A bamboo ganoderma extract and its use in the preparation of anti-inflammatory drugs

Abstract

The present application relates to the technical field of medicine, and particularly relates to a bamboo ganoderma extract and application thereof in preparation of anti-inflammatory drugs. Eight heteroterpene compounds are successfully separated and identified from ethyl acetate extraction layer of bamboo ganoderma fruiting body, including six new compounds (compounds 43-48) and two known compounds (compounds 49-50). Among the new compounds, compound 43 (methyl ganocalidin D) and compound 44 (Ganocalidonoid L) exhibit significant anti-inflammatory activity, which not only has high anti-inflammatory activity, but also is derived from natural bamboo ganoderma, indicating that it may have lower toxicity and better biocompatibility. Therefore, the compounds 43 and 44 have broad application prospects in preparation of anti-inflammatory drugs, and provide a new idea and direction for development of new, safe and effective anti-inflammatory therapy.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a bamboo Ganoderma extract and its application in the preparation of anti-inflammatory drugs. Background Technology

[0002] Inflammation is a common and important pathological process, a defensive response of the body to endogenous and exogenous stimuli. Under normal circumstances, inflammation helps clear pathogens and necrotic tissue, promotes the healing of damaged tissues, and is a natural physiological response of the body. However, when the body's anti-inflammatory capacity becomes unbalanced with the damage caused by inflammatory factors, the inflammatory response intensifies, which may lead to a series of serious diseases, such as septic shock, arthritis, sepsis, diabetes, and may even trigger a cytokine storm, leading to multiple organ failure.

[0003] In recent years, a growing body of research has shown that almost all diseases are related to inflammation, especially chronic inflammation. Many metabolic diseases, such as cancer, often begin with chronic inflammation. In the early stages of an inflammatory response, a series of complex pathophysiological changes are involved, including increased capillary permeability, the release of arachidonic acid metabolites and platelet-activating factor, and the release of pro-inflammatory factors (such as TNF-α, IL-6, IL-1, serotonin, histamine, and PGE2). These changes collectively constitute a complex inflammatory response network, in which NO (nitric oxide) plays a crucial regulatory role in the inflammatory cascade, particularly in the occurrence and signal transduction of the inflammatory response.

[0004] Given the importance of inflammation in various diseases, the development of effective anti-inflammatory drugs has become a hot topic in current medical research. Currently, commonly used anti-inflammatory drugs in clinical practice are mainly divided into two categories: steroidal anti-inflammatory drugs (steroidal anti-inflammatory drugs) and non-steroidal anti-inflammatory drugs (NSAIDs). Steroidal anti-inflammatory drugs, such as hydrocortisone, prednisone, and dexamethasone, while having significant anti-inflammatory effects, can easily lead to damage to organs such as the liver, kidneys, and stomach with long-term use, and may cause side effects such as dysbiosis, vitamin deficiencies, bleeding, and superinfections. Non-steroidal anti-inflammatory drugs, such as aspirin and acetaminophen, although widely used, also carry risks such as gastrointestinal discomfort and liver and kidney damage.

[0005] Therefore, finding natural, safe, and effective anti-inflammatory drugs has become an urgent problem to be solved. Ganoderma lucidum, a traditional Chinese medicine, possesses various biological activities, including enhancing immunity, regulating blood sugar, and protecting the liver. However, research on the active ingredients of Ganoderma lucidum for anti-inflammatory effects in vitro and in vivo remains lacking. Consequently, there are currently no Ganoderma lucidum extracts specifically formulated for anti-inflammatory purposes, which is highly detrimental to the scientific and efficient utilization and application of Ganoderma lucidum as a medicinal material. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a bamboo Ganoderma extract and its application in the preparation of anti-inflammatory drugs. This extract has broad application prospects in the preparation of anti-inflammatory drugs and provides new ideas and directions for developing novel, safe, and effective anti-inflammatory therapies.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides a bamboo Ganoderma extract, wherein the bamboo Ganoderma extract is composed of any one or more compounds 43-50, and the chemical structural formulas of compounds 43-52 are shown below:

[0009]

[0010] Furthermore, the bamboo Ganoderma extract is composed of any one or more of compounds 43-48.

[0011] Furthermore, the bamboo Ganoderma extract is composed of any one of compounds 43-44.

[0012] The present invention also provides a method for preparing the bamboo Ganoderma extract, including the step of separating it from the ethyl acetate extract of the bamboo Ganoderma fruiting body.

