury, which is one of the serious side effects of cisplatin in clinical use. In view of this, it has become an urgent issue to find an effective drug to prevent cisplatin-induced acute renal injury.

[0003] Quzhou Aurantii Fructus (QAF) refers to a dried immature fruit of Citrus aurantium 'Changshanhuyou'. The QAF was included in the 2015 edition of "Zhejiang Province Traditional Chinese Medicine Processing Specifications" in 2016 and was selected as one of the novel traditional Chinese medicine cultivation varieties belonging to the "Eight Traditional Chinese Medicines of Zhejiang" and the "Six Traditional Chinese Medicines of Quzhou" in Zhejiang Province in 2018. The QAF is cold in nature and bitter in taste and has the effects of regulating qi and relieving chest tightness, relieving stagnation and dissolving swelling, relieving cough and resolving phlegm, as well as clearing away heat and detoxification. The main components of QAF are flavonoids. Modem pharmacological studies have found that the flavonoid components in QAF have the effects of lowering blood lipids and blood glucose, anti-inflammatory, and protecting the liver. In addition, naringenin and hesperidin in QAF can increase the serum insulin level of spontaneous type 2 diabetes mellitus-loaded db / db mice under a free diet and alleviate pancreatic islet cell damage.

[0004] In the prior art, the pharmacological studies on QAF extracts mostly focus on the therapeutic applications of liver injury and lung injury. However, there are no reports on the use of the QAF extracts in cisplatin-induced renal injury. SUMMARY

[0005] In view of the problems existing in the prior art, an object of the present disclosure is to provide a novel use of QAF and a QAF extract, specifically the use of QAF and a QAF extract in preparation of a product for preventing and treating a renal injury.

[0006] Further, the renal injury includes cisplatin-induced renal function decline and an acute pathological injury of renal tissue during cancer treatment.

[0007] Further, the product significantly improves a cisplatin-induced acute renal injury, including significantly improving renal function and a pathological change of renal tissue in a mouse with acute renal injury.

[0008] Further, a mechanism by which the product improves renal injury is related to increasing expression levels of peroxidase 3 and an anti-apoptotic protein Bcl2 in the renal tissue, indicating that the product protects the renal function and improves renal injury by inhibiting oxidative stress and apoptosis.

[0009] Further, a preparation method for the QAF extract includes the following steps:

[0010] 1) immersing a QAF decoction piece in boiling water, conducting reflux extraction and filtration in sequence, and collecting an obtained primary filtrate;

[0011] 2) adding water into a filter residue to allow reflux extraction by heating, and conducting filtration to obtain a secondary filtrate; and

[0012] 3) mixing the primary filtrate of step 1) and the secondary filtrate of step 2) to obtain a mixed filtrate, evaporating the mixed filtrate to dryness under reduced pressure and recovering a solvent, and drying a residue in a vacuum drying oven to obtain the QAF extract.

[0013] Further, the QAF decoction piece and the water are at a mass ratio of 1:8, the immersing is conducted for 2 h, and the reflux extraction is conducted for 1.5 h in step 1).

[0014] Further, the filter residue and the water are at a mass ratio of 1:6, and the reflux extraction is conducted twice, 1 h for each time in step 2).

[0015] The present disclosure creatively proposes novel medical use of a QAF extract in treatment of a renal injury and opens up a novel application field for the QAF extract. The QAF shows abundant sources, low cost, safety, and no toxic side effects. The preparation process of the QAF extract has a simple route and low production cost and can be used for preparing a drug for alleviating cisplatin-induced oxidative stress and apoptosis of a renal tissue as well as improving a cisplatin-induced renal injury.

[0016] According to a first aspect of the present invention there is provided Quzhou Aurantii Fructus (QAF) and QAF extract thereof for use in the prevention and treatment of renal injury.

[0017] Preferably the renal injury comprises cisplatin-induced renal function decline and an acute pathological injury of a renal tissue during a cancer treatment.

