Extract of stone orchid, its preparation method, use and pharmaceutical composition

Abstract

The present disclosure provides a Dactylorhiza extract, a preparation method and uses thereof, and a pharmaceutical composition comprising the Dactylorhiza extract, wherein the Dactylorhiza extract is prepared by the following steps: mixing Dactylorhiza pseudobulbus with alcohol in a weight ratio of 1:5 to 1:10 to obtain a first extraction solution; wherein the alcohol is selected from the group consisting of methanol, ethanol, propanol, butanol, or any combination thereof; filtering the first extraction solution to remove chlorophyll and impurities to obtain a second extraction solution; and subjecting the second extraction solution to reverse-phase high-performance liquid chromatography to obtain the Dactylorhiza extract; wherein the Dactylorhiza extract comprises compounds dactylorhin D, dactylorhin B, 1-(4-β-D-glucopyranosyloxybenzyl)-2-isobutyl tartrate, loroglossin, or any combination thereof. The present disclosure also provides a pharmaceutical composition comprising the Dactylorhiza extract. The Dactylorhiza extract of the present disclosure has uses for preparing anti-inflammatory drugs.

Description

Technical Field

[0001] This disclosure relates to an orchid extract, and more particularly to a *Dendrobium nobile* extract, its preparation method, uses, and pharmaceutical compositions thereof. Background Technology

[0002] For thousands of years, plants have been widely used as herbs to treat diseases. Orchids are the largest family in the plant kingdom and one of the most widely distributed, and orchids have a long history of being used as medicine. For example, the stems of *Dendrobium nobile* Lindl. (Orchidaceae) have effects such as relieving thirst and calming anxiety, accelerating recovery, and reducing dry mouth (Hew et al., 2006); the stems of *Bletilla striata* have effects such as expectorant, antitussive, lung-tonifying, and hemostatic effects (Kong et al., 2003); *Gastrodia elate* has effects such as treating headaches, dizziness, spasms, migraines, rheumatism, back pain, fever, anticonvulsant effects, and lowering blood pressure (Wang et al., 2006; Sun et al., 2004; and Zhang et al., 1989); and *Pholidota chinensis* has effects such as treating coughs, stomachaches, internal bleeding, toothaches, and wounds (Wang et al., 2006).

[0003] The genus *Dendrobium*, belonging to the Orchidaceae family, is mainly distributed in East and South Asia, including southeastern China, Vietnam, and Myanmar, and primarily grows in forests at altitudes of approximately 300 to 2500 meters. Previous studies have reported that benzoxepin derivative bulbophylol B, a benzoxepin triene derivative found in *Dendrobium*, has the effects of reducing nitric oxide synthase mRNA expression, inhibiting nitric oxide production, and scavenging free radicals (Wang et al., 2007). However, to date, the active ingredients with therapeutic effects within *Dendrobium* have not yet been identified.

[0004] Therefore, identifying the active ingredients in *Dendrobium nobile* that have therapeutic effects for application in the medical field is a problem that needs to be solved in this field. Summary of the Invention

[0005] This disclosure provides a *Dendrobium nobile* extract, which is prepared by the following steps:

[0006] The pseudobulbs of *Dendrobium nobile* are mixed with alcohols in a weight ratio of 1:5 to 1:10 to obtain an extract; wherein the alcohols are selected from methanol, ethanol, propanol, butanol, or any combination thereof; the extract is filtered to remove chlorophyll and impurities to obtain a filtrate; the filtrate is subjected to reverse high-performance liquid chromatography to obtain *Dendrobium nobile* extract; wherein the *Dendrobium nobile* extract contains compounds dactylorhin D, dactylorhin B, 1-(4-β-D-glucopyranosyloxybenzyl)-2-isobutyltartrate, loroglossin, or any combination thereof.

[0007] This disclosure also provides a pharmaceutical composition comprising a therapeutically effective amount of *Dendrobium nobile* extract and a pharmaceutically acceptable excipient thereof, wherein the active ingredient of the *Dendrobium nobile* extract comprises compounds dactylorhin D, dactylorhin B, 1-(4-β-D-glucopyranoyloxybenzyl)-2-isobutyl tartrate, loroglossin, or any combination thereof.

