A new isoglycyrrhizin from carob and its preparation method and application
By extracting high-purity neo-glycyrrhizin from carob pods and preparing it into tablets, the problems of significant side effects and unclear composition of existing drugs have been solved, achieving effective treatment for liver damage and liver cancer.
Patent Information
- Application Number
- CN202310690608.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-12
- Publication Date
- 2026-02-03
- Estimated Expiration
- 2043-06-12
AI Technical Summary
Existing chemical drugs for treating hepatitis have significant side effects, while the ingredients of traditional Chinese medicine are unclear, leading to large dosages and uncertain effects. The proportions of effective components in Chinese herbal extracts are also unclear, affecting drug quality and efficacy.
High-purity neo-isoglycyrrhizin was extracted from carob pods, separated and purified using a specific process, and prepared into tablets for the treatment of liver damage and liver cancer.
High-purity neo-glycyrrhizin tablets significantly reduce liver enzyme activity, decrease liver inflammation, and block the formation of cirrhosis and liver cancer, exhibiting significant liver protection and repair effects with few side effects.
Smart Images
Figure CN116655711B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of medicine and health food, specifically to a novel isoglycyrrhizin, a flavonoid glycoside compound isolated from the dried pods of the carob tree (Ceratonia siliqua Linn.), which has the effects of protecting the liver, repairing liver damage, reducing liver inflammation, and blocking the formation of cirrhosis and liver cancer. The invention also provides a method for preparing the compound and its applications. Background Technology
[0002] The liver is a vital organ for maintaining bodily functions, participating in metabolic processes such as oxidation, reduction, hydrolysis, and conjugation, and secreting bile to aid digestion. During metabolism and digestion, it transforms toxic substances, undergoing a detoxification process. It is important to note that the liver is a fragile organ; if a large amount of toxic chemicals is ingested in a short period exceeding its capacity, it can cause acute or chronic liver damage. Liver dysfunction caused by liver damage is the common pathological basis for many liver diseases and a cause of the occurrence, development, and eventual failure of many serious liver diseases. Therefore, repairing liver damage is of great significance for the prevention and treatment of acute and chronic liver diseases, hepatitis, cirrhosis, and liver cancer.
[0003] There is currently no specific, long-term treatment for hepatitis using modern chemical drugs. Most existing drugs cannot avoid side effects. For example, biphenyl diester may cause dry mouth, nausea, and rashes, and rebound transaminase levels may occur after discontinuation; qiangli Ning may cause chest tightness, edema, and mild hypertension. Therefore, it is of great significance to leverage the traditional advantages of traditional Chinese medicine (TCM) to develop stable, safe, accurately quantified, quality-controlled, and reliably effective TCM preparations. Currently, many TCM preparations are used to treat liver diseases. However, regardless of the extraction method used, the resulting extract contains not only effective components such as volatile oils and flavonoids but also other ineffective components. This inevitably leads to larger dosages and unclear mechanisms of action for various components, which limits the market application of such drugs. Furthermore, the proportions of various effective components are unclear. This inevitably compromises the quality of these products, hindering the efficacy of TCM.
[0004] Carob pods are the dried pods of *Ceratonia siliqua* Linn., a legume belonging to the genus *Ceratonia*, which grows in the Mediterranean region. Mature pods are 10–25 cm long and mainly contain polysaccharides, cellulose, minerals, small amounts of protein, and small amounts of non-carbohydrate small molecules. Modern research shows that water extracts, flavonoids, and polyphenols from carob pods have significant anti-inflammatory and antioxidant bioactivities, but the specific pharmacodynamic basis remains unclear. Developing active ingredients from carob pods that can protect the liver, repair liver damage, reduce liver inflammation, and inhibit the formation of cirrhosis and liver cancer is feasible. Plant-derived ingredients possess specific functions, are highly effective in small quantities, and have virtually no toxic side effects. Summary of the Invention
[0005] This invention provides a novel isoglycyrrhizin derived from carob, which has hepatoprotective effects, repairs liver damage, reduces liver inflammation, and inhibits the formation of cirrhosis and liver cancer. This invention also provides a method for preparing this compound.
[0006] This invention provides a novel isoglycyrrhizin, a flavonoid compound derived from carob, extracted from the dried pods of the carob tree (scientific name: Ceratonia siliqua Linn.), a plant belonging to the genus Ceratonia in the legume family, after the seeds have been removed.
