A bamboo vinegar powder extract, a method for identifying effective components thereof, and applications thereof
Bamboo vinegar powder extract was prepared by extracting components such as pyrogallol and 4-methylcatechol from bamboo vinegar, which solved the problem of large usage of bamboo vinegar and achieved high-efficiency antioxidant and antifungal effects at low concentrations, making it suitable for a variety of products.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- XIAMEN MEDICAL COLLEGE
- Filing Date
- 2023-02-20
- Publication Date
- 2026-04-17
AI Technical Summary
Existing research indicates that bamboo vinegar is used in large quantities and is not suitable as an additive to be added in small amounts to other products for auxiliary use. Furthermore, its active ingredients do not show significant antioxidant and antifungal effects at low concentrations.
Components such as pyrogallol, 4-methylcatechol, p-hydroxyphenylethanol, p-methylbenzoic acid, and dihydrocaffeic acid were extracted from bamboo vinegar, concentrated by ethyl acetate extraction, and freeze-dried to prepare bamboo vinegar powder extract, which is used to prepare antioxidant and antifungal products.
Bamboo vinegar powder extract exhibits significant antioxidant and antifungal capabilities at low concentrations, effectively removing free radicals and inhibiting fungi. It requires less dosage than bamboo vinegar itself and is suitable for various products.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention relates to a bamboo vinegar powder extract and its applications. Background Technology
[0002] Bamboo vinegar is a brownish-red liquid produced during pyrolysis. It contains over 200 different components, including phenols, organic acids, alcohols, aldehydes, ketones, and furans. With a pH of only about 2.5-3.0, bamboo vinegar can be used for sterilization, disinfection, and as a feed additive. Past studies have found that adding bamboo charcoal and bamboo vinegar to compost materials can optimize the microbial community and improve the safety of compost products (Guo et al., 2019. Environ. Pollut. 252, 1097-1105.). Adding bamboo vinegar to feed can increase the production efficiency of chickens and ducks and promote growth (Yamauchi et al., 2010. J. Anim. Feed Sci. 19, 257–268; Ruttanavut et al., 2009. Int. J. Poultry Sci. 8, 229-236.). Furthermore, it can effectively regulate the immune system, significantly reduce the expression of cytokines (such as IL-1β and IL-6) and the production of nitric oxide (NO), while activating the NLRP3 inflammasome to produce anti-inflammatory effects (Ho et al., PloS One. 8, e75738). Bamboo vinegar in feed may also increase the concentration of IgG and IgA in serum without affecting the concentration of IgM, thus building a stronger immune system (Chu et al., 2013, Anim Sci J. 84, 113-120). Bamboo vinegar has also been used as an antibiotic alternative, effectively inhibiting microorganisms.
[0003] Existing studies have found that bamboo vinegar can effectively inhibit Cryptococcus neoformans, Candida albicans, Trichophyton mentagrophytes, Saccharomyces cerevisiae, Aspergillus niger, and Pseudomonas aeruginosa (Han Liang et al., 2011, Journal of Jiangsu University (Medical Edition), 21, 167-170+174; Wang Yu et al., 2013, Chinese Journal of Disinfection, 30, 1116-1118). However, existing studies mainly focus on the whole use of bamboo vinegar, which involves large quantities and is not suitable for use as an additive in small amounts in other products. Summary of the Invention
[0004] The main objective of this invention is to provide a bamboo vinegar powder extract.
[0005] The technical solution adopted by this invention to solve its technical problem is:
[0006] Bamboo vinegar powder extract, wherein the extract comprises at least one of the following components: pyrogallol, 4-methylcatechol, p-hydroxyphenylethanol, p-methylbenzoic acid, dihydrocaffeic acid, and 1,4-benzenediol.
[0007] In this invention, bamboo vinegar powder is extracted and concentrated with ethyl acetate, and then freeze-dried to obtain bamboo vinegar powder extract. Preferably, 10g of bamboo vinegar powder is dissolved in 50-200ml of deionized water, and then 100-400ml of ethyl acetate is added for mixing and extraction. The extracted ethyl acetate is concentrated using a rotary evaporator and then freeze-dried.
