CN111643568A - Fermentation treatment process and application of houttuynia cordata - Google Patents
Fermentation treatment process and application of houttuynia cordata Download PDFInfo
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Abstract
The invention relates to a fermentation treatment process and application of houttuynia cordata, and belongs to the technical field of Chinese herbal medicine processing. The method is characterized in that the content of methyl n-nonanone in the houttuynia cordata fermentation liquor is at least 24.6 percent, and the content of 4-terpineol is not less than 13.4 percent; the content of bornyl acetate is not less than 13.5%; the content of neryl acetate is not less than 8.7%. The prepared houttuynia cordata fermentation liquor has new application as an effective component of a medicine for inhibiting acute inflammation, and the prepared houttuynia cordata fermentation liquor has new application of methyl-n-nonanone as an effective component of a medicine for inhibiting acute inflammation. Particularly has obvious effect on the medicine for inhibiting otitis media or mastoiditis.
Description
Technical Field
The invention belongs to the technical field of Chinese herbal medicine processing, and particularly relates to a fermentation treatment process and application of houttuynia cordata.
Background
Houttuynia cordata Thunb, a herb recorded in the Chinese pharmacopoeia, is derived from dried aerial parts of houttuynia cordata Thunb (the name of Latin corporation, Houttuyniacordia Thunb.) of Saururaceae. Harvesting in summer when stem and leaf are flourishing and flower ear is more, removing impurities, and drying in the sun. The most common processing method is to remove impurities, remove residual roots, wash and cut into segments, dry in the sun directly or remove impurities, remove residual roots, wash and cut into segments, and carry out water extraction treatment. In both the sun drying and water extraction, the heat-clearing and detoxifying, antibacterial and anti-inflammatory effects are achieved by using the active ingredients such as diethyl hexanol, 2-methyl-, 2-ethyl-3-hydroxyhexyl propionic acid, butyl acetate and the like contained in the houttuynia cordata. However, the volatile oil component in the houttuynia cordata is easy to polymerize and volatilize, and the property of the volatile oil component is very unstable, so that the houttuynia cordata has the problems of poor efficacy, low content of active ingredients, low utilization rate of the houttuynia cordata and the like when being used alone.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a fermentation treatment process of houttuynia cordata, which can improve the content of methyl-n-nonanone.
Meanwhile, the application also provides a new application of the houttuynia cordata fermentation liquor prepared by the houttuynia cordata fermentation treatment process as an effective component of a medicine for inhibiting acute inflammation.
The technical scheme adopted by the application is as follows:
a fermentation treatment process of houttuynia cordata comprises the following steps:
(1) cleaning herba Houttuyniae, cutting, scalding in boiling water for 1-3min, cooling, and draining;
(2) and (3) putting the drained cordate houttuynia into a fermentation tank, adding 4-6 times of pure water, inoculating 1-3% of lactobacillus rhamnosus, sealing, fermenting at room temperature for 45-48h, and filtering to remove impurities to obtain the cordate houttuynia fermentation liquid.
More preferably, the content of the methyl-n-nonanone in the houttuynia cordata fermentation liquor is at least 24.6 percent, and the content of the 4-terpineol is not less than 13.4 percent; the content of bornyl acetate is not less than 13.5%; the content of neryl acetate is not less than 8.7%.
The new use of the houttuynia cordata fermentation liquor prepared by the houttuynia cordata fermentation treatment process as an effective component of a medicine for inhibiting acute inflammation.
The new application of methyl n-nonanone in the houttuynia cordata fermentation liquor prepared by the houttuynia cordata fermentation treatment process as an effective component of a medicine for inhibiting acute inflammation.
Compared with the prior art, the beneficial effect of this application lies in:
1) the application selects proper lactobacillus for fermentation treatment of the houttuynia cordata, so that flavonoid substances and volatile oil in the houttuynia cordata are released, and reasonable fermentation conditions are selected to ensure that the houttuynine sodium bisulfite in the houttuynia cordata is converted into methyl n-nonanone, so that the methyl n-nonanone which mainly plays an antibacterial and inflammation-inhibiting role is completely decomposed, and the acute inflammation inhibiting effect of the houttuynia cordata is greatly improved.
