Preparation method and application of cumin essential oil

Abstract

The invention relates to a preparation method and application of cumin essential oil. The method comprises the following steps: adding a cumin medicinal material into water, uniformly mixing, carrying out high-temperature distillation to obtain an oil-water mixture, and separating by adopting a separator to obtain a cumin essential oil part. An anti-inflammatory experiment of the obtained cumin essential oil shows that the cumin essential oil can significantly down-regulate an inflammatory factor nitric oxide (NO), a tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), which indicates that the cumin essential oil has an anti-inflammatory effect; the cumin essential oil provides a good application prospect for developing medicines or anti-inflammatory health-care products, cosmetics and food additives for treating inflammatory diseases by using the cumin essential oil.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a preparation method and application of cumin essential oil. Background Art

[0002] Inflammation is a common physiological response, with redness, heat, swelling, distension, and dysfunction as key clinical manifestations. When the body is stimulated by external stimuli, it activates its own defense response, making most inflammation beneficial. However, when inflammation attacks the body's own tissues, it becomes harmful, leading to numerous diseases and, in severe cases, death. Currently, medications for treating inflammation are primarily steroidal and nonsteroidal. Most of these drugs suffer from disadvantages such as long treatment cycles, significant side effects, high costs, and poor efficacy. However, natural plant-based active ingredients offer advantages in preventing and treating inflammation, including multiple targets, multiple pathways, low toxicity, and minimal side effects. However, cumin (Cuminum cyminum L.), the world's second-largest spice, is cultivated in Xinjiang, with 90% of its production concentrated there. Current research on cumin essential oil focuses primarily on its antioxidant, anti-tumor, and antibacterial properties. However, limited research has examined the anti-inflammatory activity of cumin essential oil.

[0003] Cumin (Cuminum cyminum L.), also known as fennel (Uyghur translation), cumin, fennel, and fragrant celery, belongs to the genus Cuminum (Cuminum L.) of the Umbelliferae family. As a food spice, cumin, with its rich oily components and aromatic aroma, can reduce the greasiness of meat and remove the fishy odor. Cumin has also been certified as a spice by agencies such as FEMA (Federal Emergency Management Agency) and the FDA (Food and Drug Administration), making it suitable for use in food, some cosmetics, and tobacco flavoring products. Furthermore, Traditional Chinese Medicine (TCM) believes that cumin has the effects of reducing internal heat and calming the liver, invigorating the brain and activating meridians. It is effective in treating symptoms such as dampness and cold, dispelling wind and relieving pain, and indigestion. The TCM classic "Puji Fang" mentions the use of cumin to treat indigestion and chills. Uyghur medicine also records the use of its fruit as a medicinal treatment for indigestion and stomach pain caused by cold.

[0004] Currently, research on cumin, both domestically and internationally, focuses on the development of industrial products such as cumin essential oil and cumin microcapsules. Studies have shown that the polyphenols in cumin essential oil have antioxidant, antibacterial, anticancer, and hypoglycemic effects. However, less research has been conducted on the anti-inflammatory activity of cumin essential oil. The present study demonstrates that cumin essential oil exhibits significant anti-inflammatory activity, thus demonstrating the scientific significance of preparing cumin essential oil through distillation and studying its anti-inflammatory activity. Summary of the Invention

[0005] The present invention provides a method for preparing and using cumin essential oil. This method involves adding cumin medicinal material to deionized water, mixing uniformly, and performing high-temperature distillation to obtain an oil-water mixture. This mixture is then separated using a separator to obtain the cumin essential oil fraction. Anti-inflammatory experiments on the resulting cumin essential oil revealed that the oil significantly inhibited the expression of nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), demonstrating its anti-inflammatory properties. This provides promising application prospects for the development of cumin essential oil as a pharmaceutical for treating inflammatory diseases, or as an anti-inflammatory health supplement, food, or cosmetic additive.

[0006] The preparation method of cumin essential oil of the present invention is carried out according to the following steps:

[0007] a. Weigh cumin seeds, add 3 times their weight of deionized water, mix well, place in a round-bottom flask, connect a distillation apparatus, and distill at 105°C for 8 hours to obtain an oil-water mixture;

[0008] b. Separating the oil-water mixture in step a with a separator to obtain a cumin essential oil portion.