[0008] The present invention also provides the application of the bamboo vinegar powder extract in the preparation of antioxidant products.
[0009] Furthermore, the extract comprises at least one of the following components: pyrogallol, 4-methylcatechol, dihydrocaffeic acid, and 1,4-benzenediol, which achieves a removal rate of over 90% for ABTS free radicals at a concentration of 0.005 mg / mL.
[0010] The present invention also provides the application of the bamboo vinegar powder extract in products resistant to Aspergillus terreus, Trichoderma longibrachiatum or Paecilomyces variotii, wherein the bamboo vinegar powder extract comprises at least one of 4-methylcatechol and p-methylbenzoic acid.
[0011] Furthermore, the minimum amount of the bamboo vinegar powder extract used is 0.25 mg / mL.
[0012] The present invention also provides a method for preparing bamboo vinegar powder extract, the preparation method comprising the following steps:
[0013] Step 1: Bamboo vinegar powder is extracted and concentrated with ethyl acetate, and then freeze-dried to obtain crude bamboo vinegar powder extract;
[0014] Step 2: The components were separated using a chromatographic column and eluted with 0.1% formic acid acetonitrile and 0.1% formic acid water. The retention times of the compounds were identified as follows:
[0015] 1,2,3-Trihydroxybenzene (phloroglucinol): 371 seconds
[0016] 4-Methylcatechol: 540 seconds
[0017] 4-Hydroxyphenylethanol (p-hydroxyphenylethanol: 614 seconds)
[0018] 4-Methylbenzoic acid (p-methylbenzoic acid): 519 seconds
[0019] 3,4-Dihydroxyhydrocinnamic acid (dihydrocaffeic acid): 367 seconds
[0020] Hydroquinone (1,4-benzenediol): 437 seconds
[0021] Further, in step one, every 10g of bamboo vinegar powder is dissolved in 50-200ml of deionized water, and then 100-400ml of ethyl acetate is added for mixing and extraction. The extracted ethyl acetate is concentrated using a rotary evaporator.
[0022] Furthermore, in step two, ACQUITY is used. HSS T3 1.8μm (2.1×150mm) column.
[0023] Compared with the prior art, this technical solution has the following advantages:
[0024] 1. The present invention identifies several compounds from bamboo vinegar that exhibit excellent fungal removal rates even at low concentrations.
[0025] 2. Several compounds identified from bamboo vinegar in this invention exhibit excellent antioxidant properties even at low concentrations (the lowest concentration of a single compound is 0.005 mg / mL).
[0026] 3. Compared to using bamboo vinegar itself, which requires a large amount, this invention uses an extract that, when added in small amounts, can have antioxidant or antifungal effects and can be added to various products such as pharmaceuticals. Attached Figure Description
[0027] The present invention will be further described below with reference to the accompanying drawings and embodiments.
[0028] Figure 1 The scavenging rate of bamboo vinegar powder extract against ABTS(A) and DPPH(B) free radicals.
[0029] Figure 2 The scavenging rate of nitric oxide by bamboo vinegar powder extract.
[0030] Figure 3The inhibition rate of bamboo vinegar powder extract against different fungi, such as Aspergillus terreus CICC40377(A), Trichoderma longibrachiatum CICC40380(B), and Paecilomyces variotii CICC40716(C).
[0031] Figure 4 The bamboo vinegar powder extract exhibits an antibacterial clear zone against various fungi, such as Aspergillus terreus CICC40377(A), Trichoderma longibrachiatum CICC40380(B), and Paecilomyces variotii CICC40716(C).
[0032] Figure 5 ABTS radical scavenging rates of compounds in bamboo vinegar powder extract, such as pyrogallol (1,2,3-Trihydroxybenzene, A), 4-methylcatechol (B), 4-Hydroxyphenylethanol (C), 3,4-Dihydroxyhydrocinnamic acid (D), and 1,4-hydroxyquinone (E), were analyzed.