2) According to the method, the houttuynia cordata is fermented, then the houttuynine sodium houttuyfonate is converted into ester compounds such as methyl n-nonanone, 4-terpineol, borneol acetate, neryl acetate and the like, the technical bottleneck that the conventional water extraction method does not exist in the treatment of the houttuynia cordata, such as the methyl n-nonanone, the 4-terpineol, the borneol acetate, the neryl acetate and the like is overcome, the content of the methyl n-nonanone is not less than 24.6%, the content of the 4-terpineol is not less than 13.4%, the content of the borneol acetate is not less than 13.5%, the content of the neryl acetate is not less than 8.7%, the properties of the active ingredients are stable, the anti-inflammatory activity is high, and the medicinal and edible effects of the houttuynia.
3) According to the application, the houttuynia cordata is subjected to fermentation treatment, so that the effective components for inhibiting acute inflammation after the houttuynia cordata is treated by a conventional process are changed, the effective components for inhibiting acute inflammation in the houttuynia cordata after being treated by a conventional water extraction or sun-drying processing method are the active components contained in the houttuynia cordata, such as diethyl hexanol, 2-methyl-, 2-ethyl-3-hydroxyhexyl propionic acid, butyl acetate and the like, and the methyl n-nonyl ketone which the houttuynia cordata does not contain can be extracted as an anti-inflammatory active component after the fermentation treatment, so that the houttuynia cordata belongs to the breakthrough of the technical field of houttuynia cordata treatment.
Drawings
FIG. 1 is a comparison of pH values of fermentation broths obtained at different fermentation times in the fermentation process of the present application.
FIG. 2 shows the total number of colonies of fermentation broth obtained at different fermentation times in the fermentation process of the present application.
FIG. 3 is a comparison of the swelling degree of mouse ears.
FIG. 4 is a comparison of the results of lymphocyte counts in whole blood of mice.
FIG. 5 is a comparison of the results of white blood cell count in whole blood of mice.
FIG. 6 is a comparison of the staining results of mouse acute inflammation sections.
FIG. 7 shows the results of changes in the amount of inflammatory factors involved in inflammation in mice.
FIG. 8 shows the abundance changes of intestinal flora in each experimental group of mice.
Detailed Description
The technical solution of the present application will now be further described with reference to experiments and examples.
The houttuynia cordata fermentation liquor prepared by the houttuynia cordata fermentation treatment process is used as an effective component of a medicine for inhibiting acute inflammation.
Example 1
The fermentation treatment process of the houttuynia cordata is realized by the following steps:
(1) cleaning herba Houttuyniae, cutting, scalding in boiling water for 2min, cooling, and draining;
(2) and (3) putting the drained cordate houttuynia into a fermentation tank, adding 5 times of pure water, inoculating 2% of lactobacillus rhamnosus, sealing, fermenting at room temperature for 48h, and filtering to remove impurities to obtain the cordate houttuynia fermentation liquor.
Example 2
The fermentation treatment process of the houttuynia cordata is realized by the following steps:
(1) cleaning herba Houttuyniae, cutting, scalding in boiling water for 1min, cooling, and draining;
(2) and (3) putting the drained cordate houttuynia into a fermentation tank, adding 4 times of pure water, inoculating 1% of lactobacillus rhamnosus, sealing, fermenting at room temperature for 45 hours, and filtering to remove impurities to obtain the cordate houttuynia fermentation liquor.
Example 3
The fermentation treatment process of the houttuynia cordata is realized by the following steps:
(1) cleaning herba Houttuyniae, cutting, scalding in boiling water for 3min, cooling, and draining;
(2) and (3) putting the drained cordate houttuynia into a fermentation tank, adding 6 times of pure water, inoculating 3% of lactobacillus rhamnosus, sealing, fermenting at room temperature for 46h, and filtering to remove impurities to obtain the cordate houttuynia fermentation liquor.
The new application of the methyl n-nonanone in the houttuynia cordata fermentation liquor prepared by the houttuynia cordata fermentation treatment process as an effective component of a medicine for inhibiting acute inflammation, and particularly has obvious effect on the aspect of medicines for inhibiting otitis media or medicines for inhibiting mastoiditis.
As can be seen from FIGS. 1 and 2, the experiment shows that the value is not changed greatly from the beginning to the end of the fermentation, the pH value is stabilized between 3.5 and 4.5, mainly because the houttuynia cordata produces a small amount of acidic substances during the fermentation process, but reacts with alcohol substances during the fermentation process to form esters, and then the fermentation process is continued to produce a small amount of acidic substances. The reason that the total number of the bacterial colonies is sharply reduced is that the nutrient substances in the houttuynia cordata are released and uncomfortable to the environment, and the nutrient substances suitable for the growth of the lactic acid bacteria in the houttuynia cordata are released along with the fermentation in the later stage of the fermentation, so that the bacterial colonies start to grow and reproduce after being adapted to the environment, and the total number of the bacterial colonies starts to increase.
