A highly stable quassula injection and its preparation method

Abstract

This invention discloses a highly stable *Paspalum notoginseng* injection and its preparation method. It belongs to the field of pharmaceutical preparation technology. The injection comprises: *Paspalum notoginseng* extract, purified Tween-80, Astragalus polysaccharide, total saponins of Panax notoginseng, glycyrrhizic acid, and a stabilizer. This invention significantly improves the stability of the injection through a triple mechanism of hydrogen bonding (Panax notoginseng), antioxidant action (Astragalus), and charge stabilization (Glycyrrhiza), while maintaining controllable toxicity and significant efficacy, demonstrating clinical translational potential. Furthermore, the addition of purified Tween-80 further enhances the stability.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical preparation technology, and more specifically to a highly stable quassop injection and its preparation method. Background Technology

[0002] In recent years, with the continuous development of pharmaceutical technology, traditional Chinese medicine (TCM) injections have been widely used in clinical practice due to their convenience, rapid onset of action, and good efficacy. However, the complexity of the components, instability of quality, and insufficient purity of TCM injections often lead to adverse drug reactions (ADRs) during their clinical use. Therefore, stability studies of TCM injections have become an important aspect of ensuring the safe and effective use of these drugs.

[0003] *Quercus acutissima* is an important component of many traditional Chinese medicine preparations, such as *Quercus acutissima* injection, *Quercus acutissima* compound, *Quercus acutissima* tablets, and anti-inflammatory and choleretic tablets. Among these, *Quercus acutissima* injection is extracted and processed from the dried branches or stems of *Quercus acutissima* and is clinically used to treat colds, upper respiratory tract infections, acute tonsillitis, measles, and bacterial infections. However, because existing *Quercus acutissima* injections are processed from extracts, they exhibit instability during storage and have limited efficacy. Furthermore, current *Quercus acutissima* extracts are mostly used to treat specific diseases; whether they can be combined with other traditional Chinese medicine extracts to prepare injections for the treatment of other diseases remains unknown.

[0004] Therefore, how to develop a stable and effective injection solution and its preparation method is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a highly stable quassop injection and its preparation method.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] One embodiment of the present invention provides a *Paspalum notatum* injection with high stability, comprising the following components by mass fraction:

[0008] Basas extract: 1.5%-3.0%;

[0009] Purified Tween-80: 0.5-0.6%;

[0010] Astragalus polysaccharides: 0.02%-0.1%;

[0011] Total saponins of Panax notoginseng: 0.01%-0.08%;

[0012] Glycyrrhizic acid: 0.01-0.02%;

[0013] Stabilizer: 0.1%-0.5%;

[0014] Water for injection: Balance;

[0015] Adjust the pH to 5.8-6.2 using a pH adjuster.

[0016] Preferably, the *Paprika* injection solution comprises the following components in the optimal mass fraction ratio:

[0017] Basswood extract: 2.7%;

[0018] Purified Tween-80: 0.5%;

[0019] Astragalus polysaccharide: 0.05%;

[0020] Total saponins of Panax notoginseng: 0.05%;

[0021] Glycyrrhizic acid: 0.015%;

[0022] Stabilizer: 0.2%;

[0023] Water for injection: Balance;

[0024] Adjust the pH to 6.0 using a pH adjuster.

[0025] Preferably, the preparation process of purified Tween-80 includes: degassing commercially available Tween-80 under reduced pressure at 60~80℃ to remove volatile impurities; then collecting the distillate in a molecular distillation apparatus at a controlled temperature of 120~150℃ and a vacuum degree ≤0.1Pa; the distillate is purified Tween-80 after cooling.

[0026] Preferably, the stabilizer comprises mannitol and sodium citrate in a mass ratio of 2:1.

[0027] Preferably, the pH adjuster is a 1M hydrochloric acid solution or a 2.5M sodium hydroxide solution.

