CN113713046A - Extract of Dendrobium candidum and application of two chemical components thereof as anti-inflammatory preparation - Google Patents

Abstract

The invention relates to the technical field of natural medicines, and discloses application of two plant extracts and two compounds in the field of medicines. The plant extract is extracts of two plants of dendrobium candidum and two main active ingredients thereof. The invention utilizes the extract and the main components thereof obtained by the plant extraction and separation method, can effectively resist inflammatory reaction, has no toxic or side effect and drug dependence on human bodies, has anti-inflammatory activity experiment drug effect superior to that of the prior common western medicines, can be widely applied to eliminating the inflammation of throat, tonsil and the like, and can be used for preparing oral and external anti-inflammatory medicines for treating various inflammations.

Description

Extract of Dendrobium candidum and application of two chemical components thereof as anti-inflammatory preparation

Technical Field

The invention relates to the technical field of natural medicines, in particular to dendrobium candidum and dendrobium candidum extracts and application of two chemical components thereof as anti-inflammatory preparations.

Background

Inflammation is the defense response of the body to noxious stimuli, tissue damage and infection. During inflammation, macrophages secrete a range of inflammatory factors, such as tumor necrosis factor (TNF- α), various interleukins, Prostaglandins (PG), leukotrienes, and chemokines, among others. Inflammation is accompanied by red swelling, fever, pain, dysfunction and the like, and uncontrolled or long-term inflammatory reaction can cause body injury or chronic diseases such as cancer, rheumatoid arthritis, vascular diseases and the like. At present, the medicaments for treating inflammation mainly comprise aspirin, indometacin, ibuprofen and other medicaments, and the medicaments can cause stomach injury, bronchospasm, kidney and heart injury and other adverse reactions after being taken for a long time. Therefore, the search for novel anti-inflammatory active substances is of great significance for the development of novel anti-inflammatory drugs.

Dendrobium nobile is known as the first best reputation of "Jiuda sianchau et Ohwi", is slightly cold in nature and sweet in flavor, and enters stomach and kidney meridians. Has good effects of nourishing yin, clearing heat and reducing pathogenic fire. Dendrobium Crystal (Dendrobium crystallinum Rchb. F.) and Dendrobium candidum (Dendrobium grandis Rchb. f.) are distributed on the stems of the southern Yunnan of China and the mountain forest edge or sparse forest of the southeast Asia countries such as Burma, Thailand, Laos, etc. Chemical composition research shows that the dendrobium contains rich phenolic compounds such as bibenzyl, lignin, flavone, phenanthrene and the like, and pharmacological research shows that part of the phenolic compounds have a structure with obvious anti-inflammatory activity.

Chinese herbal medicine plays an unappreciable role in the prevention and treatment of diseases of people, particularly, traditional Chinese medicine is treasure of national culture, and the direct utilization of Chinese herbal medicine and the utilization of effective chemical components thereof are also one of the important directions of the current medicine development. Therefore, it is feasible to develop anti-inflammatory drugs from Chinese herbal medicines with low toxicity and remarkable therapeutic effects.

Disclosure of Invention

In view of the above, the present invention aims to provide two dendrobium extracts and applications of main component compounds thereof, so that the extracts and the compounds have anti-inflammatory activity and anti-inflammation effects, and have high safety and no toxic or side effects.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a dendrobium extract, the main component compounds of which comprise one or more of densiflorol B and 7-methoxyphenanthrene-2, 5-diol.

In some embodiments of the invention, the method of preparation comprises: the dendrobium extract is prepared by the steps of drying and crushing plants, extracting the plants with ethanol with the 2-fold volume fraction of not less than 95% for three times, concentrating, adding water with the same volume to prepare suspension, extracting and degreasing the suspension for 3 times by using petroleum ether with the same volume, adjusting the pH value to be 9.0-10.0, filtering, extracting the suspension for 3 times by using ethyl acetate with 1-fold volume, and concentrating to obtain an ethyl acetate part.

In some embodiments of the invention, the dendrobium comprises one or more of dendrobium candidum or dendrobium candidum.

Based on the research, the invention also provides application of the dendrobium extract or the main component compound thereof in preparing preparations or medicines for preventing and/or treating inflammation.

In some embodiments of the invention, the inflammation comprises inflammation caused by lipopolysaccharide.

