CN113730522A - Application of Pu' er ginger flower volatile oil in preparation of antibacterial or anti-inflammatory drugs - Google Patents
Application of Pu' er ginger flower volatile oil in preparation of antibacterial or anti-inflammatory drugs Download PDFInfo
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- CN113730522A CN113730522A CN202111146055.XA CN202111146055A CN113730522A CN 113730522 A CN113730522 A CN 113730522A CN 202111146055 A CN202111146055 A CN 202111146055A CN 113730522 A CN113730522 A CN 113730522A
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- volatile oil
- ginger
- ginger flower
- puer
- antibacterial
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Abstract
The invention discloses application of Pu' er ginger flower volatile oil in preparation of antibacterial or anti-inflammatory drugs. According to the invention, the antibacterial or anti-inflammatory Puer ginger flower volatile oil is obtained by extracting underground rhizomes of Puer ginger flowers through steam distillation, has a remarkable antibacterial effect on gram-positive strains and gram-negative strains, and shows a broad-spectrum antibacterial property; the volatile oil remarkably inhibits the production of proinflammatory mediators (NO) and proinflammatory cytokines (TNF-alpha and IL-6) in RAW264.7 cells induced by LPS under the condition of NO cytotoxicity, has remarkable inhibiting effect on ear swelling induced by xylene, and has remarkable anti-inflammatory effect in vivo and in vitro. Therefore, the Pu' er ginger flower volatile oil has the application prospect of preparing antibacterial or anti-inflammatory drugs.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to application of Pu' er ginger flower volatile oil in preparation of antibacterial or anti-inflammatory medicines.
Background
The volatile oil is a mixture of secondary metabolites extracted from plants with volatility and strong odor (Food Chem Toxicol.2008,46: 446-475; Evi Based complete Alternate Med.2018,2018: 3149362.). It is estimated that more than 3000 essential oils have been studied, of which about 300 have important commercial value and have been used in cosmetics, perfumes, agriculture, hygiene, Food and pharmaceutical fields (Int J Food Microbiol.2004,94: 223-. In addition, volatile Oils have great potential for development in the pharmaceutical industry due to their various pharmacological activities, such as antioxidant, anti-inflammatory, antifungal, antibacterial, antiviral, anticancer, antimutagenic, antidiabetic, antiprotozoal and analgesic activities (Ind Crop prod.2014,62: 250-. The negative impact of synthetic products and antibiotics on human health and the environment has become a serious global problem, and thus healthier and natural alternatives such as essential oils are urgently needed.
There are about 93 plants of the genus Zingiber (Hedychium) of the family Zingiberaceae, which are mainly distributed in tropical and temperate regions of China, India and southeast Asia (Prakash O, Chandra M, Punetha H, Pant AK, Rawat DS. Chapter 84-Spiked Ginger Lily. in: Press V, ed. essential Oils in Food pressure, flavour and safety. London: Academic Press; 2016: 737-. Plants of the genus zingiber are widely used in the perfume, ornamental, paper, cosmetic, food and pharmaceutical industries, among which in national medicine for the treatment of nausea, leishmaniasis, asthma, bronchitis, diarrhoea, stomach diseases, influenza and snake bites and the like (sakhanokhanokho HF, Rajasekaran k.hedychium Essential Oils: Composition and uses.in: Malik S, ed.essential Oil research. cham: Springer; 2019: 49-60; Procedia chem.2014,13: 150-. Especially volatile oils from plants of the genus zingiber have great potential for development and have been used in high-grade perfumery and ethnic medicines (Procedia chem.2014,13: 150-. The volatile oils of plants of the genus Zingiber are reported to have various pharmacological activities such as antibacterial, anti-inflammatory, antifungal, antioxidant, analgesic, cytotoxic, anticholinesterase, anthelmintic and insecticidal properties (Ind Crop Prod.2018,126: 135-142; Ind Crops Prod.2018,112: 353-362; Acta trop.2021,218: 105912; medicins.2020, 7: 23).
Puerh ginger flower (Hedychium puerense Y. Qian) is a perennial herb of the genus Zingiber, and is native to Yunnan province in China (plant classification, 1996,34(4): 443-. Pu' er ginger flower is widely cultivated as ornamental plant because of its beautiful and aromatic flower. Essential oils from plants of the genus zingiber have proven to have great potential for use in the pharmaceutical field. However, no reports about the chemical components and the pharmacological activity of the Pu' er tea volatile oil exist at present, which may hinder the development and the utilization of the volatile oil.