[0009] The cumin essential oil obtained by the method is used in preparing anti-inflammatory medicines.

[0010] The cumin essential oil obtained by the method is used as an additive in preparing food.

[0011] The cumin essential oil obtained by the method is used as an additive in the preparation of cosmetics.

[0012] The cumin essential oil obtained by the method is used as an additive in the preparation of health products.

[0013] The present invention provides a preparation method and use of cumin essential oil. LPS is used to stimulate mouse mononuclear macrophages (RAW264.7 cells) to establish an acute inflammation model. Gradual doses of cumin essential oil samples at safe concentrations are added to the model cells, and then changes in nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) levels before and after the addition of cumin essential oil are detected.

[0014] An in vitro neuroinflammatory model was established by stimulating mouse microglia (BV2 cells) with lipopolysaccharide. Gradual doses of cumin essential oil at safe concentrations were added to the model cells, and the changes in nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) levels before and after the addition of cumin essential oil were detected.

[0015] The cumin essential oil obtained by the method of the present invention has the effect of inhibiting the occurrence and development of systemic inflammation and neuroinflammation in vitro, which indicates that it provides good application prospects in the treatment of inflammatory diseases or anti-inflammatory health products, food and cosmetic additives. Pharmacological experiments show that the cumin essential oil of the present invention can significantly reduce the content of nitric oxide (NO) secreted by mouse mononuclear macrophages (RAW264.7 cells) and mouse microglia (BV2 cells) after lipopolysaccharide stimulation, and significantly reduce the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β secreted by mouse mononuclear macrophages (RAW264.7 cells) after lipopolysaccharide stimulation. The results of the present invention show that cumin essential oil has a good anti-inflammatory effect and is expected to be developed into a new innovative drug or health product, cosmetic and food additive with anti-inflammatory activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The cumin essential oil of the present invention has an effect on the cell viability of mouse mononuclear macrophage RAW264.7 cells; compared with the model group, ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05;

[0017] Figure 2 The cumin essential oil of the present invention is used to determine the NO content secreted by mouse mononuclear macrophage RAW264.7 cells; compared with the model group, ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05;

[0018] Figure 3 The cumin essential oil of the present invention is used to determine the content of inflammatory factors secreted by mouse mononuclear macrophage RAW264.7 cells; A: Effect on the content of tumor necrosis factor-α (TNF-α) in the cells; B: Effect on the content of interleukin-6 (IL-6) in the cells; C: Effect on the content of interleukin-1β (IL-1β) in the cells; Compared with the model group, ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05;

[0019] Figure 4 The cumin essential oil of the present invention has an effect on the cell viability of mouse microglial cells BV2. Compared with the model group, ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05;

[0020] Figure 5 The present invention shows a determination of the NO content secreted by lipopolysaccharide-induced mouse microglia (BV2 cells) by cumin essential oil at different concentrations, wherein compared with the model group, ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below with reference to specific embodiments.

[0022] Example 1

[0023] Preparation of cumin essential oil:

[0024] a. Weigh 1 kg of cumin, add 3 L of deionized water and mix well. Place in a round-bottom flask, connect a distillation apparatus, and distill at 105°C for 8 hours to obtain an oil-water mixture.

[0025] b. Separating the oil-water mixture in step a with a separator to obtain a cumin essential oil portion.