[0018] Preferably a method for the preparation of the QAF extract comprises the following steps: 1) immersing a QAF decoction piece in boiling water, conducting reflux extraction and filtration in sequence, and collecting an obtained primary filtrate; 2) adding water into a filter residue to allow reflux extraction by heating, and conducting filtration to obtain a secondary filtrate; and 3) mixing the primary filtrate of step 1) and the secondary filtrate of step 2) to obtain a mixed filtrate, evaporating the mixed filtrate dryness under pressure and recovering a solvent, and drying a residue in a vacuum drying oven to obtain the QAF extract.

[0019] Preferably the QAF decoction piece and the water are at a mass ratio of 1:8, the immersing is conducted for 2 h, and the reflux extraction is conducted for 1.5 h in step 1).

[0020] Preferably the filter residue and the water are at a mass ratio of 1:6, and the reflux extraction in step 2) is conducted twice, 1 h for each time.

[0021] Preferably the product significantly improves a cisplatin-induced acute renal injury, comprising significantly improving renal function and a pathological change of renal tissue in a mouse with the acute renal injury.

[0022] Preferably a mechanism by which the product improves the renal injury is related to increasing expression levels of peroxidase 3 and an anti-apoptotic protein Bcl2 in the renal tissue, indicating that the product protects the renal function and improves the renal injury by inhibiting oxidative stress and apoptosis.

[0023] According to a second aspect of the present invention there is provided Quzhou Aurantii Fructus (QAF) and QAF extract thereof for use in the preparation of a product for preventing and treating renal injury.

[0024] Preferably the renal injury comprises cisplatin-induced renal function decline and an acute pathological injury of a renal tissue during a cancer treatment.

[0025] Preferably a method for the preparation of the QAF extract comprises the following steps: 1) immersing a QAF decoction piece in boiling water, conducting reflux extraction and filtration in sequence, and collecting an obtained primary filtrate; 2) adding water into a filter residue to allow reflux extraction by heating, and conducting filtration to obtain a secondary filtrate; and 3) mixing the primary filtrate of step 1) and the secondary filtrate of step 2) to obtain a mixed filtrate, evaporating the mixed filtrate dryness under pressure and recovering a solvent, and drying a residue in a vacuum drying oven to obtain the QAF extract.

[0026] Preferably the QAF decoction piece and the water are at a mass ratio of 1:8, the immersing is conducted for 2 h, and the reflux extraction is conducted for 1.5 h in step 1).

[0027] Preferably the filter residue and the water are at a mass ratio of 1:6, and the reflux extraction in step 2) is conducted twice, 1 h for each time.

[0028] Preferably the product significantly improves a cisplatin-induced acute renal injury, comprising significantly improving renal function and a pathological change of renal tissue in a mouse with the acute renal injury.

[0029] Preferably a mechanism by which the product improves the renal injury is related to increasing expression levels of peroxidase 3 and an anti-apoptotic protein Bcl2 in the renal tissue, indicating that the product protects the renal function and improves the renal injury by inhibiting oxidative stress and apoptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1 shows a chromatogram of the QAF extract in the present disclosure; NOTE: A is a standard chromatogram; B is a sample chromatogram; 1 is narirutin; 2 is naringin; 3 is hesperidin; 4 is neohesperidin;

[0031] FIG. 2 shows an influence of the QAF extract in the present disclosure on serum blood urea nitrogen (BUN) and creatinine (CREA) in cisplatin-induced acute renal injury in mice;

[0032] FIG. 3 shows H2O2 and glutathione (GSH) contents in the kidneys of mice in each group in an animal experiment of the present disclosure;

[0033] FIG. 4 shows pathological changes of renal tissues of mice in each group in an animal experiment of the present disclosure (where H&E staining of mouse kidneys in the CDDP model group shows a large number of renal tubular epithelial cell damages, mainly proximal tubule damages; there are tubular epithelial cell swelling, vacuolar degeneration, nuclear swelling, partial nuclear dissolution, and cell necrosis; compared with a cisplatin (CDDP) model group, high-dose QAF can significantly improve cisplatin-induced tubular epithelial cell damage, nuclear dissolution, and protein deposition); and

[0034] FIG. 5A and FIG. 5B show an influence of the QAF extract in the present disclosure on expression levels of peroxiredoxin 3 (PRDX3) and anti-apoptotic protein in kidney tissues of the cisplatin-induced acute renal injury model in mice. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035] The present disclosure is further described below in conjunction with the accompanying drawings in order to better understand the present technical solution.