Disclosure of Invention
The purpose of the invention is as follows: provides the application of the Pu 'er ginger flower volatile oil in preparing antibacterial or anti-inflammatory drugs, develops new application of the Pu' er ginger flower volatile oil, and provides a new choice for preparing antibacterial or anti-inflammatory drugs.
The invention also discovers that the Pu 'er ginger flower volatile oil has broad-spectrum antibacterial property, inhibits the production of proinflammatory mediators (NO) and proinflammatory cytokines (TNF-alpha and IL-6) induced by lipopolysaccharide and improves the ear swelling caused by dimethylbenzene, and identifies the chemical components of the Pu' er ginger flower volatile oil.
The technical scheme adopted by the invention is as follows:
application of Pu' er ginger flower volatile oil in preparing antibacterial or anti-inflammatory medicine is provided.
Application of Puer ginger flower volatile oil in preparing medicine for resisting gram-positive bacteria and gram-negative bacteria is provided.
Application of Puer ginger flower volatile oil in preparation of drugs for improving abnormality of proinflammatory mediator NO and proinflammatory cytokines TNF-alpha and IL-6 in RAW264.7 cells induced by Lipopolysaccharide (LPS).
Application of Pu' er ginger flower volatile oil in preparation of medicine for improving ear swelling caused by xylene is provided.
The Pu' er ginger flower volatile oil and a pharmaceutically acceptable carrier are prepared into the medicament in the dosage forms of tablets, capsules, fat emulsion, suppositories, dropping pills or ointments.
The preparation method of the Pu ' er ginger flower volatile oil comprises the steps of crushing underground roots and stems of fresh Pu ' er ginger flowers, mixing the crushed raw materials with distilled water according to a material-liquid ratio of 1: 2-1: 5g/mL, carrying out steam distillation for 3-5 h in a volatile oil extractor, collecting the volatile oil, and removing water by using anhydrous sodium sulfate to obtain the Pu ' er ginger flower volatile oil.
By adopting the technical scheme, the invention discovers the new application of the Pu' er ginger flower volatile oil in the aspect of treating bacterial infection and inflammation related diseases for the first time, provides a new choice for preparing antibacterial or anti-inflammatory medicines, and has important application value in the pharmaceutical industry.
Drawings
FIG. 1 is a GC-MS chromatogram of the volatile oil of the underground rhizomes of Puer ginger flowers;
FIG. 2 shows cytotoxicity of Pu' er ginger flower volatile oil on RAW264.7 (mouse macrophage) and L929 (mouse fibroblast);
FIG. 3 shows the effect of Pu' er ginger flower volatile oil on the cell morphology of RAW264.7 cell line induced by LPS and the production of NO, IL-6 and TNF-alpha;
FIG. 4 shows the effect of Pu' er ginger flower volatile oil on the swelling degree of mouse ears induced by xylene.
Detailed Description
The embodiment of the invention comprises the following steps: pulverizing underground rhizome of fresh Puer ginger flower (harvested place: Lincang city in Yunnan province; identified by professor moustache in Guizhou university), mixing the pulverized raw materials with distilled water according to a material-liquid ratio of 1:4, performing steam distillation on the mixed materials in a volatile oil extractor for 4h, collecting volatile oil, removing water by anhydrous sodium sulfate to obtain the volatile oil of the Puer ginger flower, and sealing and storing in a refrigerator at 4 ℃.