[0028] A second aspect of this invention provides a method for preparing a *Paspalum notatum* injection, which is prepared from the raw materials described above, and the specific process includes:

[0029] Step 1: Prepare the extract of *Paspalum notoginseng*, and then dissolve the extract of *Paspalum notoginseng*, astragalus polysaccharide, glycyrrhizic acid and total saponins of Panax notoginseng in water for injection at 60°C according to the mass fraction ratio to obtain system 1;

[0030] Step 2: Add stabilizer and purified Tween-80 to system 1, stir at 200-300 rpm to dissolve for 0.5-1 h, then add pH adjuster to adjust pH to 5.8-6.2 to obtain system 2;

[0031] Step 3: Filter system 2 through a 0.22μm microporous membrane, fill with nitrogen, and autoclave at 115℃ for 30 minutes to obtain the quassop injection.

[0032] Preferably, the preparation process of the *Paspalum notatum* extract is as follows:

[0033] Step 1: Extract the branches and leaves of the bitter tree by reflux with 70% ethanol three times, each time for 1.5 hours, and combine the extracts;

[0034] Step 2: Concentrate the extract under reduced pressure at -20~-30 MPa for 1-2 hours, pass it through a macroporous resin column AB-8, first elute impurities with 5% ethanol, then elute quassin with 80% ethanol; after concentrating the quassin solution, freeze-dry it at -30~-40℃ for 2 hours to obtain a brownish-yellow powder with a purity ≥90%.

[0035] The third aspect of this invention provides the application of the *Quercus mongolica* injection prepared by the above method in the preparation of heat-clearing and detoxifying drugs, anti-inflammatory drugs, and anti-tumor drugs.

[0036] As can be seen from the above technical solution, the mechanism of action of each component in the injection solution described in this invention is as follows:

[0037] The complex interaction between notoginsenosides and quassin: hydrogen bond complexation: the aglycone hydroxyl group of notoginsenosides forms a hydrogen bond with the ketone group of quassin (FTIR confirmed peak shift to 3420 cm⁻¹). -1 This reduces the aggregation of quassinolide molecules.

[0038] Astragalus polysaccharides provide antioxidant protection: Free radical scavenging: The phenolic hydroxyl groups of astragalus polysaccharides can capture reactive oxygen species that oxidize magnesia, resulting in a decrease in DPPH signal in ESR detection and delaying precipitation formation. The carboxyl groups of the polysaccharides bind to trace amounts of Fe in the formulation, blocking the Fenton reaction.

[0039] Glycyrrhizic acid-quassine charge neutralization: electrostatic shielding: the carboxylate group (-COO⁻) of glycyrrhizic acid and quassine (positively charged) are combined through ionic bonds (Zeta potential decreases), reducing precipitation caused by interparticle repulsion.

[0040] Therefore, this invention significantly improves the stability of the injection solution and keeps its toxicity under control through a triple mechanism of hydrogen bond complexation (Panax notoginseng), antioxidant (Astragalus membranaceus), and charge stabilization (Glycyrrhiza uralensis), thus possessing clinical translation potential; at the same time, the addition of refined Tween-80 further enhances the stability. Detailed Implementation

[0041] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0042] Example 1: *Paspalum notatum* extract: 2.7%;

[0043] Purified Tween-80: 0.5%;

[0044] Astragalus polysaccharide: 0.05%;

[0045] Total saponins of Panax notoginseng: 0.05%;

[0046] Glycyrrhizic acid: 0.015%;

[0047] Mannitol and sodium citrate in a mass ratio of 2:1: 0.2%;

[0048] Water for injection: Balance;

[0049] Adjust the pH to 6.0 with 1M hydrochloric acid solution.

[0050] The preparation process of purified Tween-80 includes: degassing commercially available Tween-80 under reduced pressure at 70°C to remove volatile impurities; then collecting the distillate in a molecular distillation apparatus at a controlled temperature of 130°C and a vacuum degree ≤0.1Pa; the distillate is purified Tween-80 after cooling.

[0051] The specific process includes:

[0052] Step 1: Prepare the extract of *Paspalum notoginseng*, and then dissolve the extract of *Paspalum notoginseng*, astragalus polysaccharide and total saponins of Panax notoginseng in water for injection at 60°C according to the mass fraction ratio to obtain system 1;

[0053] Step 2: Add mannitol, sodium citrate, and purified Tween-80 to system 1. Stir at 200-300 rpm for 0.7 h to dissolve. Then add pH adjuster to adjust pH to 6.0 to obtain system 2.

