Artemisia sieversiana volatile oil and use thereof in preparing antifungal bacteriostatic product

Abstract

The application belongs to the technical field of natural plant extracts and application, and particularly relates to azafran volatile oil and application thereof in preparation of antifungal bacteriostatic products. In the application, the azafran is soaked in water and then distilled to obtain azafran volatile oil. The obtained azafran volatile oil shows good bacteriostasis, especially in inhibiting fungal strains such as Candida albicans, Malassezia furfur, Trichophyton mentagrophytes and Propionibacterium acnes. The application develops a new use of azafran volatile oil in preparation of antifungal bacteriostatic products.

Description

Technical Field

[0001] This invention belongs to the field of natural plant extracts and their application technology, specifically relating to a volatile oil of *Nepeta cataria* and its application in the preparation of antifungal and antibacterial products. Background Technology

[0002] Tibetan catnip (Tibetan medicine name: Sadunabo) is the dried whole herb of *Nepeta angustifolia* CYWu, belonging to the Lamiaceae family. It is cool in nature and bitter in taste, and has the effect of clearing the mind and refreshing the spirit. Traditional Tibetan medicine uses it to treat delirium, convulsions, stroke, epilepsy, cerebral hemorrhage, sores, and pain. Recent studies have found that Tibetan catnip contains terpenes, organic acids, flavonoids, and other components. Pharmacological experiments have shown that Tibetan catnip has anti-inflammatory, analgesic, anti-fatigue, hypoxia-resistant, hypoglycemic, cerebral hemorrhage-improving, and tyrosinase-inhibiting activities, making it a Tibetan medicinal material with great development potential.

[0003] However, at present, research on the volatile oil of *Nepeta cataria* mainly focuses on the detection and identification of its components. For example, in the study on the chemical composition of volatile oil of *Nepeta cataria*, Zerenlam et al. successfully identified 29 components from the volatile oil of *Nepeta cataria*, among which the main components are nepeta lactone, spirododecane, and 2,4-dimethyl-1,3-pentadiene. In the comparison of volatile components between *Nepeta cataria* and *Nepeta cataria*, Hu Dandan et al. disclosed that the number of volatile components in *Nepeta cataria* is the most in the leaves (21 types) and the least in the whole herb (17 types). The main component in the stem is nepeta lactone, with a relative content of 73.24%. Eighteen volatile components were identified in *Nepeta cataria*, mainly isomenthone, menthone, and limonene.

[0004] There is relatively little research on the efficacy of volatile oil from *Schizonepeta tenuifolia*. Although existing studies have confirmed that volatile oil from *Schizonepeta tenuifolia* has good anti-inflammatory, analgesic, hypoxia-resistant, and anti-fatigue effects, there is no relevant research on whether volatile oil from *Schizonepeta tenuifolia* has good antibacterial properties.

[0005] Although Dr. Cheng Shuanghuai's team found in their study on "The Antibacterial Effect of Nepeta cataria volatile oil" that a 15% concentration of Nepeta cataria volatile oil has good antibacterial activity against Escherichia coli and Staphylococcus aureus, further research on the components of Nepeta cataria and Nepeta cataria revealed that although both are medicinal plants of the Nepeta genus, the types of volatile components they contain are quite different. For example, the volatile oil of Nepeta cataria mainly contains isomenthone, menthol, and limonene, while the volatile oil of Nepeta cataria mainly contains nepelide. The effects of volatile oils with different active ingredients are completely different.

[0006] Furthermore, the aforementioned text only discloses that the volatile oil of *Nepeta cataria* has a certain antibacterial effect against bacteria, but it is unknown whether it has a good antibacterial effect against fungi. Moreover, compared to bacteria, fungi are eukaryotic organisms with a nucleus surrounded by a nuclear membrane. Their cell walls are mainly composed of chitin and they have various organelles such as endoplasmic reticulum, Golgi apparatus, and mitochondria, exhibiting more tenacious vitality. Therefore, even if it shows good antibacterial activity against bacteria, it does not necessarily have inhibitory effect on fungi. For example, common antibiotics such as penicillins, cephalosporins, and quinolones have no inhibitory effect on fungi.

[0007] Furthermore, there are currently no reports on the antibacterial effects of the volatile oil of *Corydalis tangutica*, particularly its inhibitory effects on strains of Malassezia, Trichophyton, and Candida albicans. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a volatile oil from *Nepeta cataria* and its application in the preparation of antifungal and antibacterial products.