The Pu' er ginger flower volatile oil comprises the following chemical components: identifying the chemical components of the Pu' er ginger flower volatile oil by GC-MS. GC-MS analysis conditions: sample introduction 2 μ L, chromatography column HP-5MS (60m × 0.25mm × 0.25 μm) elastic quartz capillary column, initial temperature 70 deg.C (2 min retained), temperature 2 deg.C/min to 180 deg.C, temperature 10 deg.C/min to 310 deg.C (14 min retained), run time: 84 min; the temperature of the vaporization chamber is 250 ℃; the carrier gas is high-purity He (99.999%); the column front pressure is 18.53psi, the carrier gas flow is 1.0mL/min, the split ratio is 10: 1, solvent delay time: 6.0 min; the ion source is an EI source; the ion source temperature is 230 ℃; the temperature of the quadrupole rods is 150 ℃; electron energy 70 eV; emission current 34.6 μ A; multiplier voltage 1847V; the interface temperature is 280 ℃; the mass range is 29-500 amu. And searching and checking the Nist2017 and Wiley275 standard mass spectrograms of each peak in the total ion flow graph through a mass spectrum computer data system, determining chemical components, and measuring the relative mass fraction of each chemical component by using a peak area normalization method. The chemical components of the puer ginger flower underground rhizome volatile oil are shown in figure 1 and table 1, 34 chemical components are identified in total and account for 98.2% of the total peak area, wherein the main chemical components are Linalool (Linalool, 26.5%), beta-Pinene (beta-Pinene, 18.6%), gamma-Terpinene (gamma-Terpinene, 12.1%), 4-terpineol (Terpinen-4-ol, 7.7%), alpha-Pinene (alpha-Pinene, 5.8%), Sabinene (Sabinene, 4.9%), E-Nerolidol (E-Nerolidol, 4.1%) and p-Cymene (p-Cymene, 3.6%).
TABLE 1 chemical composition of volatile oil of underground rhizome of Puer ginger flower
aA compound: in order of elution from the HP-5MS column.
bRI (retention index) was calculated from normal alkanes (C8-C22) on an HP-5MS column.
cDatabase RI (retention index) is from NIST2017 and Wiley275 of the database.
d tr：trace(trace<0.1％)。
Pharmacological example 1: the Pu' er ginger flower volatile oil is used for testing the antibacterial activity of 3 gram-positive strains (staphylococcus aureus ATCC 6538P, enterococcus faecalis ATCC 19433 and bacillus subtilis ATCC 6633) and 3 gram-negative strains (proteus ACCC 11002, pseudomonas aeruginosa ATCC 9027 and escherichia coli CICC 10389).
The zone Diameter (DIZ) was measured by diffusion on filter paper (BioMed Res int.2021,2021: 5562461). 100. mu.L of bacterial suspension (1X 10)6CFU/mL) was spread evenly on Mueller-Hinton agar medium. Subsequently, a filter paper sheet (diameter 6mm) containing 20. mu.L of the volatile oil (100mg/mL in ethyl acetate) or streptomycin (100. mu.g/mL in distilled water) was placed on the surface of the agar medium. After incubation at 37 ℃ for 24h, the diameter of the zone of inhibition (DIZ) (including 6mm filter paper sheets) was measured and recorded.
The Minimum Inhibitory Concentration (MIC) and The Minimum Bactericidal Concentration (MBC) were determined by half-fold dilution (Front pharmacol.2020,11: 572659). 100 μ L of bacterial suspension (2X 10)5CFU/mL) and half-diluted sample solution (100 μ L) were added to each well of a 96-well plate. After incubation at 37 ℃ for 24h, resazurin (resazurin) solution was added to each well(20. mu.L, 0.1mg/mL) and incubated at 37 ℃ for 2h in the absence of light. MIC values were defined as the minimum sample concentration without color change. To determine the MBC value, medium from 96-well plates without color change (10. mu.L) was spread evenly on Mueller-Hinton agar medium and incubated at 37 ℃ for a further 24 h. MBC values are defined as the minimum sample concentration at which no bacteria grow.
The antibacterial activity of the volatile oils against gram-negative and gram-positive strains was evaluated by measuring zone of inhibition Diameter (DIZ), Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values. Streptomycin was used as a positive control. As shown in table 2, the volatile oil of zingiber Puerh blooms exhibited broad-spectrum antibacterial properties, with DIZ values ranging from 7.44 to 10.30mm, enterococcus faecalis (MIC 3.13mg/mL, MBC 3.13mg/mL), Bacillus subtilis (MIC 3.13mg/mL, MBC 3.13mg/mL), Staphylococcus aureus (MIC 6.25mg/mL, MBC 12.50mg/mL), Proteus (MIC 3.13mg/mL, MBC 6.25mg/mL), Pseudomonas aeruginosa (MIC 6.25mg/mL, MBC 12.50mg/mL) and Escherichia coli (MIC 6.25mg/mL, MBC 12.50 mg/mL). Linalool, the most important component of the volatile oil of zingiber Puerh flowers, has been proved to have excellent antibacterial activity, and can induce cell leakage and energy metabolism disorder by interfering with the function and integrity of cell membranes and cell walls (molecules.2021,26: 245). Therefore, the Pu' er ginger flower volatile oil can be used as a natural antibacterial agent in the pharmaceutical industry.