[0036] The present disclosure provides the use of a QAF extract in preparation of a product for alleviating and treating a renal injury. Specifically, the renal injury includes cisplatin-induced oxidative stress and apoptosis of renal tissue in cancer treatment, where the QAF extract is prepared by the following steps:

[0037] 470 g of a QAF decoction piece is added to water of 8 times in amount, heated and immersed for 2 h, heated to allow reflux extraction for 1.5 h, and then filtered to obtain a primary filtrate; water of 6 times in amount is added into a remaining residue, and the reflux extraction by heating is continued twice, 1 h for each time, to obtain a secondary filtrate; the two filtrates are combined to obtain a mixed filtrate, which is evaporated to dryness and the solvent is recovered, and then dried in a vacuum drying oven to remove moisture, to obtain the QAF extract.

[0038] Experimental Example

[0039] Drugs and reagents used in the experiment: QAF extract; cisplatin (Sigma, USA), hydrogen peroxide content detection kit, glutathione content determination kit (Beijing Solarbio Co., Ltd., China), P-actin primary antibody, PRDX3 primary antibody, Bcl2 primary antibody, KIMI primary antibody (Abeam, USA), PAGE gel rapid preparation kit (12.5%) (Shanghai Epizyme Biotech Co., Ltd.), SDS-PAGE loading buffer (5x) (Beyotime Biotech Co., Ltd.).

[0040] Instruments: EI0001 electrophoresis instrument (Thermo Fisher, USA), 6000exp chemiluminescence imaging system (Shanghai Qinxiang); 1658001 vertical electrophoresis tank, 170-3932 transfer electrophoresis tank (Bio-Rad, USA), MB-102 oscillating constant-temperature metal bath (Hangzhou Bioer Technology), 7100 fully automatic biochemical analyzer (Hitachi, Japan), 80i microscope (Nikon, Japan). Experimental animals: 42 C57 mice, male, SPF-grade, 7 weeks old, body weight 22 g+2 g, provided by Beijing Vital River Laboratories Co., Ltd., Laboratory Animal Production License NO: SCXK (Zhejiang) 2019-0002, Laboratory Animal License NO: SYXK (Zhejiang) 2022-0027. The mice were given free access to water, in a 12-h circadian rhythm, and at room temperature (22+1 °C). The animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Safety Evaluation Research Center of Zhejiang Academy of Medical Sciences (approval number: KY-2023-019).

[0041] Experimental method

[0042] 1. Identification of the QAF extract: chromatographic conditions: Agilent Extend Cis (5 pm, 4.6x250 nm) was used as a chromatographic column, acetonitrile-0.1% formic acid water (18:82, v / v) was used as a mobile phase for isocratic elution; where the flow rate was 1.0 mL / min; the detection wavelength was 330 nm; the column temperature was 30°C; and the injection volume was 10 pL. Preparation of reference solution: an appropriate amount of narirutin, naringin, hesperidin, and neohesperidin reference substances were taken separately, accurately weighed, and dissolved in 50% methanol, to obtain mixed reference solutions of about 60, 150, 120, and 120 pg / mL. Preparation of a test solution: 0.1 g of QAF extract (T230301), accurately weighed, put in a 25 mL volumetric flask, an appropriate amount of 50% methanol was added, ultrasonically treated for 20 min, cooled to room temperature, added with 50% methanol to a preset scale, shaken well, centrifuged, and a supernatant was collected to obtain the test solution. The chromatogram of the QAF extract is shown in FIG. 1.