[0026] Example 2

[0027] Effects of cumin essential oil on the acute inflammation model established by mouse mononuclear macrophage RAW264.7 cells stimulated by lipopolysaccharide:

[0028] After distillation and separation, high-concentration cumin essential oil is obtained, which is then fully dissolved in dimethyl sulfoxide (DMSO) solution for later use;

[0029] Mouse mononuclear macrophage RAW264.7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). 4 Mononuclear macrophages (RAW264.7) were inoculated into 96-well plates and cultured for 24 h. Cumin essential oil with gradient concentrations of 50-400 μg / mL was added to the 96-well plates containing mouse mononuclear macrophages RAW264.7 cells. LPS with a final concentration of 1 μg / mL was added to all cells except the normal cell group 1 h after the addition of the samples. After further culture for 16 h, the cell culture plates were removed and processed. The cell viability was determined using a cell counting kit (CCK-8). The cell supernatant was collected and lysed using enzyme cytometry. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β in the cell supernatant were detected using an ELISA kit. The level of nitric oxide (NO) in the cell supernatant was detected using a nitrite determination kit (Griess method). 50 μL of the cell supernatant from each group was transferred to a 96-well plate, with three replicates per group. 50 μL of Griess reagent I and Griess reagent II were added to each well. The absorbance was measured at 540 nm using a microplate reader.

[0030] Experimental results:

[0031] As shown in Table 1, Figure 1As shown, the effect of the cumin essential oil provided by the present invention on the cell viability of mouse mononuclear macrophage RAW264.7 cells after incubation was investigated in a CCK-8 test:

[0032] Table 1 Effect of cumin essential oil on the survival rate of mouse mononuclear macrophage RAW264.7

[0033]

[0034]

[0035] The results showed that cumin essential oil had no effect on the cell viability of mouse mononuclear macrophage RAW264.7 cells within the concentration range of 50-200 μg / mL, which provides a reference for screening the appropriate drug concentration in the experiment.

[0036] As shown in Table 2, Figure 2 As shown, the effect of 50-200 μg / mL cumin essential oil on the secretion of nitric oxide (NO) in acute inflammation model cells RAW264.7 was determined:

[0037] Table 2 Effect of cumin essential oil on NO secretion in mouse mononuclear macrophage RAW264.7 cells stimulated by lipopolysaccharide

[0038]

[0039] The results showed that cumin essential oil significantly inhibited the secretion of nitric oxide (NO) by inflammatory cells, and at a concentration of 200 μg / mL, the inhibition rate on nitric oxide (NO) secretion by RAW264.7 cells reached 97.06% ± 0.267%, indicating that the cumin essential oil of the present invention has anti-inflammatory activity.

[0040] As shown in Table 3, Figure 3 As shown, the effect of 50-200 μg / mL cumin essential oil on the secretion of TNF-α in acute inflammation model cells RAW264.7 is determined:

[0041] Table 3 Effect of cumin essential oil on TNF-α content in mouse mononuclear macrophage RAW264.7 cells stimulated by lipopolysaccharide

[0042]

[0043] The results showed that cumin essential oil significantly inhibited the secretion of TNF-α in inflammatory cells, and at a concentration of 200 μg / mL, it could reduce the secretion of TNF-α in RAW264.7 cells to 35.90 pg / mL.

[0044] As shown in Table 4, Figure 3As shown, the effect of 50-200 μg / mL cumin essential oil on the secretion of IL-6 in acute inflammation model cells RAW264.7 was determined:

[0045] Table 4 Effect of cumin essential oil on IL-6 content in mouse mononuclear macrophage RAW264.7 cells stimulated by lipopolysaccharide

[0046]

[0047] The results showed that cumin essential oil significantly inhibited the secretion of IL-6 by inflammatory cells, and at a concentration of 200 μg / mL, it could reduce the secretion of IL-6 by RAW264.7 cells to 10.23 pg / mL.

[0048] As shown in Table 5, Figure 3 As shown, the effect of 50-200 μg / mL cumin essential oil on the secretion of IL-1β in acute inflammation model cells RAW264.7 is determined:

[0049] Table 5 Effect of cumin essential oil on IL-1β content in mouse mononuclear macrophage RAW264.7 cells stimulated by lipopolysaccharide

[0050]

[0051] The results showed that cumin essential oil significantly inhibited the secretion of IL-1β in inflammatory cells, and at a concentration of 200 μg / mL, it could reduce the IL-1β secreted by RAW264.7 cells to 43.76 pg / mL.