[0008] This disclosure also provides the use of *Dendrobium nobile* extract in the preparation of anti-inflammatory drugs. The *Dendrobium nobile* extract is prepared by the following steps: mixing *Dendrobium nobile* pseudobulb with an alcohol in a weight ratio of 1:5 to 1:10 to obtain an extract; wherein the alcohol is selected from methanol, ethanol, propanol, butanol, or any combination thereof; filtering the extract to remove chlorophyll and impurities to obtain a filtrate; and subjecting the filtrate to reverse high-performance liquid chromatography to obtain the *Dendrobium nobile* extract; wherein the *Dendrobium nobile* extract contains compounds dactylorhin D, dactylorhin B, 1-(4-β-D-glucopyranoyloxybenzyl)-2-tartrate isobutyl ester, loroglossin, or any combination thereof.

[0009] This disclosure also provides a method for preparing a *Dendrobium nobile* extract, comprising the following steps:

[0010] The pseudobulbs of *Pyrrosia lingua* were mixed with alcohols in a weight ratio of 1:5 to 1:10 to obtain an extract; wherein the alcohols were selected from methanol, ethanol, propanol, butanol or any combination thereof; the extract was filtered to remove chlorophyll and impurities to obtain a filtrate; the filtrate was subjected to reverse high performance liquid chromatography to obtain *Pyrrosia lingua* extract.

[0011] The active ingredients contained in the disclosed *Dendrobium nobile* extract, including dactylorhin D, dactylorhin B, isobutyl 1-(4-β-D-glucopyranoylbenzyl)-2-tartrate, loroglossin, or any combination thereof, have anti-inflammatory activity and can be used to prepare anti-inflammatory drugs for treating respiratory inflammation, such as cough, associated with increased interleukin-1β. Attached Figure Description

[0012] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0013] Figure 1 The reverse high performance liquid chromatography (HPLC) results of the extract of *Dendrobium nobile* disclosed herein show the absorption peaks detected at a wavelength of 214 nm from the 1st minute to the 135th minute of the residence time. Fraction 1 was collected from the 1st minute to the 25th minute of residence time; fraction 2 from the 26th minute to the 50th minute of residence time; fraction 3 from the 51st minute to the 75th minute of residence time; fraction 4 from the 76th minute to the 100th minute of residence time; and fraction 5 from the 101st minute to the 135th minute of residence time.

[0014] Figure 2 The figure shows the results of the anti-inflammatory activity of the extract of *Prunus persica* disclosed in this paper. The mouse macrophage cell line RAW 264.7 was treated with lipopolysaccharide (LPS). "-LPS" indicates that RAW 264.7 cells were not treated with LPS; "+LPS" indicates that RAW 264.7 cells were treated with 3 μg / ml LPS; "+LPS+fraction one" indicates that RAW 264.7 cells were treated with 3 μg / ml LPS; "+LPS+fraction one" indicates that RAW 264.7 cells were treated with 3 μg / ml LPS and 100 μg / ml fraction one; "+LPS+fraction two" indicates that RAW 264.7 cells were treated with 3 μg / ml LPS and 100 μg / ml fraction two; "+LPS+fraction three" indicates that RAW 264.7 cells were treated with 3 μg / ml LPS and 100 μg / ml fraction two; "+LPS+fraction three" indicates that RAW 264.7 cells were treated with 3 μg / ml LPS and 100 μg / ml fraction two. RAW 264.7 cells were treated with 3 μg / ml LPS and 100 μg / ml fraction three; "+LPS+fraction four" indicates that RAW 264.7 cells were treated with 3 μg / ml LPS and 100 μg / ml fraction four; "+LPS+fraction five" indicates that RAW 264.7 cells were treated with 3 μg / ml LPS and 100 μg / ml fraction five; all experiments were repeated three times.