[0013] The present invention also provides the application of the bamboo Ganoderma extract in the preparation of anti-inflammatory drugs, wherein the bamboo Ganoderma extract exerts its anti-inflammatory effect by inhibiting NO and anti-inflammatory factors mIL-6 and mTNF.

[0014] The present invention also provides an anti-inflammatory pharmaceutical composition, wherein the anti-inflammatory pharmaceutical composition is prepared by adding pharmaceutically acceptable excipients or auxiliary ingredients, with the aforementioned Ganoderma lucidum extract as the active ingredient.

[0015] Furthermore, the dosage forms of the anti-inflammatory drug composition include injections, tablets, pills, and granules.

[0016] The present invention discloses the following technical effects:

[0017] This invention successfully isolated and identified eight heteroterpenoid compounds from the ethyl acetate extract of Ganoderma lucidum fruiting bodies, including six new compounds (compounds 43-48) and two known compounds (compounds 49-50). Among these new compounds, compound 43 (methyl ganocalidin D) and compound 44 (ganocalidonoid L) exhibited significant anti-inflammatory activity. Not only do they possess highly potent anti-inflammatory activity, but their origin from the natural plant Ganoderma lucidum suggests potentially lower toxicity and better biocompatibility. Therefore, compounds 43 and 44 have broad application prospects in the preparation of anti-inflammatory drugs, providing new ideas and directions for developing novel, safe, and effective anti-inflammatory therapies. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The chemical structural formulas of compounds 43-52 obtained in Example 1 of this invention are shown below.

[0020] Figure 2 The key HMBC in compound 43 obtained in Example 1 of this invention 1 H- 1 Correlation plot of H COSY and NOESY;

[0021] Figure 3 The key HMBC in compound 44 obtained in Example 1 of this invention 1 H- 1 Correlation plot of H COSY and NOESY;

[0022] Figure 4 The key HMBC of compound 45 obtained in Example 1 of this invention, 1 H- 1 Correlation plot of H COSY and NOESY;

[0023] Figure 5 The key HMBC of compound 46 obtained in Example 1 of this invention 1 H- 1 Correlation plot of H COSY and NOESY;

[0024] Figure 6 The key HMBC of compound 47 obtained in Example 1 of this invention 1H- 1 Correlation plot of H COSY and NOESY;

[0025] Figure 7 The key HMBC of compound 48 obtained in Example 1 of this invention 1 H- 1 Correlation plot of H COSY and NOESY;

[0026] Figure 8 This is a graph showing the NO inhibition rate of compounds 43-50 obtained in Example 1 of this invention;

[0027] Figure 9 This is a graph showing the mIL-6 inhibition rate of compounds 43-50 obtained in Example 1 of this invention;

[0028] Figure 10 This is a graph showing the mTNF inhibition rate of compounds 43-50 obtained in Example 1 of the present invention;

[0029] Figure 11 DEPT of compound 43 obtained in Example 1 of this invention and 13 C-NMR spectrum;

[0030] Figure 12 DEPT of compound 44 obtained in Example 1 of this invention and 13 C-NMR spectrum;

[0031] Figure 13 DEPT of compound 45 obtained in Example 1 of this invention and 13 C-NMR spectrum;

[0032] Figure 14 DEPT of compound 46 obtained in Example 1 of this invention and 13 C-NMR spectrum;

[0033] Figure 15 DEPT of compound 47 obtained in Example 1 of this invention and 13 C-NMR spectrum;

[0034] Figure 16 Compound 48 obtained in Example 1 of this invention 13 C-NMR spectrum. Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0039] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0040] Unless otherwise specified, the test methods used in the following implementations are all commonly used test methods in this field;

[0041] Unless otherwise specified, the test materials used in the following implementations are all commonly used test materials in this field.

[0042] Example 1

[0043] 1. Extraction Method

[0044] The extraction method of the bamboo Ganoderma lucidum fruiting body extract in Example 1 of this invention is as follows:

[0045] The bamboo Ganoderma (G. calidophilum) was purchased in Yunnan in October 2021. The specimen is currently preserved in the College of Chemistry and Pharmacy of Northwest A&F University. The main experimental instruments used in the experiment are shown in Table 1, and the main biochemical reagents are shown in Table 2.