[0009] The application of *Nepeta cataria* volatile oil in the preparation of antifungal and antibacterial products provided by this invention is a new use of *Nepeta cataria* volatile oil and is the key technical content to be protected by this invention. The fungus is selected from at least one of *Candida albicans*, *Malassezia*, and *Trichophyton*.

[0010] In the above applications, the concentration of volatile oil from *Nepeta cataria* in the antibacterial product, i.e., the mass fraction of volatile oil in the product, is 0.07–5%.

[0011] During the experimental research, it was found that although the antibacterial effect of the volatile oil of *Schizonepeta tenuifolia* increases with the increase of its added amount, when the added amount is too large, the volatile oil of *Schizonepeta tenuifolia* will also show strong irritation to the skin. Therefore, in this invention, the added concentration of volatile oil of *Schizonepeta tenuifolia* in the antibacterial product is controlled between 0.07% and 5%.

[0012] In the above applications provided by the present invention, preferably, the product is selected from any one of pharmaceuticals and daily chemical products.

[0013] Preferably, the product is selected from any one of ointments, lotions, sprays, liquids, emulsions, creams, and oils.

[0014] In addition, the present invention also provides a method for preparing volatile oil of *Nepeta cataria* with the above-mentioned applications, which specifically includes the following steps: crushing the above-ground parts of *Nepeta cataria*, soaking in water, distilling, and collecting the volatile oil to obtain the product.

[0015] A further preferred method for preparing the volatile oil of *Nepeta cataria* specifically includes the following steps: taking the above-ground parts of *Nepeta cataria*, crushing them into powder, adding 5 to 20 times the weight of *Nepeta cataria* in water and soaking for 0 to 12 hours, distilling for 2 to 6 hours after soaking until no more volatile oil is added, stopping heating, and collecting the volatile oil.

[0016] Preferably, the above-ground parts of the catnip are selected from at least one of the stems and leaves of the catnip.

[0017] The beneficial effects of this invention are as follows:

[0018] (1) A new use of the volatile oil of *Nepeta cataria* is disclosed, namely, the use of the volatile oil of *Nepeta cataria* in the preparation of antifungal and antibacterial products, wherein the fungi include, but are not limited to, *Candida albicans*, *Malassezia*, *Trichophyton* and other fungi;

[0019] (2) The preparation method of the volatile oil of Nepeta cataria in this invention is simple to operate and has a high extraction rate, which is convenient for large-scale production. The volatile oil of Nepeta cataria obtained by the method of this invention is identified to contain 11 kinds of volatile active ingredients, accounting for 97.92% of the total volatile oil. Among them, the contents of nepeta lactone and pseudonepeta lactone are as high as 57.61% and 16.45%, respectively. Attached Figure Description

[0020] Figure 1 The total ion chromatogram is shown for the determination of the volatile oil components of the *Nepeta cataria* prepared in Example 1 of this invention using gas chromatography-mass spectrometry.

[0021] Figure 2 The antibacterial properties of the volatile oil from *Nepeta cataria* obtained in Example 1 of this invention at different concentrations;

[0022] Figure 3 The antibacterial effect of volatile oil from the stems and leaves of *Nepeta cataria* on various bacterial strains in Examples 2-3 of the present invention is shown.

[0023] Figure 4 This is a comparative example 1 of the present invention, showing the antibacterial effect of volatile oil from the root of *Nepeta cataria* on various bacterial strains.

[0024] Figure 5 This is a comparative example 2 of the present invention, showing the antibacterial effect of different concentrations of Nepeta cataria volatile oil on various bacterial strains. Detailed Implementation

[0025] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0026] The raw material used in this invention is *Nepeta cataria*. Unless otherwise specified, all other raw materials are commercially available products.

[0027] Example 1

[0028] Preparation of volatile oil from *Schizonepeta tenuifolia*: Take 500g of the aerial parts of *Schizonepeta tenuifolia*, crush them into coarse powder, add 5 times the amount of water as the medicinal material, soak for 6 hours, then steam distill for 4 hours, observe the oil release, and stop heating when there is no increase in volatile oil. Collect the volatile oil to obtain volatile oil from *Schizonepeta tenuifolia*.