[0015] Figure 3 Compound one of the extracts of *Dendrobium nobile* disclosed herein 1¹H-NMR (400MHz, MeOH-d⁴) spectra;

[0016] Figure 4 Compound one of the extracts of *Dendrobium nobile* disclosed herein 13 C-NMR (100MHz, MeOH-d4) spectrum results;

[0017] Figure 5 This is a structural diagram of compound one contained in the extract of *Prunus cerasifera* disclosed herein;

[0018] Figure 6 Compound two of the extract of *Prunus persica* disclosed herein 1 ¹H-NMR (400MHz, MeOH-d⁴) spectra;

[0019] Figure 7 Compound two of the extract of *Prunus persica* disclosed herein 13 C-NMR (100MHz, MeOH-d4) spectrum results;

[0020] Figure 8 This is a structural diagram of compound two contained in the extract of *Prunus cerasifera* disclosed herein;

[0021] Figure 9 Compound three of the extract of *Prunus persica* disclosed herein 1 ¹H-NMR (400MHz, MeOH-d⁴) spectra;

[0022] Figure 10 Compound three of the extract of *Prunus persica* disclosed herein 13 C-NMR (100MHz, MeOH-d4) spectrum results;

[0023] Figure 11 This is a structural diagram of compound three contained in the extract of *Prunus persica* disclosed herein;

[0024] Figure 12 Compound four of the extract of *Prunus persica* disclosed herein 1The ¹H NMR (700MHz, DMSO-d6) spectra are as follows: ¹H NMR (DMSO-d6, 700MHz): 0.66 (3H,d,J 6.2Hz,H-7), 0.80 (3H,d,J=6.4Hz,H-8), 1.52 (¹H,m,H-6), 1.52, 1.72 (2H,m,H-5), 4.23 (¹H,s,H-3), 4.80 (d,J=4.2Hz,H-1”,H-1””), 4.82, 4.75 (2H,d,JA=JB 12.0Hz,H-1'A and H-1'B), 4.96, 4.86 (2H,d,JA=JB12.0Hz,H-1'”A and H-1'"B),6.97(4H,m,H-4',6'H; H-4'",6'"), 7.16(2H,d,J=8.7Hz,H-3',7'),7.23(2H,d,J 8.7Hz,H-3'",7'"),3.21, 3.26,3.29(m,H-2”,H-2”” / H-3”,H-3”” / H-5”,H-5””),3.14(m,H-4”, H-4””),3.45(m,H-6A”,H-6A””),3.66(m,H-6B”,H-6B””);

[0025] Figure 13 Compound four of the extract of *Prunus persica* disclosed herein 13 The C-NMR (175MHz, DMSO-d6) spectra are as follows: 24.7 (C-8), 23.9 (C-7), 24.0 (C-6), 44.5 (C-5), 61.2 (C-6”, C-6””), 66.6 (C-1’, C-1’”), 70.3 (C-4”, C-4””), 73.7 (C-2”, C-2””), 76.5 (C-3), 76.9 / 77.3 (C-3”, C-3””; C-5”, C-5””), 80.0 (C-2). 100.8(C-1”,C-1””),116.7(C-4’,6’;C-4”’,6”’),129.4(C-2’,C-2’”),130.4 / 130.6(C-3',7';C-3"',7'"),157.6(C-5',C-5'"),171.2(C-4),173.7(C-1);

[0026] Figure 14 The HSQC spectrum (700 Hz) of compound 4 contained in the extract of *Dendrobium nobile* disclosed herein is shown.

[0027] Figure 15The HMBC spectrum (700 Hz) of compound four contained in the extract of *Prunus persica* disclosed herein; and

[0028] Figure 16 This is a structural diagram of compound four contained in the extract of *Prunus persica* disclosed herein. Detailed Implementation

[0029] The following specific embodiments are used to illustrate the disclosure of this specification. After reading the disclosure of this specification, those skilled in the art can easily understand its advantages and effects.

[0030] Unless otherwise stated herein, the singular forms “a” and “the” used in the specification and the appended claims include the plural individuals.

[0031] Unless otherwise stated herein, the term "or" as used in the specification and the appended claims includes the meaning of "and / or".