TABLE 2 antibacterial Activity of Pu her ginger flower underground rhizome volatile oil
aThe strain is as follows: enterococcus faecalis (Enterococcus faecalis, ATCC 19433), Bacillus subtilis (ATCC 6633), Staphylococcus aureus (Staphylococcus aureus, ATCC 6538P), Proteus vulgaris (ACCC 11002), Pseudomonas aeruginosa (ATCC 9027), Escherichia coli (CICC 10389).
bDIZ: the diameter (mm) of the zone of inhibition includes the diameter (6mm) of the filter paper sheet. With ethyl acetateDiluted volatile oil solution (test volume: 20. mu.L, 100 mg/mL); a streptomycin solution diluted with distilled water (test volume: 20. mu.L, 100. mu.g/mL) was used as a positive reference.
cMIC: a minimum inhibitory concentration; MBC: minimum germicidal concentration.
Pharmacological example 2: influence of Pu' er ginger flower volatile oil on RAW264.7 induced by LPS (in vitro anti-inflammatory experiment)
The cytotoxic effect of the volatile oil of zingiber Puerh ginger (EO) on RAW264.7 (mouse macrophage) and L929 (mouse fibroblast) was evaluated by MTT (J Immunol methods.1983,65: 55-63). RAW264.7 and L929 cells were cultured in DMEM medium and RPMI 1640 medium, respectively, and supplemented with 10% fetal bovine serum, 100. mu.g/mL streptomycin, 100U/mL penicillin and 2mM glutamine. Dissolving the Pu' er ginger flower volatile oil in DMSO, and then continuously diluting with a culture medium by half times, wherein the final DMSO concentration is not higher than 0.5%. Cell suspension (100. mu.L) was added to 96-well plates (2X 10)4cells per well) and 5% CO at 37 deg.C2Culturing in an incubator for 24 h. Subsequently, diluted Pu-erh ginger flower volatile oil solution (100 μ L) is added into each well for further culture for 24 h. Then, 10 μ LMTT solution (5mg/mL in PBS) was added and incubated for an additional 4 h. After final dissolution of formazan crystals in DMSO (150 μ Ι _), absorbance was measured at 490nm to assess cell viability.
As shown in FIG. 3, cytotoxicity was measured on RAW264.7 and L929 cells by MTT assay after incubation of various doses of volatile oil of flowers of Zingiber Puerhans (0, 31.25, 62.5, 125, 250, 500 μ g/mL) for 24 h. Compared with untreated cells, the Puer ginger flower volatile oil with the concentration of 31.25-250 mug/mL does not generate obvious cytotoxicity on RAW264.7 and L929 cells. Therefore, the non-cytotoxic concentration of the Pu' er ginger flower volatile oil is 31.25-250 mug/mL and is used for subsequent experiments.
RAW264.7 cell suspension (100. mu.L) was added to 96-well plates (2X 10)4cells per well) for 24 h. Subsequently, the cells were exposed to 100 μ L of fresh medium containing the volatile oil of flowers of Zingiber Puerhans. After 2h incubation, 100. mu.L of Lipopolysaccharide (LPS) solution was added to each well to a final concentration of 1. mu.g/mL and incubated for 24 h. Through a Leica DMi8 microscope (Leica microsystems)tems, Germany) observed cell morphology changes in RAW264.7 cells. After collecting the supernatant by centrifugation, the amount of accumulated NO in the supernatant was measured using a NO detection kit (Beyotime, Shanghai, China). Proinflammatory cytokines (IL-6 and TNF- α) of RAW264.7 cells were measured using respective ELISA assay kits (Multi Sciences Biotech co., ltd., Hangzhou, China). Dexamethasone (DXM, 20. mu.g/mL) was used as a positive control.
The anti-inflammatory effect of the Puer ginger flower volatile oil is detected by an LPS-induced RAW264.7 cell inflammatory cell model. After the Pu' er ginger flower volatile oil treatment, the morphological change of RAW264.7 cells is observed under a microscope contrast (figure 3A). The cells of the control group were round and smooth in surface. The volume of RAW264.7 cells treated by LPS is enlarged and the shape is irregular, while the change of cell morphology of the Pu' er ginger flower volatile oil treatment group is smaller.