[0043] As shown in FIG. 1, the amount of sample extracted was 179.6 g, the yield was 38.2%, and the content determination results showed that the contents of narirutin, naringin, hesperidin, and neohesperidin were 13.07, 141.05, 7.38, and 91.27 mg / g, respectively.

[0044] 2. Animal grouping, modeling, and drug administration

[0045] 42 male C57 mice were randomly divided into 7 groups based on their body weight, with 6 mice in each group, including: a control group (control), a QAF administration group (2.5 g / kg, QAF), a cisplatin model group (CDDP, 15 mg / kg), a cisplatin + dexamethasone group (5 mg / kg, CDDP+Dex), a cisplatin + QAF low-dose group (0.625 g / kg QAF, CDDP+LQAF), a cisplatin + QAF medium-dose group (1.25 g / kg, CDDP+MQAF), and a cisplatin QAF high-dose group (2.5 g / kg, CDDP+HQAF). The QAF aqueous solution was intraperitoneally administered for 7 consecutive days, twice a day. On the 5th day of intraperitoneal administration, the cisplatin model group, the cisplatin + QAF low-, medium-, and high-dose groups were intraperitoneally injected with cisplatin (15 mg / kg) to establish the model, and the cisplatin + Dex group was intraperitoneally injected with Dex (5 mg / kg) one hour before the model establishment; the control group and the QAF group were intraperitoneally injected with an equal amount of normal saline. Tissue and blood samples were collected and stored from animals. 72 h after modeling, the mice were fasted but water was allowed for 12 h in advance, anesthetized with 0.5% sodium pentobarbital, blood was collected from the abdominal aorta, serum was separated, kidneys were separated and weighed, and stored at -80°C.

[0046] Serum biochemical index monitoring: the automatic biochemical analyzer was used to detect the BUN and CREA contents in mouse serum. The effects of QAF extract on serum BUN and CREA in cisplatin-induced acute renal injury in mice are shown in FIG. 2. As shown in FIG. 2, compared with the control group, the BUN level in the CDDP model group was significantly increased, and compared with the model group, the BUN level of CDDP+HQAF (2.5 g / kg) was significantly reduced, MP<0.05, ##P<0.05; compared with the control group, the CREA level in the mouse kidney was significantly increased in the CDDP model group, and compared with the model group, the CREA level of CDDP+HQAF (2.5 g / kg) was significantly reduced, **P<0.05, **P<0.05.

[0047] Monitoring of renal tissue homogenate indicators: the H2O2 and GSH contents in renal tissue homogenate were detected according to the kit instructions. The results are shown in FIG. 3. From FIG. 3, compared with the H2O2 content in control group, the CDDP model H2O2 content was significantly higher than that of the control group, compared with the CDDP model group, CDDP+HQAF (2.5 g / kg) group H2O2 content had significantly decreased significantly, *P<0.05; compared with the control group of mice, the GSH content of the CDDP model group had increased significantly. Compared with the CDDP model group, the CDDP+HQAF (2.5 g / kg) group GSH content decreased significantly, **P<0.05.

[0048] Renal tissue pathological observation: renal tissue was fixated with 10% formaldehyde solution, embedded in paraffin, sectioned, stained with hematoxylin&eosin (H&E), and observed. The results are shown in FIG. 4. From FIG. 4, H&E staining showed that the renal tissue cortex and medulla of the blank control group and the single-dose group had normal proportions, normal arrangement of glomeruli and tubules, clear structure, no abnormal changes such as swelling, degeneration, and necrosis. The CDDP model group had severe protein casts in the cortex and medulla, diffuse degeneration and necrosis of tubules, almost no normal tubules, swelling, fragmentation, and dissolution of tubular epithelial cell nuclei, and cytoplasm disintegration. The QAF low-dose group had moderate protein casts in the cortex and medulla, large-area necrosis of tubules, swelling, fragmentation, and dissolution of tubular epithelial cell nuclei, and cytoplasm disintegration, but a small number of relatively normal tubules were visible. The QAF medium-dose group had mild protein casts in the cortex, multifocal tubular necrosis, necrosis and shedding of tubular epithelial cells, nuclear condensation, nuclear fragmentation, and nuclear dissolution, and more normal tubules were seen locally. The QAF high-dose group and the Dex-positive control group had mild cortical protein casts and focal tubular necrosis. The area of necrosis was smaller than that of the medium and low-dose groups, and the number of normal tubules increased.