[0007] Neo-glycyrrhizin is a known compound. Naturally derived neo-glycyrrhizin is primarily extracted from licorice and burdock root, while neo-glycyrrhizin derived from carob tree has not been addressed in existing research. Furthermore, existing studies have suggested that licorice and burdock root extracts have antioxidant and anti-inflammatory effects, and component analysis has revealed the presence of neo-glycyrrhizin in these extracts, demonstrating the overall effect of the extract. This application utilizes advanced technology to isolate and purify neo-glycyrrhizin from carob tree, achieving a very high extraction rate and purity exceeding 99.50%. Subsequent research has revealed that neo-glycyrrhizin derived from carob tree possesses hepatoprotective properties, repairs liver damage, reduces liver inflammation, and inhibits the formation of cirrhosis and liver cancer.
[0008] The specific preparation method of the above-mentioned novel isoglycyrrhizin compounds derived from carob is as follows:
[0009] The seedless carob pods are crushed, preferably crushed to 60-120 mesh.
[0010] Then, after repeated reflux extraction with ethanol solutions of different concentrations at a mass ratio of 2-10, the extracts are combined and concentrated into a paste. Preferably, reflux extraction with ethanol is performed 1-4 times, with the concentration of the ethanol solution used each time being 60%-100%. More preferably, reflux extraction is performed in two stages: the first reflux extraction is performed with an ethanol concentration of 80-100% for 2-3 extractions, and the second reflux extraction is performed with an ethanol concentration of 60-80% for 1-2 extractions.
[0011] Then, the mixture is diluted and dispersed with 1-5 times the amount of purified water, adsorbed by macroporous adsorption resin D101, and eluted with distilled water and ethanol solution to recover non-sugar small molecules from the solvent. Preferably, the solvent is first washed with distilled water 1-3 times, and then washed with 80%-100% ethanol solution 1-3 times, each washing lasting 0.5-2.0 hours.
[0012] The non-carbohydrate small molecules were then dispersed in a 35%-65% methanol solution and extracted 4-6 times each with petroleum ether and ethyl acetate, respectively, to obtain the ethyl acetate fraction. Each extract was analyzed by HPLC.
[0013] Preferably, the chromatographic column is an ACE 5C18 column (4.6x250mm, 5μm); the mobile phase system is water (A)-methanol (B); the flow rate is 1.0mL / min; the column temperature is 30℃; the injection volume is 10μL; the detection wavelength is 254nm; and the gradient elution program is 0-10min: 10-30%B, 10-40min: 30-60%B, and 40-50min: 60-95%B.
[0014] The ethyl acetate fraction was eluted by gradient elution using silica gel column chromatography, preferably with a 200-300 mesh silica gel column. Two elution systems were used: petroleum ether-ethyl acetate and dichloromethane-methanol. The volume ratio of petroleum ether-ethyl acetate was 50:50-0:100, and the volume ratio of dichloromethane-methanol was 88:12-0:100. TLC analysis was performed, and similar fractions were combined. The operation was repeated 1-3 times.
[0015] The eluted fractions are then separated again by an ODS column, preferably with a mobile phase of methanol-water 15:85-100:0.
[0016] Then, the separation is performed by Sephadex LH-20 column chromatography, preferably using dichloromethane-methanol (volume ratio 1:1) as the eluent.
[0017] The semi-preparative liquid chromatography (SLC) was used for separation and purification to obtain neo-glycyrrhizin. Preferably, the semi-preparative liquid chromatography was performed using a YMC-PackODS-A column.
[0018] This invention, through research, reveals that the extraction and separation of compounds from plants often results in very low extraction rates and significant purification difficulties due to numerous impurities and the instability of most compounds. However, the extraction method proposed in this invention achieves a high extraction rate and high purity (99.50%-99.99%) for novel isoglycyrrhizin compounds extracted from carob pods.
[0019] The present invention also provides a tablet made from a novel isoglycyrrhizin compound derived from carob pods.
[0020] Specifically, it contains a novel isoglycyrrhizin compound derived from carob pods, an active pharmaceutical ingredient, a binder, a diluent, a disintegrant, and a lubricant.
[0021] This invention also provides the application of the novel isoglycyrrhizin compound obtained from the above-mentioned carob pods.
[0022] The novel isoglycyrrhizin compound prepared by this invention has the effects of protecting the liver, repairing liver damage, reducing liver inflammation, and blocking the formation of cirrhosis and liver cancer. Attached Figure Description
[0023] Figure 1 This refers to the activity level of alanine aminotransferase (ALT).
[0024] Figure 2 This refers to the activity level of aspartate aminotransferase (AST).
[0025] Figure 3 This represents the level of superoxide dismutase (SOD).
[0026] Figure 4 This represents the activity level of malondialdehyde (MDA).
[0027] Figure 5 For structural
[0028] Figure 6 This is a hydrogen nuclear magnetic resonance spectrum.
[0029] Figure 7 This is a carbon NMR spectrum. Detailed Implementation
[0030] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.