[0029] 1.1 The components of the volatile oil of *Corydalis yanhusuo* obtained in this example were analyzed and determined by gas chromatography-mass spectrometry (GC-MS).

[0030] GC conditions: Instrument model: GC-2030AM (Shimadzu); Column: SP-I-5SIL MS 0.25mm×0.5μm×30m.

[0031] EI source temperature: 250℃; scanning range: 30-500 m / z; acquisition mode: full scan mode; injection port temperature: 280℃; injection volume: 1 μL; electron energy: 70 eV; column oven temperature program: 50℃ for 3 min, ramp to 150℃ at 5℃ / min, ramp to 250℃ at 10℃ / min, hold for 7 min; carrier gas: high-purity helium, carrier gas flow rate: 1 mL / min, split ratio: 1:10; the total ion chromatogram of the volatile oil of *Nepeta cataria* was obtained, see attached. Figure 1 .

[0032] The spectral data were analyzed by searching the NIST17-1.lib standard mass spectrometry library to identify each compound. The mass fraction of each component was determined by peak area normalization. The results are shown in Table 1.

[0033] Table 1. GC-MS analysis results of volatile oil components of *Schizonepeta tenuifolia*.

[0034]

[0035]

[0036] The results showed that 11 volatile components were identified in the obtained volatile oil of *Nepeta cataria*, accounting for 97.92% of the total volatile oil. They were mainly divided into esters, terpenes, alkenes, ketones and alkanes. Among them, the components with higher content were nepeta lactone, pseudonepeta lactone, (4aR,5R,9aS)-2,5,9,9-tetramethyl-4,4a,6,7,8,9a-hexahydro-3H-benzo[7]cyclohepten-5-ol and α-bisabolene-Z, with proportions of 57.61%, 16.45%, 7.42% and 6.43%, respectively.

[0037] 1.2 The bioactivity of the volatile oil of *Corydalis tangutica* obtained in this embodiment was determined.

[0038] (1) Determination of inhibition zone

[0039] The diameter of the inhibition zone for each sample was determined using the agar perforation method.

[0040] On a clean bench, sterilized tryptic soy agar (TSA), Sabouraud agar (SDA), potato dextrose agar (PDA), Clostridium perfringens agar, and modified Dixon agar were cooled to approximately 60°C. 20 mL of each medium was poured into a petri dish and allowed to solidify horizontally. An Oxford cup (6 mm inner diameter, 10 mm height) was placed on the surface of the agar medium, and a hole was gently punched under pressure. The medium was removed from the well using a sterile needle. 0.1 mL of the test bacterial suspension was transferred to the corresponding plate and spread evenly using a spreader to prepare bacterial plates. The prepared *Nepeta cataria* volatile oil sample (5% *Nepeta cataria* volatile oil DMSO solution, 80 μL per well) was then injected sequentially. The petri dishes were then incubated at 37°C for 24-72 hours. The diameter of the inhibition zone was measured using calipers. A blank control group was prepared using 0.9% sterile sodium chloride solution.

[0041] The results of the inhibition zone determination of the volatile oil of *Nepeta cataria* obtained in this embodiment are shown in the appendix. Figure 2 And Table 2 below.

[0042] Table 2 Results of inhibition zone determination (n=3, )

[0043]

[0044] (2) Determination of minimum inhibitory concentration (MIC)

[0045] The minimum inhibitory concentration of each sample was determined using a two-fold serial dilution method.

[0046] Solutions of different samples were serially diluted with TSB liquid medium, modified Dixon liquid medium, and potato dextrose liquid medium to prepare solutions of specific concentrations. Indicator bacteria in the logarithmic growth phase were serially diluted to 10⁻⁶ using TSB liquid medium, modified Dixon liquid medium, and potato dextrose liquid medium, respectively. 5 For each CFU / mL bacterial suspension, 100 μL of the bacterial culture was added to wells 2-12 of a 96-well plate. An additional 100 μL of serial dilution was added to each well. The first well contained 100 μL of liquid culture medium and 100 μL of bacterial suspension as a blank control. Each strain was tested three times. After mixing, the 96-well plate was incubated at 37°C for the specified time. Bacterial growth was observed. Each experiment was repeated three times, and the lowest concentration at which no colonies were observed in three trials was defined as the MIC.