[0033] Figure 6 Analysis of DPPH radical scavenging rates of compounds in bamboo vinegar powder extract, such as pyrogallol (1,2,3-Trihydroxybenzene, A), 4-methylcatechol (B), 3,4-dihydroxyhydrocinnamic acid (C), and 1,4-hydroxyquinone (D).
[0034] Figure 7The fungal inhibition rates of compounds in bamboo vinegar powder extract, such as 1,2,3-trihydroxybenzene, 4-methylcatechol, 4-hydroxyphenylethanol, 4-methylbenzoic acid, D-mandelic acid, 3,4-dihydroxyhydrocinnamic acid, maleic acid, and 1,4-hydroquinone, are as follows: Aspergillus terreus CICC40377(A), Trichoderma longibrachiatum CICC40380(B), and Paecilomyces variotii CICC40716(C).
[0035] Figure 8 The antibacterial transparent zone is a compound in bamboo vinegar powder extract, such as 4-methylcatechol and 4-methylbenzoic acid.
[0036] Figure 9 Cytotoxicity analysis of compounds in bamboo vinegar powder extract, such as 4-methylcatechol and 4-methylbenzoic acid: A shows the effect of 4-methylcatechol on human immortalized keratinocytes (HaCaT); B shows the effect of 4-methylbenzoic acid on human immortalized keratinocytes (HaCaT); C shows the effect of 4-methylcatechol on macrophages (RAW 264.7); D shows the effect of 4-methylbenzoic acid on macrophages (RAW 264.7). Detailed Implementation
[0037] Example 1: Preparation of Bamboo Vinegar Powder Extract
[0038] 10g of bamboo vinegar powder (Jiangyin Zhongju Biotechnology Co., Ltd.) was dissolved in 100mL of deionized water and then mixed with ethyl acetate for extraction. The extracted ethyl acetate was concentrated using a rotary evaporator and then dried using a freeze dryer to obtain the bamboo vinegar powder extract.
[0039] Example 2 Antioxidant Analysis of Bamboo Vinegar Powder Extract
[0040] 1. Evaluation of DPPH free radical inhibition: Prepare a 10mM DPPH methanol solution, then dilute this solution to a 1mM DPPH solution. Take bamboo vinegar powder extracts of different concentrations, add 1mM DPPH solution, vortex to mix, and let stand at room temperature for 30 minutes. Analyze the absorbance at 517nm using a microplate reader.
[0041] 2. Assessment of ABST free radical inhibition: The ABTS free radical scavenging rate was analyzed according to the Total Antioxidant Capacity Assay Kit (ABTS method) (Shanghai Beyotime Biotechnology Co., Ltd.). The ABTS working solution was diluted to an OD734 of about 0.7, and then bamboo vinegar powder extract of different concentrations was added. The absorbance at 734 nm was analyzed by microplate reader.
[0042] Example 3: Analysis of the inhibition of nitric oxide formation by bamboo vinegar powder extract
[0043] Take 50 μL of 20 mM sodium nitroprusside (nitric oxide donor), add 50 μL of bamboo vinegar powder extract at different dilutions, incubate at 25℃ for 60 minutes, then add 100 μL of Griess Reagent I and Griess Reagent II mixed reagent from the nitric oxide detection kit (Beyotime Biotechnology Co., Ltd.), and analyze the absorbance at 540 nm using an ELISA reader.
[0044] Example 4 Antifungal Analysis
[0045] 1. Antifungal ability assessment: PDB medium containing fungal spores (Aspergillus terreus CICC40377, Trichodermalongibrachiatum CICC40380, Paecilomyces variotii CICC40716) was introduced into 24-well plates. Different concentrations of bamboo vinegar powder extract or bamboo vinegar powder extract compounds were added. After incubation at 30°C for 48 hours, the growth of fungi was measured under OD600 spectrophotometer conditions.
[0046] 2. The disc diffusion method was used to analyze fungi, and the inhibitory efficacy was evaluated based on the size of the clear zone formed. Fungal culture medium (PDA medium) containing fungal spores was poured into petri dishes using the pour method. Different concentrations of bamboo vinegar powder extract or bamboo vinegar powder extract compounds were added to 6mm diameter circular paper discs.