As shown in figures 3B-3D, the Pu-erh ginger flower volatile oil remarkably inhibits the production of proinflammatory mediators (NO) and proinflammatory cytokines (TNF-alpha and IL-6) in RAW264.7 cells induced by LPS. Dexamethasone (DXM, 20. mu.g/mL) was used as a positive reference. As shown in FIG. 3B, the volatile oil of flower of Zingiber Puerhans has significantly reduced NO production compared with LPS group (7.88 + -0.27 μ M). Particularly, the NO yield in RAW264.7 cells pretreated by Puer ginger flower volatile oil with the dosages of 62.5, 125 and 250 mu g/mL is respectively reduced to 1.01 +/-0.05 mu M (93.84 +/-0.93%), 0.98 +/-0.06 mu M (94.16 +/-1.07%), 0.61 +/-0.03 mu M (99.23 +/-0.26%) and is lower than DXM (2.11 +/-0.15 mu M,78.76 +/-2.31%). In addition, the volatile oil of flower of Helianthus Puerhans in the concentration of 31.25 μ g/mL (2910.24. + -. 62.13pg/mL), 62.5 μ g/mL (2689.24. + -. 68.85pg/mL), 125 μ g/mL (2649.58. + -. 80.41pg/mL) and 250 μ g/mL (131.58. + -. 3.51pg/mL) significantly reduced the production of TNF- α in RAW264.7 cells induced by LPS, compared to the LPS group (3335.91. + -. 216.38pg/mL) (FIG. 3C). Particularly, the inhibition rate of the Pu' er ginger flower volatile oil at 250 mu g/mL (97.14 +/-0.11%) exceeds DXM (67.36 +/-3.67%, 20 mu g/mL). As shown in FIG. 3D, IL-6 accumulation in the supernatant of the Pu' er ginger flower volatile oil-pretreated group was significantly reduced compared to LPS group (832.51. + -. 29.29 pg/mL). Particularly, when the concentration of the Pu' er ginger flower volatile oil is 62.5 mu g/mL (441.36 +/-30.58 pg/mL,46.98 +/-3.67%), 125 mu g/mL (417.90 +/-36.80 pg/mL,49.80 +/-4.42%) and 250 mu g/mL (146.36 +/-1.35 pg/mL,82.42 +/-0.16%), the inhibition effect on IL-6 secretion induced by LPS is more than that of DXM (551.63 +/-17.62 pg/mL,33.74 +/-2.12%). Proinflammatory factors (NO, IL-6 and TNF-alpha) play a crucial role in inflammatory diseases, and inhibition of their production has become a therapeutic strategy for inflammation-related diseases (J Ethnopharmacol.2013,149: 162-168). Linalool, the most major component of the volatile oil of the flower of Zingiber Puerh Hedychium, has been shown to inhibit the secretion of IL-6 and TNF-alpha induced by LPS in vitro and in vivo, and to be a potential candidate for treating inflammation-related diseases (J Surg Res.2013,180: E47-E54). The results show that the Pu' er ginger flower volatile oil can be used as a natural anti-inflammatory agent.
Pharmacological example 3: influence of Pu' er ginger flower volatile oil on xylene-induced ear swelling (in vivo anti-inflammatory experiment)
The inflammatory mice are molded by adopting a mouse auricle swelling method, 60 Kunming mice are randomly divided into 6 groups, each half is male and female, and each group comprises 10 mice; respectively establishing a blank control group, a model group, a dexamethasone group (7.5mg/kg) and low (3.75g/kg), medium (7.5mg/kg) and high (15mg/kg) dose groups of Pu' er ginger flower volatile oil (EO). The medicine is administrated once a day, the volume of the medicine liquid is 20mL/kg, and the medicine liquid is administrated by gastric gavage for 7 days continuously. The grouping situation is specifically as follows:
blank control group: the same volume of physiological saline containing DMSO was administered daily;
model group: the same volume of physiological saline containing DMSO was administered daily;
positive drug group (Dexamethasone, DXM): 7.5mg/kg is administered daily;
low dose group: 3.75mg/kg is administered daily;
the medium dose group: 7.5mg/kg is administered daily;
high dose group: 15mg/kg was administered daily.