[0032] Unless otherwise stated herein, the term "therapeutic effective amount" as used in the specification and the appended claims means the amount of the provided *Prunus persica* extract that can achieve a therapeutic effect.

[0033] Unless otherwise stated herein, the term "pharmaceutically acceptable excipient" as used in the specification and the appended claims means any pharmaceutically acceptable common excipient, including but not limited to binders, fillers and disintegrants.

[0034] This disclosure provides an extract of *Prunus cerasifera*, which is prepared by the following steps: mixing *Prunus cerasifera* pseudobulbs with alcohols in a weight ratio of 1:5 to 1:10 to obtain an extract; wherein the alcohols are selected from methanol, ethanol, propanol, butanol, or any combination thereof; filtering the extract to remove chlorophyll and impurities to obtain a filtrate; and subjecting the filtrate to reverse high-performance liquid chromatography to obtain the *Prunus cerasifera* extract; wherein the *Prunus cerasifera* extract contains compounds dactylorhin D, dactylorhin B, 1-(4-β-D-glucopyranosyloxybenzyl)-2-isobutyltartrate, loroglossin, or any combination thereof.

[0035] In one specific embodiment, the upper limit of the concentration of methanol, ethanol, propanol, or butanol can be 100% (v / v), 95% (v / v), 90% (v / v), 85% (v / v), 80% (v / v), 75% (v / v), 70% (v / v), 65% (v / v), 60% (v / v), 55% (v / v), 50% (v / v), 45% (v / v), 40% (v / v), or 35% (v / v), and the lower limit of the concentration of methanol, ethanol, propanol, or butanol can be 5% (v / v), 10% (v / v), 15% (v / v), 20% (v / v), 25% (v / v), or 30% (v / v). 35% (v / v), 40% (v / v), 45% (v / v), 50% (v / v), 55% (v / v), 60% (v / v), 65% (v / v), 70% (v / v), 75% (v / v), 80% (v / v), or 85% (v / v). For example, the methanol, ethanol, propanol, or butanol has a concentration range of 5% to 100% (v / v), 75% to 100% (v / v), or 85% to 100% (v / v). In another specific embodiment, the methanol, ethanol, propanol, or butanol has a concentration of 95% (v / v).

[0036] This disclosure also provides a pharmaceutical composition comprising a therapeutically effective amount of *Dendrobium nobile* extract and a pharmaceutically acceptable excipient thereof, wherein the active ingredient of the *Dendrobium nobile* extract comprises compounds dactylorhin D, dactylorhin B, 1-(4-β-D-glucopyranoyloxybenzyl)-2-isobutyl tartrate, loroglossin, or any combination thereof.

[0037] This disclosure also provides the use of *Prunus persica* extract in the preparation of anti-inflammatory drugs, wherein the *Prunus persica* extract is prepared by the following steps:

[0038] The pseudobulbs of *Dendrobium nobile* are mixed with alcohols in a weight ratio of 1:5 to 1:10 to obtain an extract; wherein the alcohols are selected from methanol, ethanol, propanol, butanol, or any combination thereof; the extract is filtered to remove chlorophyll and impurities to obtain a filtrate; the filtrate is subjected to reverse high-performance liquid chromatography to obtain *Dendrobium nobile* extract; wherein the active ingredients of the *Dendrobium nobile* extract include compounds dactylorhin D, dactylorhin B, 1-(4-β-D-glucopyranoyloxybenzyl)-2-tartrate isobutyl ester, loroglossin, or any combination thereof.

[0039] In one specific embodiment, the anti-inflammatory drug is used to treat respiratory inflammation. In another specific embodiment, the respiratory inflammation is associated with an increase in interleukin-1β. In yet another specific embodiment, the respiratory inflammation includes coughing.

[0040] This disclosure also provides a method for preparing a *Dendrobium nobile* extract, the method comprising the following steps:

[0041] The pseudobulbs of *Pyrrosia lingua* were mixed with alcohols in a weight ratio of 1:5 to 1:10 to obtain an extract; wherein the alcohols were selected from methanol, ethanol, propanol, butanol or any combination thereof; the extract was filtered to remove chlorophyll and impurities to obtain a filtrate; the filtrate was subjected to reverse high performance liquid chromatography to obtain the *Pyrrosia lingua* extract.