The houttuynia cordata fermentation liquor obtained by processing the houttuynia cordata by the process line and the process conditions and the houttuynia cordata treatment liquor obtained by the conventional water extraction process are respectively detected by a GC-MS method, and the specific results are as follows:
determination conditions for GC-MS: taking a 5.0g sample and a 20ml sample bottle, sealing, placing the sample bottle in a heater at 70 ℃ for balancing for 40min, inserting an aged extraction head into the sealed sample bottle, incubating at 70 ℃ for 30min, extracting at a head space for 40min, then analyzing at 230 ℃ for 3min at a gas chromatography injection port, and analyzing volatile components by adopting GC-MS.
GC conditions were as follows: quartz capillary chromatography column HP-5MS (30m × 0.25mm × 0.25 μm); the initial temperature is 40 ℃, the retention time is 2.5min, the temperature is increased to 240 ℃ at 5 ℃/min, then the temperature is increased to 250 ℃ at 10 ℃/min, and the retention time is 7.5 min; no split flow sample injection, He as carrier gas, flow rate: 1 mL/min.
MS conditions: the ionization mode EI has electron energy of 70eV, ion source temperature of 230 ℃, injection port temperature of 230 ℃, transmission line temperature of 250 ℃ and mass range of 35-400 amu. The results are shown in tables 1-5 below:
TABLE 1 comparison of the ingredients in the fermentation broth with the ingredients in the aqueous extract
TABLE 2 comparison of the relative contents of alcoholic compounds in the fermentation broth and in the aqueous extract
TABLE 3 comparison of the relative contents of the aldehydes and ketones in the fermentation broth and in the water extract
TABLE 4 comparison of the relative contents of the acid compounds in the fermentation broth and in the aqueous extract
TABLE 5 comparison of the relative contents of the ester compounds in the fermentation broth and in the water extract
TABLE 6 comparison of the relative amounts of other compounds in the fermentation broth and in the aqueous extract
As can be seen from the comparison of tables 1-6, the main substances in the water extract of the control group are alkane compounds and a small amount of acid, alcohol and ester substances, after lactic acid fermentation, a large amount of methyl n-nonanone, substances with low fragrance threshold values of alcohol, aldehyde, acid and ester and a small amount of alkane compounds with high fragrance threshold values are detected in the houttuynia cordata, and the volatile substances with high content are the antibacterial and anti-inflammatory substances, namely volatile oil and flavonoid substances in the houttuynia cordata, and the increase of the substances indicates that the beneficial substances in the houttuynia cordata can be released by lactic acid bacteria fermentation, and the substances just enable the anti-inflammatory effect of the houttuynia cordata to be exerted to the maximum.
From the comparison results, no methyl-n-nonanone was detected in the houttuynia cordata, and a large amount of methyl-n-nonanone was detected in the houttuynia cordata fermented by lactic acid bacteria, indicating that the lactic acid bacteria can decompose the methyl-n-nonanone, which is the main component of the antibacterial and anti-inflammatory activity, in the houttuynia cordata, and the content of the methyl-n-nonanone is the highest.
The applicant also obtains the anti-inflammatory effect of the houttuynia cordata fermentation liquor on mice of an ear swelling inflammation model caused by phorbol ester (TPA), and the specific experiment is as follows:
(1) animals: 50 clean-grade ICR mice are fed freely during the experiment, the temperature of the feeding environment is 20-24 ℃, the relative humidity is 40% -70%, and the light and the shade alternate for 12 hours.
(2) Establishment and administration of mouse ear swelling inflammation model
Randomly dividing into 5 groups, including blank control group (water), herba Houttuyniae injection group (drug control), herba Houttuyniae high dose group, herba Houttuyniae middle dose group, and herba Houttuyniae low dose group, each group containing 10 drugs; the gavage is administered 1 time per day for 7 days, and the dose of the gavage is 0.1mL, 0.2mL, 0.4mL, 30min after the last administration, acetone is used as solvent, and the dose of phorbol ester used by each mouse is 2.5 μ g/ear.
(3) Observation of ear swelling and histopathology
After 6h of dosing, ear thickness of each group of mice was measured with a digital thickness meter. The same part of each ear was punched out with a punch (d ═ 6mm) to calculate the degree of swelling in the right ear/left ear. Ear tissues were fixed with 4% neutral formaldehyde, dehydrated, embedded in paraffin, cut into 10 μm slices, stained with hematoxylin-eosin (HE), and the pathological changes of the ear tissues of each group of mice were observed under a microscope.