[0047] Place the culture dish on a solid fungal culture medium and continue culturing at 30°C to observe the formation of the inhibition zone.
[0048] Example 5: Compound Analysis of Bamboo Vinegar Powder Extract
[0049] Different metabolites were identified using LC-MS (liquid chromatography-mass spectrometry). The chromatographic instrument used was a Thermo Vanquish, and the instrument was ACQUITY. HSS T3 1.8 μm (2.1 × 150 mm) column, positive ion mode, mobile phase: 0.1% formic acid acetonitrile (A) and 0.1% formic acid water (B); gradient elution program: 0–1 min, 2% A; 1–9 min, 2%–50% A; 9–12 min, 50%–98% A; 12–13.5 min, 98% A; 13.5–14 min, 98%–2% A; 14–20 min, 2% A. Negative ion mode, mobile phase: acetonitrile (C) and 5 mM formic acid water (D); gradient elution program: 0–1 min, 2% C; 1–9 min, 2%–50% C; 9–12 min, 50%–98% C; 12–13.5 min, 98% C; 13.5–14 min, 98%–2% C; 14–17 min, 2% C. The mass spectrometer used was Thermo Q Exactive Plus, with an electrospray ionization (ESI) source, in both positive and negative ion ionization modes. The positive ion spray voltage was 3.50 kV, and the negative ion spray voltage was -2.50 kV. A full scan was performed at a resolution of 70,000, with a scan range of 100–1,000 m / z. Secondary fragmentation was performed using HCD at a collision voltage of 30 eV. Dynamic exclusion was used to remove unnecessary MS / MS information. Analysis was conducted using public databases such as HMDB, massbank, mzclound, and the self-built standard database of Suzhou Genewiz Biotechnology Co., Ltd.
[0050] Since the identified compounds were already commercially available drugs, they were subsequently purchased directly for functional analysis of individual compounds.
[0051] Example 6 Cytotoxicity Analysis
[0052] Safety assessment was performed using the MTT assay. RAW264.7 and HaCaT cells were cultured for 24 hours at 37°C and 5% CO2 to assess the cytotoxicity of different concentrations of 4-methylcatechol and 4-methylbenzoic acid. Different concentrations of 4-methylcatechol and 4-methylbenzoic acid were added to the cells and reacted for 24 hours. After the reaction time was complete, 6 μL of MTT (5 mg / mL) solution was added, and the reaction was carried out at 37°C and 5% CO2 for 4 hours. The culture medium was then removed, and 150 μL of DMSO was added to dissolve the precipitate. The absorbance at 570 nm was analyzed using a microplate reader.
[0053] Experimental results:
[0054] 1. The antioxidant capacity of the bamboo vinegar powder extract of the present invention was evaluated, and it showed an antioxidant capacity of over 90% at a concentration of 0.025 mg / mL.
[0055] The above describes the ability to remove ABTS free radicals. Even at a concentration of 0.00625 mg / mL, it still exhibits a removal rate of 44.8%. Figure 1 ).
[0056] 2. The antioxidant capacity of the bamboo vinegar powder extract of the present invention was evaluated. At a concentration of 0.05 mg / mL, it exhibited an antioxidant capacity exceeding 80%.
[0057] The above describes the ability to remove DPPH free radicals. Even at a concentration of 0.0125 mg / mL, it still maintains a removal rate of 35%. Figure 1 ).
[0058] 3. Nitric oxide is considered to be one of the factors involved in inflammation; therefore, effective removal of nitric oxide can serve as a basis for anti-inflammatory effects. The bamboo vinegar powder extract of this invention shows an increasing trend in nitric oxide removal rate with increasing concentration, but its nitric oxide removal rate is only 16.6% at a concentration of 0.1 mg / mL. Figure 2 ).
[0059] 4. The antifungal activity of the bamboo vinegar powder extract of this invention was evaluated. At a concentration of 1.25 mg / mL, it achieved a removal rate of over 50% against *Aspergillusterreus* CICC40377, *Trichoderma longibrachiatum* CICC40380, and *Paecilomyces variotii* CICC40716. Figure 3The inhibitory effect on the fungal clear zone was noticeable at a dose of 4 mg. Figure 4 ).