[0046] Table 1 Main Experimental Instruments

[0047]

[0048]

[0049] Table 2 Main Biochemical Reagents

[0050]

[0051] Colorimetric reagents: vanillin-sulfuric acid ethanol solution; iodine colorimetric reagent

[0052] The preparation of the fat-soluble extract from the fruiting body of Ganoderma lucidum is as follows:

[0053] Take 25 kg of Ganoderma calidophilum fruiting bodies, dry them, pulverize them, soak them in 75% ethanol for 24 hours, concentrate them under reduced pressure, and repeat three times. After concentrating the total ethanol extract, suspend it in water, extract it four times with ethyl acetate, and concentrate it under reduced pressure to obtain 411.5 g of the fat-soluble extract.

[0054] 2. Structural identification of heteroterpenoids

[0055] Ten heteroterpenoid compounds were isolated from the ethyl acetate extract of Ganoderma lucidum fruiting bodies, namely compounds 43-50. Among them, compounds 43-48 are new compounds, and compounds 49-50 are known compounds. The known compounds are: compound 49 (gancochlearol I) and compound 50 (applanatumol E). The chemical structural formulas of compounds 43-50 are shown below. Figure 1 As shown. The DEPT spectra of compounds 43-48 above are as follows. Figure 11-16 As shown.

[0056] Compound 43 is a yellow oily substance with m / z [M+H]+403.1743 and molecular formula C. 22 H 26O7 has an unsaturation degree of 10. ¹H-NMR reveals a 1,2,4-trisubstituted benzene ring (δH 6.79, d, J = 8.91 Hz H⁻⁶; δH 7.00, dd, J = 2.99, 8.94 Hz H⁻⁵; δH 7.08, d, J = 2.97 Hz H⁻⁶). ¹³C-NMR and DEPT data confirm the presence of 3 sp³ methyl groups, 4 sp³ methylene groups, 6 sp² methine groups, and 9 sp² quaternary carbons. Literature review indicates that compound 43 has a similar structure to compound ganocalidin D (Huang et al. 2016), the difference being that compound 43 has an additional methyl group (δH 3.65). Based on the correlation between δH 3.65 and δC 170.3 in HMBC, this additional methyl group is located at the C-16' position. In the NOESY spectrum, the signals of H-2' (δH 6.88) and H-4' (δH 2.48) are correlated, proving that the carbon-carbon double bonds of C-2' and C-3' are Z-configured; the signals of Me-14' (δH 1.65) and H-5' (δH 2.29) are correlated, proving that the carbon-carbon double bonds of C-6' and C-7' are Z-configured; the signals of Me-13' (δH 1.76) and H-9' (δH 2.29) are correlated, proving that the carbon-carbon double bonds of C-10' and C-11' are E-configured, as detailed below. Figure 2 As shown, compound 43 was named methyl ganocalidin D.

[0057] Compound 44 is a yellow oily substance with m / z [M+H]+405.1908 and molecular formula C. 22 H 28O7 has an unsaturation degree of 9. ¹H-NMR reveals a 1,2,4-trisubstituted benzene ring (δH 6.79, d, J = 8.89 Hz H-6; δH 7.00, dd, J = 2.95, 8.93 Hz H-5; δH 7.10, d, J = 2.89 Hz H-3). ¹³C-NMR and DEPT data confirm the presence of 3 sp³ methyl groups, 5 sp³ methylene groups, 1 sp³ methine group, 5 sp² methine groups, and 8 sp² quaternary carbons. A comprehensive comparison of the one-dimensional spectra reveals that compound 44 and compound 43 have similar structures, the difference being that compound 44 lacks one carbon-carbon double bond. HMBC spectroscopy indicates that this missing carbon-carbon double bond is located at the C-10' and C-11' positions. The correlation between the signals of H-2' (δH 6.88) and H-4' (δH 2.48) in the NOESY spectrum proves that the carbon-carbon double bonds of C-2' and C-3' are Z-configured; the correlation between the signals of Me-14' (δH 1.60) and H-5' (δH 2.26) proves that the carbon-carbon double bonds of C-6' and C-7' are Z-configured, as detailed below. Figure 3 As shown, compound 44 was named Ganocalidonoid L.