[0031] The semi-preparative liquid chromatography used in the following examples was a YMC-Pack ODS-A column, with column I having an inner diameter of 20 mm and column II having an inner diameter of 10 mm.
[0032] Preparation method of Example 1
[0033] This embodiment provides a novel isoglycyrrhizin derived from carob, and its preparation method is as follows:
[0034] (1) Take 200g of dried carob tree seeds and pods, crush them to obtain coarse powder with a particle size of less than 2mm; add 2L of 95% ethanol solution to the coarse carob tree seeds and pods and reflux extract 5 times (1.5h each time), then reflux extract 5 times with 1L of 70% ethanol (1.5h each time), combine the extracts, recover the solvent and concentrate to obtain 200mL of extract.
[0035] (2) Add purified water to 1000 mL to disperse the extract. Adsorb the extract through macroporous adsorption resin D101, wash with distilled water for 3 column volumes to remove impurities such as sugars, then wash with 95% ethanol for 3 column volumes, and recover the solvent to obtain 5 g of non-sugar small molecule fractions.
[0036] (3) The obtained non-carbohydrate small molecule fractions were dispersed with 2 parts by weight of 50% methanol, and extracted 5 times each with petroleum ether and ethyl acetate. After the solvent was recovered, 0.31 parts by weight of petroleum ether fraction, 1.52 parts by weight of ethyl acetate fraction and 3.17 parts by weight of water fraction were obtained respectively.
[0037] (4) The ethyl acetate fraction was separated by 200-mesh silica gel column chromatography using a gradient elution system of petroleum ether-ethyl acetate and dichloromethane-methanol. The volume ratios of petroleum ether-ethyl acetate were 50:50, 30:70, and 0:100, and the volume ratios of dichloromethane-methanol were 88:12, 50:50, and 0:100. Thin-layer chromatography (TLC) was used for identification, and similar fractions were combined and labeled Fr.AJ according to increasing polarity.
[0038] (5) The fraction Fr.I was subjected to reverse chromatographic column of ODS, eluted with methanol-water gradient (volume ratio of 15:85, 45:55, 75:25, 100:0), identified by TLC thin layer, and similar fractions were combined and labeled as I1-I5 from smallest to largest polarity.
[0039] (6) The above fraction I4 was separated by Sephadex LH-20 column chromatography with dichloromethane-methanol (volume ratio 1:1) as the eluent to obtain fractions 4a-4c.
[0040] (7) The above fraction 4c was separated by semi-preparative liquid chromatography (column I, methanol:water = 48:52, absorption wavelength 210 nm), and then purified by semi-preparative liquid chromatography (column II, methanol:water = 48:52, absorption wavelength 210 nm) to obtain neo-glycyrrhizin (t). R =31.57min). The structural formula is as follows: Figure 5 As shown. Its nuclear magnetic resonance (NMR) spectrum is attached. Figure 6-7 : Figure 6 for 1 H NMR spectrum Figure 7 for 13C10 NMR spectrum.
[0041] The purified isoglycyrrhizin content reached a high level, with a purity of 99.95%.
[0042] Example 2
[0043] Using the novel isoglycyrrhizin obtained in Example 1 as a raw material, and adding pharmaceutical excipients, tablets were prepared.
[0044] The specific components are shown in Table 1:
[0045] Active drugs Neo-isoglycyrrhizin 40mg adhesives Sodium hydroxymethylcellulose 13mg diluent microcrystalline cellulose 35mg Disintegrant dry starch 25.5mg lubricant magnesium stearate 1.5mg
[0046] The tablet preparation method is as follows: the active drug and the binder are mixed by an equal-volume incremental method, and then added to a 10% acetone solution and mixed evenly; the diluent and disintegrant are mixed evenly, and then added to the above solution and mixed evenly to prepare wet granules; the wet granules are dried at 60°C until the solvent is completely removed, and then granulated; then the lubricant is added and mixed evenly, and finally compressed into tablets.
[0047] Example 3
[0048] Using the novel isoglycyrrhizin obtained in Example 1 as a raw material, and adding pharmaceutical excipients, tablets were prepared.
[0049] The specific components are shown in Table 2:
[0050] Active drugs Neo-isoglycyrrhizin 20mg adhesives Sodium hydroxymethylcellulose 11mg diluent microcrystalline cellulose 35mg
[0051] Disintegrant dry starch 25.5mg lubricant magnesium stearate 1.5mg
[0052] The tablet preparation method is as follows: the active drug and the binder are mixed by an equal-volume incremental method, and then added to a 10% acetone solution and mixed evenly; the diluent and disintegrant are mixed evenly, and then added to the above solution and mixed evenly to prepare wet granules; the wet granules are dried at 60°C until the solvent is completely removed, and then granulated; then the lubricant is added and mixed evenly, and finally compressed into tablets.