[0054] Step 3: Filter system 2 through a 0.22μm microporous membrane, fill with nitrogen, and autoclave at 115℃ for 30 minutes to obtain the quassop injection.

[0055] The preparation process of the *Paspalum notatum* extract is as follows:

[0056] Step 1: Extract the branches and leaves of the bitter tree by reflux with 70% ethanol three times, each time for 1.5 hours, and combine the extracts;

[0057] Step 2: Concentrate the extract under reduced pressure at -25 MPa for 1.5 h, pass it through a macroporous resin column AB-8, first elute impurities with 5% ethanol, then elute quassin with 80% ethanol; after concentrating the quassin solution, freeze-dry it at -35℃ for 2 h to obtain a brownish-yellow powder with a purity ≥90%.

[0058] Example 2: A highly stable *Paspalum notatum* injection, comprising the following components by mass fraction:

[0059] Basas extract: 1.5%;

[0060] Purified Tween-80: 0.6%;

[0061] Astragalus polysaccharide: 0.02%;

[0062] Total saponins of Panax notoginseng: 0.01%;

[0063] Glycyrrhizic acid: 0.02%;

[0064] Mannitol and sodium citrate in a mass ratio of 2:1: 0.5%;

[0065] Water for injection: Balance;

[0066] Adjust the pH to 5.8 with 2.5M sodium hydroxide solution.

[0067] The preparation process of purified Tween-80 includes: degassing commercially available Tween-80 under reduced pressure at 80°C to remove volatile impurities; then collecting the distillate in a molecular distillation apparatus at a controlled temperature of 120°C and a vacuum degree ≤0.1Pa; the distillate is purified Tween-80 after cooling.

[0068] The specific preparation process includes:

[0069] Step 1: Prepare the extract of *Paspalum notoginseng*, and then dissolve the extract of *Paspalum notoginseng*, astragalus polysaccharide, glycyrrhizic acid and total saponins of Panax notoginseng in water for injection at 60°C according to the mass fraction ratio to obtain system 1;

[0070] Step 2: Add mannitol, sodium citrate and purified Tween-80 to system 1, stir at 200-300 rpm for 0.5 h to dissolve, then add pH adjuster to adjust pH to 5.8 to obtain system 2;

[0071] Step 3: Filter system 2 through a 0.22μm microporous membrane, fill with nitrogen, and autoclave at 115℃ for 30 minutes to obtain the quassop injection.

[0072] The preparation process of the *Paspalum notatum* extract is as follows:

[0073] Step 1: Extract the branches and leaves of the bitter tree by reflux with 70% ethanol three times, each time for 1.5 hours, and combine the extracts;

[0074] Step 2: Concentrate the extract under reduced pressure at -30 MPa for 2 hours, pass it through a macroporous resin column AB-8, first elute impurities with 5% ethanol, then elute quassin with 80% ethanol; after concentrating the quassin solution, freeze-dry it at -30℃ for 2 hours to obtain a brownish-yellow powder with a purity ≥90%.

[0075] Example 3: *Paspalum notatum* extract: 3.0%;

[0076] Purified Tween-80: 0.5%;

[0077] Astragalus polysaccharide: 0.1%;

[0078] Total saponins of Panax notoginseng: 0.08%;

[0079] Glycyrrhizic acid: 0.01%;

[0080] Mannitol to sodium citrate in a mass ratio of 2:1: 0.1%-0.5%;

[0081] Water for injection: Balance;

[0082] Adjust the pH to 6.2 with 1M hydrochloric acid solution.

[0083] The preparation process of purified Tween-80 includes: degassing commercially available Tween-80 under reduced pressure at 80°C to remove volatile impurities; then collecting the distillate in a molecular distillation apparatus at a controlled temperature of 150°C and a vacuum degree ≤0.1Pa; the distillate is purified Tween-80 after cooling.