(4) Measurement of Whole blood of mouse
Whole blood of the mouse is taken from the eyeball of the mouse, and the blood index of the mouse is measured.
(5) Determination of TNF-alpha, IL-6, IL-1 beta levels in ear tissues
Respectively taking the ear tissues of the same part, weighing the ear tissues, adding physiological saline according to the ratio of mass (g) to volume (mL) of 1: 9, shearing the tissues under the ice bath condition to prepare 10% tissue homogenate, centrifuging for 10min at 3000r/min, collecting supernatant, detecting the levels of TNF-alpha, IL-6 and IL-1 beta in the ear tissues of each group of mice by adopting an ELISA method, and specifically operating according to the instruction of a corresponding kit.
(6) Determination of intestinal flora in mice
Collecting feces in intestinal tracts of mice, freezing and storing at-20 ℃ for high-throughput detection. And (4) analyzing results:
1. swelling of the ear as a result
As can be seen from fig. 3, compared with the houttuynia cordata injection, the mice with three doses of the houttuynia cordata fermentation liquid have reduced ear weight and ear swelling degree, which indicates that the houttuynia cordata fermentation liquid has a certain dose-effect relationship to the mouse acute inflammation model. And the houttuynia cordata fermentation liquor with medium dosage has obvious inhibition effect on acute inflammation of mice compared with other groups. 2. Several primary indicators of whole blood data in mice
As can be seen from fig. 4 and 5, the white blood cell count and the neutrophil in the whole blood of the mice are increased, which reflects that the mice have inflammation, and compared with the houttuynia cordata injection group, the three dose groups of the houttuynia cordata fermentation liquid have an effect on treating the inflammation; and the blood platelet and Procalcitonin (PCT) in the blood of the mouse are reduced, which shows that the houttuynia cordata fermentation liquor has an inhibition effect on acute inflammation.
3. Results of he stained sections of mice
As can be seen from fig. 6, the houttuynia cordata injection group had a significantly increased ear thickness, significant intercellular and intracellular edema, significant inflammatory cells, and dilated capillaries, whereas the three dose groups had insignificant phenomena, indicating that the houttuynia cordata fermentation broth had an effect of suppressing inflammation, particularly, otitis media or mastoiditis, as compared to the blank group.
4. Results of TNF-alpha, IL-6, IL-1 beta level assay in ear tissue
As can be seen from FIG. 7, the levels of TNF-. alpha.IL-6 and IL-1. beta. in the ear tissues of the model mice were significantly reduced as compared with the blank group; compared with the houttuynia cordata injection group, the levels of TNF-alpha, IL-6 and IL-1 beta in the ear tissues of the mice in the high, medium and low dose groups are basically consistent, and the contents of the three in the medium dose group are lower.
5. Analysis of intestinal flora in mice
As can be seen from fig. 8, the abundance of the five groups of mouse intestinal flora shows that the number of probiotics in the mice is increased after the mice use the houttuynia cordata fermentation liquid, the harmful bacteria are reduced, and the houttuynia cordata fermentation liquid has an obvious effect on acute inflammation.
Claims (4)
1. A fermentation treatment process of houttuynia cordata is characterized by comprising the following steps:
(1) cleaning herba Houttuyniae, cutting, scalding in boiling water for 1-3min, cooling, and draining;
(2) and (3) putting the drained cordate houttuynia into a fermentation tank, adding 4-6 times of pure water, inoculating 1-3% of lactobacillus rhamnosus, sealing, fermenting at room temperature for 45-48h, and filtering to remove impurities to obtain the cordate houttuynia fermentation liquid.
2. The fermentation treatment process of houttuynia cordata as claimed in claim 1, wherein the content of methyl n-nonanone in the houttuynia cordata fermentation liquor is at least 24.6%, and the content of 4-terpineol is not less than 13.4%; the content of bornyl acetate is not less than 13.5%; the content of neryl acetate is not less than 8.7%.
3. A new use of the fermentation broth of houttuynia cordata as claimed in claim 1 as an effective ingredient of a drug for suppressing acute inflammation.
4. The use of claim 3, wherein the new use of methyl-n-nonanone in the fermentation broth of houttuynia cordata as an active ingredient of a drug for inhibiting acute inflammation is provided in the process of fermentation treatment of houttuynia cordata as claimed in claim 1.
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