In some embodiments of the invention, the preventing and/or treating inflammation comprises: relieving multiple symptoms of redness, swelling, fever, and pain, and/or inhibiting the production of one or more of tumor necrosis factor (TNF-alpha), interleukin, Prostaglandin (PG), Nitric Oxide (NO), and Reactive Oxygen Species (ROS)

In some embodiments of the invention, the principal constituent compounds include one or more of Densiflorol B and 7-methoxyphenanthrene-2, 5-diol.

The invention also provides a preparation for preventing and/or treating inflammation, which comprises the dendrobium extract or the main component compound thereof and an acceptable auxiliary agent;

the main component compounds comprise one or more of densiflorol B and 7-methoxyphenanthrene-2, 5-diol.

The invention also provides a medicament for preventing and/or treating inflammation, which comprises the dendrobium extract or the main component compound thereof and acceptable auxiliary materials;

the main component compounds comprise one or more of densiflorol B and 7-methoxyphenanthrene-2, 5-diol.

In some embodiments of the invention, the dosage form of the drug comprises one or more of a tablet, a capsule, a powder injection or a suspension.

The research shows that the dendrobium candidum and the dendrobium candidum extract can inhibit LPS (LPS) to induce RAW264.7 cells to generate NO, and the two phenanthrene compounds obtained by the plant extraction and separation method also have the same effect and low toxic and side effects on a human body, so that the plant ethanol extract and the compounds thereof can be used for preparing oral and external anti-inflammatory medicaments with anti-inflammatory effects.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

Fig. 1 shows a phytochemical preparation process of two dendrobium extracts and two compounds.

Detailed Description

The invention discloses dendrobium huoshanense and dendrobium cyrtonema extract and application of two chemical components thereof as anti-inflammatory preparations, and a person skilled in the art can realize the anti-inflammatory preparations by appropriately improving process parameters by referring to the contents. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The invention provides the following technical scheme:

the structure of the compound is:

the chemical name of the medicine is as follows:

7-hydroxy-2-methoxy phenanthrene-1, 4-dione Densiflorol B

7-methoxy-2, 5-dihydroxyphenanthrene 7-Methoxyphenanthrene-2,5-diol

7-hydroxy-2-methoxy phenanthrene-1, 4-dione Densiflorol B red amorphous powder, EIS-MS M/z: [ M + Na]⁺277, formula C₁₅H₁₀O₄。¹³C-NMR(DMSO-d₆125 MHz): 188.4,180.2,158.3,157.6,138.9,132.3,129.6,128.2,126.8,123.3,122.5,121.8,111.1,109.8,56.4. A phenanthrene substance extracted and separated from dendrobium huoshanense or dendrobium congolense is a known compound, and no inflammatory activity report is found.

7-methoxy-2, 5-dihydroxyphenanthrene 7-Methoxyphenanthrene-2, 5-diol: brown amorphous powder, EIS-MS M/z: [ M + Na]⁺267 of formula C₁₅H₁₂O₃。¹³C-NMR(DMSO-d₆125 MHz): 166.8,159.7,105.6,155.7,135.7,134.7,126.8,126.6,124.0,121.1,119.0,118.5,112.4,103.6,55.8. A phenanthrene substance extracted and separated from dendrobium huoshanense or dendrobium congolense is a known compound, and no inflammatory activity report is found.

The phytochemical preparation method comprises the following steps: air drying and pulverizing herba Dendrobii (with the scientific name of Dendrobium crystallinum Rchb. F.) and herba Dendrobii (with the scientific name of Dendrobium grandiflorum Rchb. f.), leaching with ethanol water solution with volume fraction of not less than 95%, preferably leaching for 3 times, filtering the obtained leaching solution, mixing, and concentrating under reduced pressure to obtain ethanol extract.

Adding water into the ethanol extract to prepare suspension, sequentially extracting with petroleum ether and ethyl acetate, and concentrating the ethyl acetate extract to prepare an extract. The ethyl acetate extract is divided into 10 sections by silica gel column chromatography, the position of the compound is determined by standard substance, and the key separation is carried out. The part of the compound is subjected to repeated chromatographic separation by normal phase silica gel, reverse phase silica gel, gel and liquid phase to obtain a monomer compound. (the flow chart is shown in figure 1)