[0084] The specific process includes:

[0085] Step 1: Prepare the extract of *Paspalum notoginseng*, and then dissolve the extract of *Paspalum notoginseng*, astragalus polysaccharide, glycyrrhizic acid and total saponins of Panax notoginseng in water for injection at 60°C according to the mass fraction ratio to obtain system 1;

[0086] Step 2: Add stabilizer and purified Tween-80 to system 1, stir at 200-300 rpm for 1 hour to dissolve, then add pH adjuster to adjust pH to 6.2 to obtain system 2;

[0087] Step 3: Filter system 2 through a 0.22μm microporous membrane, fill with nitrogen, and autoclave at 115℃ for 30 minutes to obtain the quassop injection.

[0088] The preparation process of the quassula extract is as follows:

[0089] Step 1: Extract the branches and leaves of the bitter tree by reflux with 70% ethanol three times, each time for 1.5 hours, and combine the extracts;

[0090] Step 2: Concentrate the extract under reduced pressure at -30 MPa for 2 hours, pass it through a macroporous resin column AB-8, first elute impurities with 5% ethanol, then elute quassin with 80% ethanol; after concentrating the quassin solution, freeze-dry it at -40℃ for 2 hours to obtain a brownish-yellow powder with a purity ≥90%.

[0091] The toxicity of the *Quercus mongolica* injections prepared in Examples 1-3 above was verified:

[0092] Acute toxicity test (LD50) 50 (Measurement)

[0093] Animal model: ICR mice (half male and half female, n=60) were randomly divided into 6 groups (dose: 10, 20, 40, 80, 160, 320 mg / kg).

[0094] Methods: Single intravenous injection via tail vein, observation for 14 days, recording the number of deaths and toxic reactions (convulsions, respiratory depression, etc.).

[0095] result:

[0096] LD 50 215.4 mg / kg (95% confidence interval: 198.6–233.7 mg / kg);

[0097] Toxic reactions: Transient shortness of breath occurred at ≥230 mg / kg (recovered within 30 min).

[0098] Long-term toxicity study (repeated dosing over 28 days)

[0099] Animal model: SD rats (n=80), divided into 4 groups (0, 5, 15, 45 mg / kg / d, intravenous injection).

[0100] Testing indicators:

[0101] Hematology: WBC, RBC, platelets;

[0102] Biochemical indicators: ALT, AST, BUN, Cr;

[0103] Histopathology: HE staining of heart, liver, spleen, lungs, and kidneys.

[0104] result:

[0105] Safe dosage: 15 mg / kg / day (no abnormalities observed in HE staining of heart, liver, spleen, lungs, and kidneys; ALT, AST, BUN, and Cr all within normal ranges);

[0106] High-dose group (45 mg / kg): Mild hepatocyte vacuolation (pathological score 1.2±0.3);

[0107] ALT showed a transient increase (day 14: 68.5±5.2 U / L, normal value ≤40 U / L), with the rest being normal.

[0108] Example 4: Based on the established toxicity of the *Paspalum notatum* injection of the present invention, comparative experiments were conducted based on the core principles of the present invention, and the stability and anti-inflammatory activity of each experimental group were evaluated:

[0109] Comparative Example 1: Compared with Example 1, the total saponins of Panax notoginseng were removed, and the mass fraction of Astragalus polysaccharides was increased to 0.1%; the total mass fraction of active ingredients remained unchanged.

[0110] Comparative Example 2: Compared with Example 1, astragalus polysaccharide was removed, and the mass fraction of glycyrrhizic acid was increased to 0.065%; the total mass fraction of active ingredients remained unchanged.

[0111] Comparative Example 3: Compared with Example 1, glycyrrhizic acid was removed, increasing the mass fraction of total saponins of Panax notoginseng to 0.065%; while maintaining the total mass fraction of active ingredients unchanged;

[0112] Comparative Example 4: Compared with Example 1, commercially available Tween-80 was used instead of purified Tween-80;

[0113] Comparative Example 5: β-cyclodextrin was selected as the stabilizer;

[0114] The preparation process described above is the same as in Example 1.

[0115] The precipitation rate, anti-inflammatory activity, and irritation of Examples 1, 1 to 5 were determined:

[0116] Method for determining sedimentation rate:

[0117] 1. Dispense 5 mL of the injection solution into vials and place them in constant temperature incubators at 4℃, 25℃, and 40℃ respectively;

[0118] 2. Take samples monthly, centrifuge at 3000 rpm for 10 min, and dry the precipitate at 105℃ to constant weight;

[0119] 3. Perform three parallel measurements.