[0042] In one specific embodiment, the reverse high performance liquid chromatography uses a chromatographic column with 18-alkylsilane bonded phase or octylsilane bonded phase as the packing material, and performs gradient extraction with water containing 0.1% trifluoroacetic acid / acetonitrile containing 0.1% trifluoroacetic acid in a volume ratio of 95:5 to 65:35.

[0043] This disclosure illustrates details through examples of embodiments. However, the interpretation of this disclosure should not be limited to the following description of embodiments.

[0044] Example 1: Extraction method of Prunus persica extract

[0045] 9.25 kg of *Dendrobium nobile* pseudobulbs (obtained from the Shenzhen Key Laboratory of Orchid Conservation and Utilization, Shenzhen, China) were washed, sliced, freeze-dried, and ground into powder, then stored at -80°C for later use. 20 g of *Dendrobium nobile* pseudobulbs were extracted with 200 mL of 95% ethanol. The 95% ethanol suspension was then concentrated under reduced pressure by rotary evaporation to obtain 10 mL of dark green extract. This 10 mL dark green extract was diluted with 30 mL of double-distilled water (ddH2O) and filtered through a 0.22 μm filter membrane to remove chlorophyll and impurities, yielding a clear brown extract. The brown extract was passed through an 18-alkylsilane bonded phase (C18) column (SUPELCO, 25 cm × 10 mm, 5 μm, USA) using a high performance liquid chromatograph (Hitachi D-7000; pump: Hitachi L-7100; autosampler: Hitachi L-7200; VU-VIS detector: Hitachi L-7420) and subjected to gradient extraction with water containing 0.1% trifluoroacetic acid (TFA) and acetonitrile (ACN) containing 0.1% TFA in different proportions. The gradient extraction conditions were as follows: at a flow rate of 3 mL / min, the C18 column was extracted with water + 0.1% TFA:ACN + 0.1% TFA in a ratio of 95:5 to 65:35 from minute 0 to minute 130; and from minute 131 to minute 135, the C18 column was extracted with 100% ACN + 0.1% TFA. Finally, the sample was passed through a 214 nm UV-VIS detector to obtain five fractions (fraction one to fraction five).

[0046] The results are as follows Figure 1 As shown, fraction one was collected with a residence time of 1 to 25 minutes; fraction two was collected with a residence time of 26 to 50 minutes; fraction three was collected with a residence time of 51 to 75 minutes; fraction four was collected with a residence time of 76 to 100 minutes; and fraction five was collected with a residence time of 101 to 135 minutes. The results showed that higher absorption peaks mainly appeared from residence times of 0 to 16 minutes, 53 to 74 minutes, and 102 to 135 minutes. Furthermore, the absorption peaks in fractions one, three, and five were significantly higher than those in fractions two and four, indicating that this gradient extraction can separate many polar compounds.

[0047] Example 2: Anti-inflammatory activity analysis of fractions one through five

[0048] Based on the reference Wu et al. (2007), bioactivity analysis was performed on fractions one through five. Mouse macrophage cell line RAW 264.7 (obtained from the American Type Culture Collection) was injected with 1 × 10⁻⁶ cells per milliliter. 4 Cells were seeded into 96-well plates and then incubated for 24 hours in Dulbecco's modified eagle medium (DMEM) supplemented with 1% fetal bovine serum and 5% dimethyl sulfoxide (DMSO) at 37°C. Each well contained 1×10⁶ cells. 4 Cells / mL and 100 μL of culture medium. Anti-inflammatory activity was assessed using a rat interleukin-1β (IL-1β) enzyme-linked immunosorbent assay (ELISA, purchased from International Bioresources, California, USA). Lipopolysaccharide (LPS) at a concentration of 2 μL / mL was added to the culture medium in each well of a 96-well plate for 24 hours to induce an inflammatory response. Then, fractions one through five at a concentration of 100 μL / mL were added to each well for 16 hours. Untreated LPS and untreated fractions one through five served as negative controls, while LPS-treated cells served as positive controls. All experiments were repeated three times. Finally, the absorbance at OD 450 nm was measured in each well using a spectrophotometer.