[0047] (3) Determination of minimum bactericidal concentration (MBC)

[0048] Take 100 μL of the solution from the wells of the above MICs and spread it evenly on tryptic soy agar (TSA), Sabouraud agar (SDA), potato dextrose agar (PDA), clostridium-enriched agar, or modified Dixon agar. Incubate the plates at 37°C for the specified time. The lowest concentration in 96 wells corresponding to a plate with no more than 5 colonies is defined as the MBC. Perform 3 parallel controls for each sample group, and take the average value of the results.

[0049] The MIC and MBC results of the volatile oil of *Nepeta cataria* for five standard strains are shown in Table 3.

[0050] Table 3. MIC and MBC measurement results (n=3)

[0051]

[0052] The data in Table 3 above show that the obtained *Schizonepeta tenuifolia* volatile oil has a strong bactericidal effect on all of the above-mentioned strains except *Propionibacterium acnes*. Among them, *Schizonepeta tenuifolia* volatile oil has the best antifungal and bactericidal effect on fungi, especially *Malassezia furfur*, with a MIC of 0.078% and an MBC of 0.156%. *Schizonepeta tenuifolia* volatile oil has a secondary bactericidal effect on Gram-positive bacteria, but a better antibacterial effect, with a MIC of 0.078%. However, its antibacterial effect on *Propionibacterium acnes*, a typical Gram-positive anaerobic bacterium closely related to acne, is poor, with an antibacterial effect between 0.3125% and 2.5%. Therefore, *Schizonepeta tenuifolia* volatile oil can be used in the preparation of antifungal and antibacterial products.

[0053] Example 2

[0054] Unlike Example 1, only the stems of *Nepeta cataria* were used to extract the volatile oil from *Nepeta cataria*. All other operations were the same as in Example 1. Finally, the concentration of the obtained volatile oil from *Nepeta cataria* was diluted to 5%.

[0055] Example 3

[0056] Unlike Example 1, only the leaves of *Nepeta cataria* were used to extract the volatile oil from *Nepeta cataria*. All other operations were the same as in Example 1. Finally, the concentration of the obtained volatile oil from *Nepeta cataria* was diluted to 5%.

[0057] In Examples 2-3, different parts of *Nepeta cataria* were used for extraction. The antibacterial properties of the obtained *Nepeta cataria* volatile oil against different types of bacteria and fungi are shown in the appendix. Figure 3 As shown in Table 4 below, the values ​​of the inhibition zone are specifically listed.

[0058] Table 4. Results of the determination of the antibacterial zone of volatile oil from different extracts of *Schizonepeta tenuifolia* (n=3). )

[0059]

[0060] Appendix Figure 3 Based on the data in Table 4 above, it can be seen that in Examples 2-3, the volatile oils obtained from the stems and leaves of *Nepeta cataria* all exhibited a certain degree of antibacterial activity. Among them, the volatile oil from the stems of *Nepeta cataria* showed significant antibacterial activity against *Trichophyton mentagrophytes*, while the antibacterial activity against other types of bacteria or fungi was not significantly different. The volatile oil from the leaves of *Nepeta cataria* showed superior antibacterial activity against several strains, including *Candida albicans*, *Malassezia furfur*, and *Trichophyton mentagrophytes*.

[0061] Comparative Example 1

[0062] Unlike Example 1, only the roots of *Nepeta cataria* were used to extract the volatile oil. All other operations were the same as in Example 1, and the concentration of the obtained volatile oil was diluted to 5%. The antibacterial effect of the *Nepeta cataria* volatile oil derived from the roots is shown in the appendix. Figure 4 As shown.

[0063] From the appendix Figure 4 The results show that the volatile oil prepared from the roots of *Nepeta cataria* did not exhibit an inhibition zone and showed almost no antibacterial activity.

[0064] Comparative Example 2

[0065] Unlike Example 1, catnip volatile oil was prepared using catnip as the raw material, and the preparation method of catnip volatile oil was the same as in Example 1.

[0066] Meanwhile, the volatile oil from *Nepeta cataria* obtained in this comparative example was diluted to concentrations of 5% and 20% respectively, and antibacterial properties were tested. The antibacterial effects of the volatile oil at different concentrations are shown in the appendix. Figure 5 The specific values ​​of the inhibition zone are shown in Table 5 below.