For example: 10kg of Dendrobium candidum (with the scientific name of Dendrobium crystallinum Rchb. F.) and an ethyl acetate part (8.7g) are subjected to silica gel reduced pressure column chromatography, and gradient elution is carried out by using a mixed solution of chloroform-methanol (1: 0-0: 1) to obtain 10 fractions Fr.1-Fr.10; fr.8 (chloroform-methanol 3:2 part) is subjected to reverse phase silica gel column chromatography, and gradient elution is carried out by methanol-water (30-100 percent) to obtain 12 fractions Fr.8-1-Fr.8-12; fr.8-5 (75% methanol-water fraction) is eluted by gel column chromatography, 100% methanol is used as a mobile phase, and 5 fractions Fr.8-5-1-Fr.8-5-5 are combined in sequence by thin layer chromatography detection; fr.8-5-5 by silica gel column chromatography, with petroleum ether-acetone (5:1) as mobile phase, and removing impurities by thin layer chromatography to obtain monomer compound densiflorol B (1, 58 mg); fr.8-5-4 was separated by semi-preparative HPLC using 68% methanol-water as the mobile phase to give the compound 7-methoxy-2, 5-dihydroxyphenanthrene (2, 126mg, retention time 14.2 min). 10kg of Dendrobii officmalis caulis (scientific name: Dendrobium grandissimum Rhhb. f.) Densiprolol B (1, 96mg), 7-methoxy-2, 5-dihydroxyphenanthrene (2, 112mg) was obtained from an ethyl acetate fraction (8.1 g).

The invention provides application of the extract and two phenanthrene compounds in a medicine for relieving inflammatory reaction.

Preferably, the inflammation is inflammation caused by lipopolysaccharide.

Preferably, the relieving of the inflammatory response includes relieving symptoms of redness, swelling, heat, pain, etc., and inhibiting production of one or more of tumor necrosis factor (TNF- α), interleukin, Prostaglandin (PG), Nitric Oxide (NO), and Reactive Oxygen Species (ROS).

In the application of preparing the inflammation medicament, the extract and the compound provided by the invention can be added with pharmaceutically acceptable auxiliary materials such as a disintegrating agent, a lubricating agent, an adhesive, a dispersing agent and the like under the dosage of effective amount.

The dosage form of the anti-inflammatory drug is not particularly limited, and can be tablets, capsules, powder injection, suspensions and other dosage forms known by persons skilled in the art.

The invention discovers two new extracts and two compounds with good anti-inflammatory activity, and researches show that the dendrobium huoshanense and dendrobium cyrtomii extract and the compound shown in the formula I can inhibit LPS (LPS) to induce RAW264.7 cells to generate NO, so that the compound can be used for preparing medicines with anti-inflammatory effect. The extract and the compound are derived from dendrobium, and the verification proves that the extract and the compound have small side effect, are safe and have good application prospect in the development of anti-inflammatory drugs.

In the application of the dendrobium huoshanense and dendrobium cyrtomii extract and the two chemical components thereof as anti-inflammatory preparations, the used raw materials and reagents can be purchased from the market.

The invention is further illustrated by the following examples:

example 1 preparation of extract

The method comprises the steps of taking plants of Dendrobium candidum (with the scientific name of Dendrobium crystauinum Rchb. F.) and Dendrobium candidum (with the scientific name of Dendrobium gratissimum Rchb. f.) to be dried and crushed, extracting with ethanol with the 2-fold integral number not less than 95% for three times, concentrating to be turbid (with the volume of 1/10), extracting and degreasing with petroleum ether with the same volume for 3 times, filtering by adjusting the pH value (9.0-10.0), extracting with ethyl acetate with the 1-fold integral number for three times, and concentrating to obtain an ethyl acetate part serving as the target extract of the invention. The ethyl acetate part is divided into 10 sections by silica gel column chromatography (200 meshes, 300 meshes, chloroform-methanol 1: 0-0: 1), the part of the compound is determined by standard substances, and the key separation is carried out. The part of the compound is subjected to repeated chromatographic separation by normal phase silica gel, reverse phase silica gel, gel and liquid phase to obtain a monomer compound.

10kg of Dendrobium candidum (with the scientific name of Dendrobium crystallinum Rchb. F.) and an ethyl acetate part (8.7g) are subjected to silica gel reduced pressure column chromatography, and gradient elution is carried out by using a mixed solution of chloroform-methanol (1: 0-0: 1) to obtain 10 fractions Fr.1-Fr.10; fr.8 (chloroform-methanol 3:2 part) is subjected to reverse phase silica gel column chromatography, and gradient elution is carried out by methanol-water (30-100 percent) to obtain 12 fractions Fr.8-1-Fr.8-12; fr.8-5 (75% methanol-water fraction) is eluted by gel column chromatography, 100% methanol is used as a mobile phase, and 5 fractions Fr.8-5-1-Fr.8-5-5 are combined in sequence by thin layer chromatography detection; fr.8-5-5 by silica gel column chromatography, with petroleum ether-acetone (5:1) as mobile phase, and removing impurities by thin layer chromatography to obtain monomer compound densiflorol B (1, 58 mg); fr.8-5-4 was separated by semi-preparative HPLC using 68% methanol-water as the mobile phase to give the compound 7-methoxy-2, 5-dihydroxyphenanthrene (2, 126mg, retention time 14.2 min).