[0049] The results are as follows Figure 2 As shown, the activities of each interleukin-1β in LPS-induced mouse macrophage line RAW 264.7 cells after treatment with fractions one through five were 61.5%, 46.1%, 13.4%, 15.3%, and 83.9%, respectively. This indicates that fractions three and four exhibited significantly stronger inhibitory activity against interleukin-1β compared to fractions one, two, and five, suggesting that the compounds in fractions three and four can effectively reduce inflammatory responses. Based on Figure 1 The absorption peak of fraction 3 is significantly higher than that of fraction 4. Therefore, fraction 3 was selected for further compound analysis.

[0050] Example 3: Purification of four compounds (compounds one through four) from fraction three by HPLC.

[0051] Four compounds were collected from fraction 3 of 18.4 mg and passed through Hypersil. TMBDS C18 column chromatography (250 mm × 4.6 mm) was performed, followed by extraction with H2O / ACN at a flow rate of 1 mL / min and a volume ratio of 85:15 to 75:25. Finally, the four compounds in fraction three were purified by UV-VIS at 214 nm, yielding 5.7 mg of compound I, 2.9 mg of compound II, 4.3 mg of compound III, and 1.9 mg of compound IV. Compound 1 was extracted for 8 minutes with H2O + 0.1% TFA: ACN + 0.1% TFA at a volume ratio of 85:15 to 80:20; Compound 2 was extracted for 10 minutes with H2O + 0.1% TFA: ACN + 0.1% TFA at a volume ratio of 85:15 to 80:20; Compound 3 was extracted for 8 minutes with H2O + 0.1% TFA: ACN + 0.1% TFA at a volume ratio of 80:15 to 80:20; and Compound 4 was extracted for 10 minutes with H2O + 0.1% TFA: ACN + 0.1% TFA at a volume ratio of 80:15 to 75:25.

[0052] Example 4: Nuclear magnetic resonance spectrum (NMR)

[0053] The structures of compounds one through four were analyzed using 400 Hz (Bruker) and 700 Hz (Bruker) NMR. Before analysis, compounds one through four were dissolved in DMSO-D6 or MeOH-d4 solutions and transferred to NMR tubes. The hydrogen NMR spectra of compounds one through three are shown below. 1 H-NMR) and nuclear magnetic resonance carbon spectroscopy ( 13 The results of C-NMR were obtained from 400 Hz NMR to obtain the structures of compounds up to compound three. Compound four... 1 H-NMR and 13 The results of C-NMR, as well as two types of two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR spectroscopy), namely heteronuclear single quantum correlation (HSQC) and heteronuclear multiple-bond correlation spectroscopy (HMBC), were obtained from 700 Hz NMR to obtain the structure of compound four.

[0054] use 1 H-NMR and 13C-NMR spectroscopy analysis of the structure of compound one. For example... Figure 3 As shown, 1 H-NMR spectroscopy indicates that the group of four aromatic protons (δ) is responsible for the signal. H 7.08 and 7.33), one hydroxymethylene group (δ H 5.04 and 5.19), one methylene (δ H 1.66 and 2.06), two methyl groups (δ) H The signals (0.75, 0.92) and the signals of two specific glucose units (δ) H 4.89, 4.96). For example... Figure 4 As shown, 13 C-NMR spectroscopy revealed that the two acetyl groups (δ) were responsible for the outcome. C 174.7, 175.3), two methyl groups (δ) C 25.1, 25.6), one hydroxymethylene group (δ C 69.2), one methylene group (δ) C 48.1), one methine (δ) C The signal of 25.8), and a group of para-substituted aryl carbons (δ C 118.6 and 132.3). Based on the above results, compound one was identified as dactylorhin D, which has the following properties: Figure 5 The structure shown has the chemical formula C. 27 H 40 O 17 And its molecular weight is 636 g / mol.