[0067] Table 5. Results of inhibition zone determination of volatile oil from *Nepeta cataria* at different concentrations (n=3, ...). )

[0068]

[0069] Table 5 and appendices Figure 5The experimental results showed that a 5% concentration of Nepeta cataria volatile oil only exhibited a certain degree of antibacterial activity against Trichophyton mentagrophytes, and almost no antibacterial activity against other strains. When the concentration of Nepeta cataria volatile oil was 20%, it showed a certain degree of antibacterial activity against the above-mentioned strains. However, in practical applications, if the concentration of Nepeta cataria volatile oil is too high, it will cause significant irritation to human skin. Therefore, within the safe range of use, Nepeta cataria volatile oil does not have good antibacterial activity against common fungal strains.

[0070] In addition to the above embodiments, the present invention also conducted the following experiments to verify the influence of process parameters during the extraction of volatile oil from *Nepeta cataria* on the antibacterial effect of the obtained volatile oil. The specific operations of each embodiment are as follows:

[0071] Example 4

[0072] The preparation of the volatile oil of *Nepeta cataria* differs from that in Example 1 in that the mass of water added is 20 times the mass of *Nepeta cataria*, and then steam distillation is carried out directly for 2 hours. All other operations are the same as in Example 1 to obtain the volatile oil of *Nepeta cataria*. Finally, the concentration of the obtained volatile oil of *Nepeta cataria* is diluted to 5%.

[0073] Example 5

[0074] The preparation of the volatile oil of *Nepeta cataria* differs from that in Example 1 in that the mass of water added is 15 times the mass of *Nepeta cataria*, and after soaking for 12 hours, steam distillation is carried out for 5 hours. The remaining operations are the same as in Example 1 to obtain the volatile oil of *Nepeta cataria*. Finally, the concentration of the obtained volatile oil of *Nepeta cataria* is diluted to 5%.

[0075] Example 6

[0076] The preparation of the volatile oil of *Nepeta cataria* differs from that in Example 1 in that the mass of water added is 10 times the mass of *Nepeta cataria*. After soaking for 3 hours, steam distillation is carried out for 3 hours. The remaining operations are the same as in Example 1 to obtain the volatile oil of *Nepeta cataria*. Finally, the concentration of the obtained volatile oil of *Nepeta cataria* is diluted to 5%.

[0077] Example 7

[0078] The preparation of the volatile oil of *Nepeta cataria* differs from that in Example 1 in that the mass of water added is 12 times the mass of *Nepeta cataria*. After soaking for 8 hours, steam distillation is carried out for 6 hours. The remaining operations are the same as in Example 1 to obtain the volatile oil of *Nepeta cataria*. Finally, the concentration of the obtained volatile oil of *Nepeta cataria* is diluted to 5%.

[0079] The volatile oils of *Nepeta cataria* obtained by the methods of the above embodiments all exhibit certain antibacterial and antifungal activities. Furthermore, under the same conditions, their antibacterial activities are significantly higher than those of *Nepeta cataria* volatile oil and volatile oil derived from the roots of *Nepeta cataria*.

[0080] Of course, the antibacterial activities of the volatile oils of *Nepeta cataria* obtained in the above embodiments also have certain differences. The main reason for this phenomenon may be the difference in extraction process parameters, which leads to differences in the extraction rate of volatile oils of *Nepeta cataria*, and thus results in some differences in antibacterial activity. However, since the main active ingredients that play an antibacterial role can be extracted through the above process, based on a comprehensive consideration of factors such as production cost, energy consumption and effect, the process parameters in Example 1 are determined to be the best extraction process for volatile oils of *Nepeta cataria*.

Claims

1. The application of a volatile oil from *Nepeta cataria* in the preparation of antifungal and antibacterial products, characterized in that, The fungus is selected from at least one of Candida albicans, Malassezia, and Trichophyton. The volatile oil from *Nepeta cataria* is prepared using the following method: Take the above-ground parts of Nepeta cataria, crush them into powder, add 5 to 20 times the weight of water and soak for 6 to 12 hours. After soaking, distill for 2 to 6 hours until no more volatile oil increases. Stop heating and collect the volatile oil. The above-ground parts of *Nepeta cataria* are selected from at least one of the stems and leaves of *Nepeta cataria*.

2. The application of the volatile oil of *Nepeta cataria* as described in claim 1 in the preparation of antifungal and antibacterial products, characterized in that... The concentration of volatile oil from *Nepeta cataria* in the antibacterial product is 0.07-5%.

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