10kg of Dendrobii officmalis caulis (scientific name: Dendrobium grandissimum Rhhb. f.) Densiprolol B (1, 96mg), 7-methoxy-2, 5-dihydroxyphenanthrene (2, 112mg) was obtained from an ethyl acetate fraction (8.1 g).

Example 2: capsules for preparing the compounds of the invention

Weighing the following raw materials (unit: g):

the target extracts or two separated main components, namely Densiflorol B and 7-methoxyphenanthrene-2,5-diol, prepared from two kinds of dendrobium according to the process are respectively 50 g.

Adding 100mL of ethanol into the extract or the compound, filtering, adding carboxymethyl starch sodium, mixing, spray drying to obtain granules, and encapsulating in No. 1 capsule to obtain capsule.

Example 3: tablets for preparing the compounds of the invention

Weighing the following raw materials (unit: g):

the target extracts or two separated main components, namely Densiflorol B and 7-methoxyphenanthrene-2,5-diol, prepared from two kinds of dendrobium according to the process are respectively 50 g.

Adding 100mL of ethanol into the extract or the compound, filtering, adding carboxymethyl starch sodium, mixing, spray drying to obtain granules, and tabletting.

Example 4: pellets for preparing the compounds of the invention

Weighing the following raw materials (unit: g):

the target extracts or two separated main components, namely Densiflorol B and 7-methoxyphenanthrene-2,5-diol, prepared from two kinds of dendrobium according to the process are respectively 50 g.

Adding 100mL of ethanol into the extract or the compound, filtering, adding carboxymethyl starch sodium, mixing, spray drying to obtain granules, adding pure oleum Sesami or Mel, mixing, and making into pill by centrifugal pill making machine.

Example 5: in vitro anti-inflammatory Activity of the extracts and Compounds of the invention

Dissolving all samples to be detected and positive control by using DMSO; the extract or the monomer compound provided by the embodiment 1 of the invention is firstly roughly sieved, and the maximum value of the final concentration is generally 200 mu M, which can be properly adjusted; if it measures IC₅₀The concentration is diluted again and detected by the same method. In the case of crude samples, the final concentration is generally 200. mu.g/mL at the maximum, and can be adjusted as appropriate. The cytotoxicity (200. mu.g/mL inhibition) of the extracts and compounds was first determined by the MTT method at a concentration of 200. mu.g/mL or. mu.M.

Anti-inflammatory Activity test (inhibitory Activity of Compound on NO production by RAW264.7 cells induced by LPS)

RAW264.7 (mouse monocyte macrophage leukemia cells) was selected and seeded on 96-well flat-bottomed cell culture plates at 100. mu.L concentration of 5X 10⁴cells/mL, cultured at 37 ℃ in 5% CO₂Under the condition of humidity of more than 90%, adding 50 mu L of prepared compound solution to be tested after 24h, continuing culturing under the condition, adding 50 mu L of prepared LPS (final concentration is 1 mu g/mL) solution after 1h, taking 100 mu L of supernatant from each hole after 24h, adding 100 mu L of Griess reagent into each hole, and uniformly mixing by a cross method. And (3) measuring and recording the absorbance of each hole under the wavelength of 540nm of a microplate reader, and calculating the NO inhibition rate according to the following formula. The control group is quercetin, the negative control group is DMSO, and the compound to be tested is half-diluted by 5 concentration gradients. The concentration of the test compound is plotted on the abscissa and the inhibition ratio on the ordinate, and the IC of the test compound is determined by plotting₅₀The value is obtained.

Inhibition ratio (%) ═ C₂-C₁)/(C₂-C₀)×100％；

In the formula: c₀、C₁、C₂Blank control group (without LPS), experimental group, and negative (with LPS) control group measured at 540nmAbsorbance of the panel. Calculating the inhibition rate at each concentration, drawing a compound concentration-inhibition rate curve chart, and calculating the half Inhibition Concentration (IC) of the compound on the NO production induced by LPS (LPS) -induced RAW264.7₅₀Value).