[0055] use 1 H-NMR and 13 C-NMR spectroscopy was used to analyze the structure of compound two. For example... Figure 6 As shown in Figure 7, 1 H-NMR spectroscopy revealed that the two groups of four aromatic protons (δ) were responsible for the phenomenon. H 7.08, 7.29 and 7.08, 7.26), two hydroxymethylene groups (δ H 5.08 and 5.13), one methylene (δ H 1.68 and 2.09), two methyl groups (δ) H The signals (0.76, 0.90) and the signals (δ) of the three glucose units were also observed. H 4.56, 2.28-3.91). 13 C-NMR spectroscopy revealed that the two hydroxymethylene (δ-) groups were responsible for the [the following]. C 68.5). Based on the above results, compound two was determined to be dactylorhin B, which has the following properties: Figure 8The structure shown has the chemical formula C. 40 H 56 O 23 And its molecular weight is 904 g / mol.

[0056] use 1 H-NMR and 13 C-NMR spectroscopy analysis of the structure of compound three. For example... Figure 9 As shown in Figure 10, 1 H-NMR spectroscopy indicates that the group of four aromatic protons (δ) is responsible for the signal. H 7.08 and 7.34), two hydroxymethylene groups (δ H 5.07 and 5.14), one methylene (δ H 1.68 and 1.86), two methyl groups (δ) H The signals (0.77, 0.92) and the signal of a group of glucose units (δ) H 3.4-3.9). 13 C-NMR spectroscopy indicates that the β-hydroxymethylene group is responsible for the δ-hydroxymethylene group. C 68.5). Based on the above results, compound three was determined to be 1-(4-β-D-pyranoseloxybenzyl)-2-tartrate isobutyl ester, with the structure shown in Figure 11, and its chemical formula is C. 21 H 30 O 12 And its molecular weight is 474 g / mol.

[0057] use 13 The structure of compound four was further analyzed using C-NMR and 2D-NMR spectroscopy (HSQC and HMBC). Figure 12 As shown in Table 1, 1 H-NMR spectroscopy revealed that the two groups of four aromatic protons (δ) were responsible for the phenomenon. H 6.97, 7.16 and 6.97, 7.23), two hydroxymethylene groups (δ H 4.75, 4.82 and 4.86, 4.96), two methyl groups (δ) H The signals (0.66, 0.80) and the signals of two special glucose units (δ) H 4.80, 4.80). For example Figure 13 As shown in Table 1, 13 C-NMR spectroscopy revealed that the two acetyl groups (δ) were responsible for the outcome. C 171.2, 173.7), two methyl groups (δ) C 23.9, 24.7), three methylene groups (δ) C 44.5, 66.6, 66.6), one methine (δ) CThe signal of 24.0), and two groups of four aryl carbons (δ) C 116.7, 130.4 and 116.7, 130.6). (e.g.) Figure 14 As shown, the HSQC experimental results confirm that the signal is generated by the presence of two glucose units, which indicates that the compound has a tetraδ-glucose content. H / δ C The correlation between 4.8 / 100.8 and aromatic proton pairs and carbon pairs is shown. The above data clearly indicate a close association between compound four and its malic acid derivatives, militarin and loroglossin, tartaric acid. Figure 15 As shown, HMBC experimental results reveal a clear correlation between compound four and the C-2 (δC 80.0) of tartaric acid skeletonine. Furthermore, a correlation is also observed at C-5' and C-5-5' (δC 80.0). C Carbon at position 157.6) and at δ H 4.80(H-1”) and δ H The aromatic proton at position 4.80 (H-1””) shows long-range correlation, indicating that the two aromatic rings are connected to two sugar units respectively. Based on the above results, compound four was identified as Loroglossin, which has the following properties: Figure 16 The structure shown has the chemical formula C. 34 H 46 O 18 And its molecular weight is 742 g / mol.

[0058] Table 1 Compound 4 1 H-NMR and 13 C-NMR data (DMSO-d6, 700MHz)

[0059]

[0060] As can be seen from the above results, the compounds dactylorhin D, dactylorhin B, 1-(4-β-D-glucopyranosyloxybenzyl)-2-isobutyltartrate, loroglossin or any combination thereof contained in the extract of *Dendrobium nobile* disclosed herein have significant anti-inflammatory activity.