[0058] Compound 45 is a yellow oily substance with m / z [M+H]+375.1804 and molecular formula C. 21 H 26 O6 has an unsaturation degree of 9. ¹H-NMR reveals a 1,2,4-trisubstituted benzene ring (δH 6.40, d, J = 2.89 Hz H⁻³; δH 6.55, dd, J = 2.95, 8.64 Hz H⁻⁵; δH 6.62, d, J = 8.63 Hz H⁻⁶). ¹³C-NMR and DEPT data confirm the presence of 2 sp³ methyl groups, 5 sp³ methylene groups, 2 sp³ methine groups, 5 sp² methine groups, and 7 sp² quaternary carbons. Comparing the one-dimensional and two-dimensional data of compound 45 with known compound 49, the only difference is that compound 45 has one more carboxyl group (δC 180.7) but one less hydroxyl group. Based on the correlation signal between Me-13' and δC 180.7 in HMBC, the carboxyl group is located at C-12'. The correlation between the signals of H-2' (δH 7.28) and H-4' (δH 4.30) in the NOESY spectrum proves that the carbon-carbon double bonds of C-2' and C-3' are Z-configured; the correlation between the signals of Me-14' (δH 1.50) and H-5' (δH 2.23) proves that the carbon-carbon double bonds of C-6' and C-7' are Z-configured, as detailed below. Figure 4 As shown, compound 45 was named Ganocalidonoid M.

[0059] Compound 46 is a yellow oily substance with m / z [M+H]+391.1749 and molecular formula C. 21 H 26 O7 has an unsaturation degree of 9. ¹H-NMR reveals a 1,2,4 trisubstituted benzene ring (δH 6.76, d, J = 8.82 Hz H⁻⁶; δH 6.92, d, J = 11.46 Hz H⁻⁵; δH 7.09, d, J = 3.00 Hz H⁻³). Comparison of ¹³C-NMR and DEPT spectra shows that compound 46 has a similar structure to compound 45—it lacks one sp³ methylene group. The mass spectra of the two compounds differ by exactly one oxygen atom mass (16), indicating that compound 46 has one more hydroxyl group than compound 45. The correlation between the signals of H-2' (δH 6.90) and H-4' (δH 2.45) in the NOESY spectrum proves that the carbon-carbon double bonds of C-2' and C-3' are Z-configured; the correlation between the signals of Me-14' (δH 1.61) and H-5' (δH 2.30) proves that the carbon-carbon double bonds of C-6' and C-7' are Z-configured, as detailed below. Figure 5 As shown, compound 46 was named GanocalidonoidN.

[0060] Compound 47 is a yellow oily substance with m / z [M+Na]+299.0351 and molecular formula C. 16 H 22 O4, with an unsaturation degree of 6. 1H-NMR reveals a 1,2,4-trisubstituted benzene ring (δH 6.74, d, J = 8.87 Hz H-6; δH 6.99, brs, H-5; δH 7.06, brs, H-3). 13C-NMR and DEPT data confirm the presence of 2 sp3 methyl groups, 4 sp3 methylene groups, 1 sp3 methine group, 4 sp2 methine groups, and 5 sp2 quaternary carbons. This compound is very similar to compound 2-[(2'E)-7'-hydroxy-3',7'-dimethyloct-2'-enyllbenzene-1.4-diol] found by Rueda et al. (Ruedae et al. 1998) in the Mediterranean tunicate Aplydium sp., the difference being that compound 47 has one more carboxyl group (δC 180.9) and one less hydroxyl group than compound 2-[(2'E)-7'-hydroxy-3',7'-dimethyloct-2'-enyllbenzene-1.4-diol. Based on the correlation between Me-9' (δH 1.08) and δC 180.9 in HMBC, the carboxyl group can be determined to be at C-8'. The correlation between the signals of Me-10' (δH 1.59) and H-5' (δH 1.39) in the NOESY spectrum proves that the carbon-carbon double bond at C-3' and C-4' is Z-configured, specifically as follows... Figure 6 As shown, compound 47 was named GanocalidonoidO.

[0061] Compound 48 is a yellow solid with m / z [M+Na]+375.1046 and molecular formula C. 16 H 16 O9 has an unsaturation degree of 9. ¹H-NMR data reveals a 1,2,4 trisubstituted benzene ring (δH 6.77, dd, J = 4.88, 8.39 Hz H⁻⁶; δH 6.99, dt, J = 3.40, 8.92 Hz H⁻⁵; δH 7.21, d, J = 2.94 Hz H⁻⁶). ¹³C-NMR and DEPT data confirm the presence of 3 sp³ methylene groups, 2 sp³ methine groups, 2 sp³ quaternary carbons, 3 sp² methine groups, and 6 sp² quaternary carbons. Comparison of the compound's one-dimensional and two-dimensional data with known compound 50 reveals similar structures. The difference lies in that compound 48 has two fewer methyl groups than compound 50. Furthermore, the benzene ring, two carbonyl groups, and two rings occupy 8 out of the 9 unsaturation degrees. Combined with mass spectrometry data, this indicates that compound 48 has one more carbonyl group than compound 50, and this carbonyl group does not show a peak in the NMR spectrum. Based on the HMBC spectrum, it can be determined that the two missing methyl groups are the two hydroxymethyl groups in compound 50 that have lost their methyl groups and become hydroxyl groups. Specifically, as shown below... Figure 7 As shown, compound 48 was named Ganocalidonoid P.