TABLE 1 in vitro anti-inflammatory Activity of extracts and Compounds

Test sample	IC50	Cytotoxic Activity (200. mu.g/mL inhibition)
			Dendrobium devonianum extract	68.40±1.30μg/mL	-23.14％
Extract of Dendrobii officmalis caulis	87.62±3.60μg/mL	-6.32％
			7-hydroxy-2-methoxyphenanthrene-1, 4-diones	4.30±0.17μM	-31.22％
7-methoxy-2, 5-dihydroxyphenanthrene	5.63±0.32μM	-15.84％
			Quercetin	14.18±0.11μM	13.51％

The results in Table 1 show that the extracts of two dendrobe species show strong anti-inflammatory activity, IC₅₀Respectively reach 68.40 +/-1.30 and 87.62 +/-3.60 mu g/mL; the main component compound also has better performance than the prior positive control drug quercetin, IC₅₀The values respectively reach 4.30 +/-0.17 and 5.63 +/-0.32 mu M, the cytotoxicity is very low, and the CC is₅₀Greater than 200 μ M, thereby providing the compound with better safety. The results show that the two dendrobium extracts and the main component compounds thereof have good in-vitro anti-inflammatory activity and good safety.

Example 6: toxicity Studies of the Compounds of the invention

1. Acute toxicity study

The drug (the extract prepared in example 2 and the compound powder in the capsule) of the invention is taken from Kunming mice and administered by intragastric administration at the dose of 1g/kg, and is continuously administered for 4 times in 1 day with the interval of 6h each time, and then normal diet is given, and each organ is subjected to pathological examination, and no organ lesion is found.

2. Long term toxicity study

The drug of the present invention (the compound powder in capsules prepared in example 2) was administered to Kunming mice by intragastric administration in medium and large dose groups (1g/kg, 2g/kg) 1 time per day for 14 days, and then the body weight, cardiac function, liver function, kidney function, electrocardiogram and other indicators were measured, and no significant abnormality was observed in the administration groups compared with the control group. The pathological examination of heart, liver, kidney, spleen, lung, stomach, duodenum, large intestine, small intestine, adrenal gland and genital organs, and the administration group has no obvious toxic change compared with the control group. The results show that the extract and the compound are safe and nontoxic, and can be used clinically.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. The dendrobium extract is characterized in that the main component compounds of the dendrobium extract comprise one or more of DensiflorolB and 7-methoxyphenanthrene-2, 5-diol.

2. The dendrobe extract of claim 1, wherein the preparation method comprises: the dendrobium extract is prepared by the steps of drying and crushing plants, extracting the plants with ethanol with the 2-fold volume fraction of not less than 95% for three times, concentrating, adding water with the same volume to prepare suspension, extracting and degreasing the suspension for 3 times by using petroleum ether with the same volume, adjusting the pH value to be 9.0-10.0, filtering, extracting the suspension for 3 times by using ethyl acetate with 1-fold volume, and concentrating to obtain an ethyl acetate part.

3. The extract of dendrobii nobile of claim 1 or 2, wherein the dendrobii nobile comprises one or more of dendrobii officmalis or dendrobii cupulosa.

4. Use of the dendrobe extract or its main component compound according to any one of claims 1 to 3 in the preparation of a preparation or medicament for preventing and/or treating inflammation.

5. The use of claim 4, wherein the inflammation comprises lipopolysaccharide-induced inflammation.

6. The use of claim 4, wherein the prevention and/or treatment of inflammation comprises: relieving one or more of redness, swelling, heat, pain, and/or inhibiting the production of one or more of the proinflammatory factors tumor necrosis factor (TNF-alpha), interleukin, Prostaglandin (PG), Nitric Oxide (NO), and Reactive Oxygen Species (ROS).

7. The use as claimed in any one of claims 4 to 6, wherein the principal component compounds include one or more of densiflorol B and 7-methoxyphenanthrene-2, 5-diol.

8. An agent for preventing and/or treating inflammation, comprising the dendrobe extract or its main component compound according to any one of claims 1 to 3, and an acceptable adjuvant;

the main component compounds comprise one or more of densiflorol B and 7-methoxyphenanthrene-2, 5-diol.

9. A medicament for preventing and/or treating inflammation, which comprises the dendrobium extract or the main component compound thereof as claimed in any one of claims 1 to 3, and acceptable auxiliary materials;

the main component compounds comprise one or more of densiflorol B and 7-methoxyphenanthrene-2, 5-diol.

10. The medicament of claim 9, wherein the dosage form of the medicament comprises one or more of tablets, capsules, powder injections or suspensions.

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