[0061] The above embodiments are illustrative of the principles and effects of this disclosure and are not intended to limit this disclosure. Any person skilled in the art can modify the above embodiments without departing from the spirit and scope of this disclosure. Therefore, the scope of protection of this disclosure should be as set forth in the claims.

[0062] All references listed below are incorporated herein by way of citation.

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[0066] Wang J.,Wang L.,Kitanaka S.,2007.Stilbene and dihydrophenanthrenederivatives from Pholidota chinensis and their nitric oxide inhibitory andradical-scavenging activities.J.Nat.Med.61,381-6.

[0067] Sun X.F.,Wang W.,Wang D.Q.,Du G.Y.,2004.Research progress ofneuroprotective mechanisms of Gastrodia elata and its preparation.ChinaJournal of Chinese Materia Medica 29,292-5.

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[0069] Wu T.F.,Hsu C.Y.,Huang H.S.,Chou S.P.,Wu H.,2007.Proteomic Analysisof Pycnogenol Effects in RAW 264.7Macrophage Reveals Induction of Cathepsin DExpression and Enhancement of Phagocytosis.J. Agric.Food Chem 55,9784-91.

Claims

1. A kind of extract of *Prunus persica*, characterized in that: The extract of *Prunus persica* was prepared by the following steps: The pseudobulbs of *Dendrobium nobile* are mixed with alcohols in a weight ratio of 1:5 to 1:10 to obtain an extract; wherein the alcohols are selected from methanol, ethanol, propanol, butanol or any combination thereof. The extract was filtered to remove chlorophyll and impurities, resulting in a filtrate. as well as The filtrate was subjected to reverse high-performance liquid chromatography to obtain the extract of *Dactylorhinus edulis*; wherein the extract contains compounds dactylorhin D, dactylorhin B, and... loroglossin; This reverse high performance liquid chromatography uses a column packed with 18-alkylsilane-bonded or octylsilane-bonded phases, and performs gradient extraction with a volume ratio of water containing 0.1% trifluoroacetic acid to acetonitrile containing 0.1% trifluoroacetic acid at 95:5 to 65:

35.

2. The extract of *Dendrobium nobile* as described in claim 1, characterized in that: The methanol, ethanol, propanol, or butanol have a concentration range of 5% to 100% by volume.

3. The extract of *Dendrobium nobile* as described in claim 1, characterized in that: The methanol, ethanol, propanol, or butanol have a concentration range of 85% to 100% by volume.

4. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises a therapeutically effective amount of the *Prunus persica* extract as described in claim 1 and pharmaceutically acceptable excipients thereof.

5. The use of the extract of *Prunus persica* as described in claim 1 for the preparation of an anti-inflammatory drug.

6. The use as described in claim 5, characterized in that: This anti-inflammatory drug is used to treat respiratory inflammation.

7. The use as described in claim 6, characterized in that: This respiratory inflammation is associated with an increase in interleukin-1β.

8. The use as described in claim 6, characterized in that: This respiratory inflammation includes coughing.

9. A method for preparing an extract of *Prunus persica*, characterized in that: The preparation method includes the following steps: The pseudobulbs of *Dendrobium nobile* are mixed with alcohols in a weight ratio of 1:5 to 1:10 to obtain an extract; wherein the alcohols are selected from methanol, ethanol, propanol, butanol or any combination thereof. The extract was filtered to remove chlorophyll and impurities, resulting in a filtrate. as well as The filtrate was subjected to reverse high-performance liquid chromatography to obtain the extract of *Dactylorhinus edulis*; wherein the extract contains compounds dactylorhin D, dactylorhin B, and... loroglossin; This reverse high performance liquid chromatography uses a column packed with 18-alkylsilane-bonded or octylsilane-bonded phases, and performs gradient extraction with a volume ratio of water containing 0.1% trifluoroacetic acid to acetonitrile containing 0.1% trifluoroacetic acid at 95:5 to 65:35.

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