[0062] Compounds 43-48 1 The H-NMR data are shown in Table 1. 13 The C-NMR data are shown in Table 2.

[0063] Table 1. Compounds 43-48 1 H-NMR data

[0064]

[0065]

[0066] Table 2 Compounds 43-48 13 C-NMR data

[0067]

[0068]

[0069] Experimental Example 1

[0070] Nitric oxide (NO), a non-adrenergic, non-cholinergic (NALC) neurotransmitter, is a major neurotransmitter in humans and animals. It is synthesized by nitric oxide synthase (NOS) through the enzymatic hydrolysis of L-arginine to L-citrulline (Jourd'Heuil et al. 1999). Inducible nitric oxide synthase (iNOS) synthesizes micromolar concentrations of NO and is neurotoxic. Microglia, resident macrophages in the brain, constitute approximately 10% of the nervous system's cells. In neurodegenerative diseases, microglia are overactivated, producing abundant neurotoxic pro-inflammatory mediators, including iNOS (leading to excessive NO production).

[0071] In this experiment, a cell model was established by inducing BV-2 to become overactivated using lipopolysaccharide (LPS). The NO produced by BV-2 under the action of compounds 43-48 extracted in Example 1 at a concentration of 20 μM was detected using a NO detection kit. The inhibition rate was calculated, and the anti-neuroinflammatory activity of the compounds was evaluated.

[0072] The final results show the NO inhibition rates of different compound treatments as follows: Figure 8 As shown in the figure, compounds 43 and 44 exhibited NO inhibition rates exceeding 50% at a concentration of 20 μM, demonstrating good anti-neuroinflammatory activity.

[0073] The effects of different compounds on the anti-inflammatory factor mIL-6, such as Figure 9 As shown in the figure, compounds 43 and 44 exhibited inhibition rates of over 50% against mIL-6, specifically 57.0 ± 8.0% and 53.0 ± 13.7%, respectively.

[0074] The effects of different compounds on the anti-inflammatory factor mTNF, such as Figure 10 As shown in the figure, compounds 43 and 44 inhibited mTNF at rates of over 40%, specifically 43.4 ± 1.7% and 49.0 ± 1.6%, respectively.

[0075] In summary, this embodiment successfully isolated and identified eight heteroterpenoid compounds from the ethyl acetate extract of Ganoderma lucidum fruiting bodies, including six new compounds (compounds 43-48) and two known compounds (compounds 49-50). Among these new compounds, compound 43 (methyl ganocalidin D) and compound 44 (ganocalidonoid L) exhibited significant anti-inflammatory activity. Not only do they possess highly potent anti-inflammatory activity, but their origin from the natural plant Ganoderma lucidum suggests potentially lower toxicity and better biocompatibility. Therefore, compounds 43 and 44 have broad application prospects in the preparation of anti-inflammatory drugs, providing new ideas and directions for developing novel, safe, and effective anti-inflammatory therapies.

[0076] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A bamboo Ganoderma extract, characterized in that, The bamboo Ganoderma extract is composed of any one of compounds 43*-44*, and the chemical structural formulas of compounds 43*-44* are shown below: 。 2. The method for preparing bamboo Ganoderma extract as described in claim 1, characterized in that, This includes the step of separating the extract from the ethyl acetate extract layer of Ganoderma lucidum fruiting body.

3. The application of the bamboo Ganoderma extract as described in claim 1 in the preparation of anti-inflammatory drugs, characterized in that, The bamboo Ganoderma extract exerts its anti-inflammatory effect by inhibiting NO and anti-inflammatory factors mIL-6 and mTNF.

4. An anti-inflammatory pharmaceutical composition, characterized in that, The anti-inflammatory drug composition is prepared by adding pharmaceutically acceptable excipients to the bamboo Ganoderma extract of claim 1 as the active ingredient.

5. The anti-inflammatory pharmaceutical composition according to claim 4, characterized in that, The dosage forms of the anti-inflammatory drug composition include injections, tablets, pills, and granules.

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