The experiment is started after the mice are adapted for 3d, each administration group is continuously administered with the gastric lavage for 7 days and is administered once a day, wherein the normal group is administered with the same volume of physiological saline containing DMSO for the gastric lavage, and the experiment is carried out 1h after the last administration. Except for the blank control group, 30 microliter of dimethylbenzene is uniformly coated on the front and back surfaces of the left ear of the mouse, and the right ear is not treated and is used for self control; after the mice were inflamed for 30min, the mice were sacrificed by dislocation, both ears of the mice were cut off, and the ears were punched out at the same positions of the left and right ears of the mice using an electric ear swelling punch with a diameter of 8mm, and weighed. Recording the weights of the left and right ear sheets, and calculating swelling degree and ear swelling inhibition rate.
Swelling degree-left ear quality-right ear quality
Ear edema inhibition (%) is (degree of swelling in model group-degree of swelling in administered group)/degree of swelling in model group × 100%
As shown in Table 3 and FIG. 4, the xylene-induced ear swelling of mice is evident, and the Pu' er ginger flower volatile oil has a certain dose-dependent inhibition effect on the xylene-induced ear swelling of mice. Compared with a model group (swelling degree: 14.89 +/-1.58 mg), the dexamethasone group (DXM, swelling degree: 7.26 +/-1.09) has obvious inhibition effect (p is less than 0.01), and the Pu' er ginger flower volatile oil has obvious inhibition effect (p is less than 0.05) in high (swelling degree: 6.55 +/-1.06 mg), medium (swelling degree: 8.86 +/-0.96 mg) and low (swelling degree: 9.56 +/-0.83 mg) dose groups; particularly, the high-dose group of the Pu' er ginger flower volatile oil has equivalent effect with the dexamethasone group, and the swelling degree and the inhibition rate have no significant difference (p is more than 0.05). In conclusion, the Pu' er ginger flower volatile oil has a remarkable inhibition effect on ear swelling induced by dimethylbenzene, and has a remarkable anti-inflammatory effect in vivo.
TABLE 3 inhibitory Effect of Pu her ginger flower volatile oil on xylene-induced ear swelling (Mean + -SD, n ═ 10)
Note: different letters in the same column represent significant differences (p < 0.05); (-) means none.
By combining the pharmacological examples, the Pu' er ginger flower volatile oil has obvious antibacterial activity on gram-positive strains (staphylococcus aureus, enterococcus faecalis and bacillus subtilis) and gram-negative strains (proteus, pseudomonas aeruginosa and escherichia coli), and shows broad-spectrum antibacterial property; the Puer ginger flower volatile oil remarkably inhibits the production of proinflammatory mediators (NO) and proinflammatory cytokines (TNF-alpha and IL-6) in RAW264.7 cells induced by LPS under the condition of NO cytotoxicity, has remarkable inhibition effect on ear swelling induced by dimethylbenzene, and has remarkable anti-inflammatory effect in vivo and in vitro.
Claims (6)
1. Application of Pu' er ginger flower volatile oil in preparing antibacterial or anti-inflammatory medicine is provided.
2. The use of claim 1, wherein: application of Puer ginger flower volatile oil in preparing medicine for resisting gram-positive bacteria and gram-negative bacteria is provided.
3. Use according to claim 1, characterized in that: application of Puer ginger flower volatile oil in preparation of drugs for improving abnormality of proinflammatory mediator NO and proinflammatory cytokines TNF-alpha and IL-6 in RAW264.7 cells induced by Lipopolysaccharide (LPS).
4. Use according to claim 1, characterized in that: application of Pu' er ginger flower volatile oil in preparation of medicine for improving ear swelling caused by xylene is provided.
5. The use as claimed in claim 1, wherein the volatile oil of flower of Puer ginger and pharmaceutically acceptable carrier are formulated into tablet, capsule, fat emulsion, suppository, dripping pill or ointment.
6. A method for preparing the Pu' er ginger flower volatile oil as claimed in claim 1, which is characterized in that: smashing underground roots and stems of fresh Puer ginger flowers, mixing the smashed raw materials with distilled water according to a material-liquid ratio of 1: 2-1: 5g/mL, performing steam distillation for 3-5 h in a volatile oil extractor, collecting volatile oil, and removing water by anhydrous sodium sulfate to obtain the Puer ginger flower volatile oil.
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