[0053] Example 4
[0054] Using the novel isoglycyrrhizin obtained in Example 1 as a raw material, and adding pharmaceutical excipients, tablets were prepared.
[0055] The specific components are shown in Table 3:
[0056] Active drugs Neo-isoglycyrrhizin 10mg adhesives Sodium hydroxymethylcellulose 8mg diluent microcrystalline cellulose 35mg Disintegrant dry starch 25.5mg lubricant magnesium stearate 1mg
[0057] The tablet preparation method is as follows: the active drug and the binder are mixed by an equal-volume incremental method, and then added to a 10% acetone solution and mixed evenly; the diluent and disintegrant are mixed evenly, and then added to the above solution and mixed evenly to prepare wet granules; the wet granules are dried at 60°C until the solvent is completely removed, and then granulated; then the lubricant is added and mixed evenly, and finally compressed into tablets.
[0058] Comparative Example
[0059] This comparative example provides a tablet that differs from Example 2 in that it does not contain an active drug, while the other ingredients and preparation method are the same as in Example 1.
[0060] Experimental studies on the repair and protection of liver damage
[0061] Forty-two 8-week-old mice were selected and divided into seven groups of six each, including a blank control group, a model group, groups 1-4 of the examples, and a comparative group. Except for the blank control group, the mice in the other groups were induced with APAP in vitro to establish an APAP-induced acute liver injury model.
[0062] The blank control group and the model group were administered an equal volume of physiological saline by gavage. The Example 1 group was administered the novel isoglycyrrhizin (200 mg / kg) prepared in Example 1 by gavage. The groups in Examples 2-4 were administered tablets (1 tablet) prepared in Examples 2, 3, and 4, respectively. Standard feed and free access to water were provided, once daily for 7 consecutive days. Two hours after the last administration, all groups except the blank control group were intraperitoneally injected with 350 mg / kg of standard APAP solution (solvent: physiological saline). The blank control group was intraperitoneally injected with the same volume of physiological saline.
[0063] After a 12-hour fast with no water restriction, blood was drawn from the orbital rim, centrifuged, and serum physiological and biochemical indicators were measured, including the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The mice were then euthanized, and their livers were removed to detect the levels of superoxide dismutase (SOD) and malondialdehyde (MDA).
[0064] The results are as follows Figure 1 , 2 As shown in Figures 3 and 4, the model group showed a highly significant difference compared to the blank control group, indicating successful modeling. The novel isoglycyrrhizin prepared in Example 1 and tablets containing the novel isoglycyrrhizin prepared in Example 1 significantly reduced the activities of AST, ALT, and MDA in mice, and significantly increased the SOD level in mice. The novel isoglycyrrhizin extracted from carob pods exhibits liver-protective and liver-injury-reducing effects.
[0065] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.
Claims
1. A method for extracting a novel isoglycyrrhizin compound from carob pods, characterized in that, The compound has the structure shown in Formula I: The specific steps of the extraction method are as follows: (1) Remove the seeds and crush the carob pods to 60-120 mesh, reflux with 2-10 times the amount of 60-100% ethanol solution for 1-4 times, combine the extracts and concentrate into an extract. (2) Dilute and disperse with 1-5 times purified water, adsorb by macroporous adsorption resin D101, and then elute with distilled water and 80-100% ethanol solution to remove impurities such as sugars and recover the solvent to obtain non-sugar small molecules. (3) Disperse the above non-carbohydrate small molecules with 35%-65% methanol solution, and extract them with petroleum ether and ethyl acetate 4-6 times respectively. Recover the ethyl acetate fraction, and analyze each extract by HPLC. (4) The ethyl acetate fraction was eluted by gradient chromatography on a 200-300 mesh silica gel column with two elution systems: petroleum ether-ethyl acetate and dichloromethane-methanol. (5) Separate again by ODS column, with a mobile phase volume ratio of methanol-water = 15:85-100:0; (6) Separation was performed by Sephadex LH-20 column chromatography with dichloromethane-methanol as the eluent at a volume ratio of 1:
1. (7) Separation and purification were performed using a semi-preparative liquid chromatography column with a YMC-Pack ODS-A column.
2. The extraction method of the new isoglycyrrhizin compound from carob pods according to claim 1, in step (4), the volume ratio of petroleum ether to ethyl acetate as eluent is 50:50-0:100, and the volume ratio of dichloromethane to methanol is 88:12-0:
100.
3. The extraction method of the new isoglycyrrhizin compound from carob pods according to claim 2, wherein the volume ratio of the semi-preparative liquid mobile phase in step (7) is methanol:water = 48:52, and the absorption wavelength is 210nm.