[0049] Immunoblotting was conducted to detect the expression of PRDX3, Bcl2, and KIMI proteins in renal tissue: mouse renal tissue was added to Western and IP cell lysate and homogenized, and the protein was extracted and separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membrane, blocked at room temperature, and then primary antibody (dilution ratio: 1:1000) was added, and incubated at 4°C overnight. After washing, the secondary antibody (anti-rabbit IgG and anti-mouse IgG, dilution ratio: 1:1000) was added and incubated on a shaker at room temperature for 1 h. After washing, the enhanced chemiluminescence (ECL) colorimetric solution was used for color development, and the bands were photographed with a gel imager. Image J software was used for statistical analysis, and the relative expression of the target protein was calculated by dividing the gray value of the target protein by the gray value of the internal reference protein.

[0050] The influence of QAF extract on the expression of antioxidant proteins and anti-apoptotic proteins in renal tissue of mice with acute renal injury induced by cisplatin is shown in FIG. 5A and FIG. 5B. As shown in FIG. 5A and FIG. 5B: compared with the Bcl2 and PRDX3 proteins in the control group, the expression of Bcl2 and PRDX3 proteins in the DDP model group was significantly reduced; and compared with the CDDP model group, the expression of Bcl2 and PRDX3 in the CDDP+HQAF (2.5 g / kg) group was significantly increased, **p<0.05.

[0051] In the present disclosure, the QAF intervention can significantly improve the renal function of mice with cisplatin-induced acute renal injury, including reducing the increase in serum CREA and BUN levels induced by cisplatin, improving renal tissue pathological changes, and increasing the expression of peroxidase 3 (PRDX3) and anti-apoptotic protein Bcl2 in renal tissue. These results suggested that the QAF had an effect in improving cisplatin-induced acute renal injury. The experimental results show that in cisplatin-induced acute renal injury, QAF can protect renal function by inhibiting oxidative stress and apoptosis, providing direction for the potential mechanism of QAF in preventing cisplatin-induced acute renal injury.

Claims

AT IS CLAIMED IS:

1. Quzhou Aurantii Fructus (QAF) and QAF extract thereof for use in the prevention and treatment of cisplatin-induced acute renal injury;wherein the QAF extract is obtained through the following steps:I) immersing a QAF decoction piece in boiling water, conducting reflux extraction and fdtration in sequence, and collecting an obtained primary filtrate;2) adding water into a filter residue to allow reflux extraction by heating, and conducting filtration to obtain a secondary filtrate; and3) mixing the primary filtrate of step 1) and the secondary filtrate of step 2) to obtain a mixed filtrate, evaporating the mixed filtrate dryness under pressure and recovering a solvent, and drying a residue in a vacuum drying oven to obtain the QAF extract.

2. The Quzhou Aurantii Fructus (QAF) and QAF extract thereof for use according to claim 1, wherein the renal injury comprises cisplatin-induced renal function decline and an acute pathological injury of a renal tissue during a cancer treatment.

3. The Quzhou Aurantii Fructus (QAF) and QAF extract thereof for use according to claim 1, wherein the QAF decoction piece and the water are at a mass ratio of 1:8, the immersing is conducted for 2 h, and the reflux extraction is conducted for 1.5 h in step 1).

4. The Quzhou Aurantii Fructus (QAF) and QAF extract thereof for use according to claim 1 or claim 3, wherein the filter residue and the water are at a mass ratio of 1:6, and the reflux extraction in step 2) is conducted twice, 1 h for each time.

5. The Quzhou Aurantii Fructus (QAF) and QAF extract thereof for use according to claim 1, wherein the renal injury is prevented or treated by increasing expression levels of peroxidase 3 and an anti-apoptotic protein Bcl2 in the renal tissue.13

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