[0060] 5. The most abundant compounds identified in bamboo vinegar powder extract include various phenols, benzene derivatives, and organic acids.
[0061] Examples include pyrogallol, 4-methylcatechol, p-hydroxyphenylethanol, p-methylbenzoic acid, D-mandelic acid, dihydrocaffeic acid, maleic acid, and 1,4-benzenediol (Table 1).
[0062] The retention times of the identified compounds are as follows:
[0063] 1,2,3-Trihydroxybenzene (phloroglucinol): 371 seconds
[0064] 4-Methylcatechol: 540 seconds
[0065] 4-Hydroxyphenylethanol (p-hydroxyphenylethanol): 614 seconds
[0066] 4-Methylbenzoic acid (p-methylbenzoic acid): 519 seconds
[0067] 3,4-Dihydroxyhydrocinnamic acid (dihydrocaffeic acid): 367 seconds
[0068] Hydroquinone (1,4-benzenediol): 437 seconds.
[0069] 6. The antioxidant capacity of the compounds identified in this invention was evaluated, among which pyrogallol, 4-methylcatechol, p-hydroxyphenylethanol, dihydrocaffeic acid, and 1,4-dibenzenediol all showed significant ability to scavenge ABTS free radicals. Figure 5 Among them, pyrogallol, 4-methylcatechol, dihydrocaffeic acid, and 1,4-benzenediol can achieve a removal rate of over 90% for ABTS free radicals at a concentration of 0.005 mg / mL.
[0070] 7. The antioxidant capacity of the compounds identified in this invention was evaluated, among which pyrogallol, 4-methylcatechol, dihydrocaffeic acid, and 1,4-dibenzenediol all showed significant ability to scavenge DPPH free radicals. Figure 6 At a concentration of 0.005 mg / mL, these four compounds exhibited a DPPH scavenging rate exceeding 50%, with pyrogallol reaching a scavenging rate of 86.9%.
[0071] 8. Based on the antifungal analysis of compounds in bamboo vinegar powder extract ( Figure 7,8), among which 4-methylcatechol and p-methylbenzoic acid have broad-spectrum antifungal effects, and have very good antifungal ability against Aspergillus terreus CICC40377, Trichodermalongibrachiatum CICC40380, and Paecilomyces variotii CICC40716.
[0072] 9. At a concentration of 1 mg / mL, p-methylbenzoic acid showed survival rates of 96.8% for macrophages (RAW 264.7) and 88.4% for human immortalized keratinocytes (HaCaT), indicating that this compound has no toxic effects on macrophages and human immortalized keratinocytes and can be used for medical antifungal applications. Figure 9 ).
[0073] From the above comparison, it can be seen that, in terms of antifungal ability, see [reference needed]. Figure 3 The crude extract of bamboo vinegar exhibited significant antibacterial effects at a concentration of 1.25 mg / mL. (See also...) Figure 7 The compounds obtained by the identification showed significant antibacterial effects at concentrations as low as 0.25 mg / mL.
[0074] For a comparison of antioxidant capacity, see [link to relevant documentation]. Figure 1 The crude extract of bamboo vinegar exhibited a DPPH free radical removal capacity exceeding 35% at a concentration of 0.0125 mg / mL. (See also...) Figure 6 The four compounds identified had a DPPH scavenging rate of over 50% at a minimum concentration of 0.005 mg / mL.
[0075] Table 1. Identification of components in bamboo vinegar powder extract
[0076]
[0077] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.
Claims
1. A bamboo vinegar powder extract in resistance to Aspergillus terrestris Aspergillus terreus Trichoderma longifolia Trichoderma longibrachiatum or Penicillium Paecilomyces variotii Its application in the product is characterized by, The bamboo vinegar powder extract includes at least one of 4-methylcatechol and p-methylbenzoic acid.
2. The application according to claim 1, characterized in that, The minimum dosage is 0.25 mg / mL.