[0052] Example 3

[0053] Effects of cumin essential oil on lipopolysaccharide-stimulated mouse microglial cells BV2:

[0054] Mouse microglial cells (BV2) were cultured in DMEM containing 10% fetal bovine serum (FBS) and 2.5×10 4 Mouse microglial BV2 cells were seeded in 96-well plates and cultured for 24 hours. After being treated with 6.25-50 μM cumin essential oil, all groups except the normal control group were added with lipopolysaccharide at a final concentration of 1 μg / mL. After 24 hours of culture, the cell culture plates were removed and processed. The nitric oxide (NO) content in the cell supernatant was detected using a nitrite assay kit (Griess). 50 μL of the cell supernatant from each group was added to a 96-well plate, and three replicates were made for each group. 50 μL of Griess reagent I and Griess reagent II were added to each well. The absorbance was measured at 540 nm using a microplate reader, and the nitric oxide (NO) content in the cell supernatant was calculated.

[0055] Experimental results:

[0056] As shown in Table 6, Figure 4 As shown, the mouse microglial cells BV2 provided by the present invention were examined in the CCK-8 test:

[0057] Table 6 Effect of cumin essential oil on the survival rate of mouse microglial cells BV2

[0058]

[0059] The results showed that cumin essential oil had no effect on the cell viability of mouse microglial BV2 cells within the concentration range of 25-50 μg / mL, providing a reference for screening the appropriate drug concentration in the experiment.

[0060] As shown in Table 7, Figure 5 As shown, the effect of 6.25-50 μg / mL cumin essential oil on the secretion of nitric oxide (NO) in acute inflammation model cells BV2 was determined:

[0061] Table 7 Effect of cumin essential oil on NO secretion in mouse microglial cells BV2 stimulated by lipopolysaccharide

[0062]

[0063] The results showed that cumin essential oil significantly inhibited the secretion of nitric oxide (NO) by inflammatory cells, and at a concentration of 50 μg / mL, the inhibition rate on nitric oxide (NO) secretion by BV2 cells reached 63.44% ± 4.218%. This indicates that the cumin essential oil of the present invention has the effect of inhibiting the occurrence and development of neuroinflammation.

[0064] From the above results, it can be seen that the inhibition rate of cumin essential oil on the secretion of nitric oxide (NO) by mouse macrophages RAW264.7 at a safe concentration of 200 μg / mL can reach 97.06%±0.267%, and its IC50 value for the inhibition rate of nitric oxide (NO) secreted by mouse macrophages RAW264.7 is 38.39±4.115 μg / mL. At the same time, the effects of cumin essential oil on the expression of related inflammatory factors TNF-α, IL-6, and IL-1β were further tested. The present invention found that cumin essential oil at a safe concentration of 200 μg / mL could reduce the secretion of TNF-α from 100.3 pg / mL to 35.90 pg / mL in mouse macrophage RAW264.7 cells; reduce the secretion of IL-6 from 176.7 pg / mL to 10.23 pg / mL in mouse macrophage RAW264.7 cells; and reduce the secretion of IL-1β from 149.8 pg / mL to 43.76 pg / mL in mouse macrophage RAW264.7 cells. In addition, the present invention found that cumin essential oil at 50 μg / mL could inhibit the secretion of nitric oxide (NO) by cells BV2 by up to 63.44% ± 4.218%, and its IC50 value for the inhibition of nitric oxide (NO) was approximately 25.73 ± 3.665 μg / mL.

Claims

1. A method for preparing cumin essential oil, characterized in that Follow these steps: a. Weigh cumin seeds, add 3 times their weight of deionized water, mix well, place in a round-bottom flask, connect a distillation apparatus, and distill at 105°C for 8 h to obtain an oil-water mixture; b. Separating the oil-water mixture in step a with a separator to obtain a cumin essential oil portion.

2. Use of cumin essential oil obtained by the method as claimed in claim 1 in the preparation of anti-inflammatory drugs.

3. Use of the cumin essential oil obtained by the method according to claim 1 as an additive in preparing food.

4. Use of the cumin essential oil obtained by the method according to claim 1 as an additive in the preparation of cosmetics.

5. Use of the cumin essential oil obtained by the method according to claim 1 as an additive in the preparation of health products.

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