[0120] Precipitation rate (%) = (mass of precipitate / total mass of injection solution) × 100; Statistical results after 6 months at 40℃;

[0121] Anti-inflammatory activity (TNF-α):

[0122] 1. RAW264.7 cells were seeded in 96-well plates and stimulated with LPS (1 μg / mL);

[0123] 2. Pretreatment with different 10 μg / mL injection solutions for 2 h;

[0124] 3. ELISA was used to detect the concentration of TNF-α in the cell supernatant.

[0125] Inhibition rate (%) = (1 - experimental group TNF-α / model group TNF-α) × 100;

[0126] Provocative evaluation:

[0127] 1. New Zealand rabbits (n=6) were injected intravenously via the ear vein at a dose of 0.1 mL / kg;

[0128] 2. Observe at 1h, 24h, and 48h after injection: diameter of redness and swelling (mm), vascular permeability (Evans blue exudate).

[0129] 3. According to the Chinese Pharmacopoeia, the score is 0 (no reaction) to 4 (severe necrosis). Stimulation score = ∑ (redness and swelling + exudation + necrosis score) / number of animals.

[0130] Scoring criteria:

[0131] 0-1: Mild; 1-2: Acceptable; 2-4: Significantly irritating.

[0132] The results are shown in Table 1.

[0133] Table 1

[0134]

[0135] As shown in Table 1, the three components of Astragalus polysaccharide, Panax notoginseng saponins, and glycyrrhizic acid in this invention have a synergistic effect, which can significantly improve the stability of the injection, while also increasing the anti-inflammatory effect, eliminating inflammatory factors, and enhancing immunity.

[0136] The various embodiments in the specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0137] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A highly stable *Paspalum notatum* injection solution, characterized in that, According to the mass fraction ratio, it includes the following components: Basas extract: 1.5%-3.0%; Purified Tween-80: 0.5-0.6%; Astragalus polysaccharides: 0.02%-0.1%; Total saponins of Panax notoginseng: 0.01%-0.08%; Glycyrrhizic acid: 0.01-0.02%; Stabilizer: 0.1%-0.5%; Water for injection: Balance; Adjust the pH to 5.8-6.2 using a pH adjuster; The preparation process of the purified Tween-80 includes: degassing commercially available Tween-80 under reduced pressure at 60~80℃ to remove volatile impurities; Then, in a molecular distillation apparatus, the temperature is controlled at 120~150℃ and the vacuum degree is ≤0.1Pa, and the distillate is collected; after cooling, the distillate is the purified Tween-80. The stabilizer comprises mannitol and sodium citrate in a mass ratio of 2:

1.

2. The *Paspalum notatum* injection with high stability according to claim 1, characterized in that, According to the optimal mass fraction ratio, it includes the following components: Basswood extract: 2.7%; Purified Tween-80: 0.5%; Astragalus polysaccharide: 0.05%; Total saponins of Panax notoginseng: 0.05%; Glycyrrhizic acid: 0.015% Stabilizer: 0.2%; Water for injection: Balance; Adjust the pH to 6.0 using a pH adjuster.

3. The *Paspalum notatum* injection with high stability according to claim 1, characterized in that, The pH adjuster is a 1M hydrochloric acid solution or a 2.5M sodium hydroxide solution.

4. A method for preparing the *Paspalum notatum* injection according to any one of claims 1-3, characterized in that, The specific process includes: Step 1: Prepare the extract of *Paspalum notoginseng*, and then dissolve the extract of *Paspalum notoginseng*, astragalus polysaccharide, glycyrrhizic acid and total saponins of Panax notoginseng in water for injection at 60°C according to the mass fraction ratio to obtain system 1; Step 2: Add stabilizer and purified Tween-80 to system 1, stir at 200-300 rpm to dissolve for 0.5-1 h, then add pH adjuster to adjust pH to 5.8-6.2 to obtain system 2; Step 3: Filter system 2 through a 0.22μm microporous membrane, fill with nitrogen, and autoclave at 115℃ for 30 minutes to obtain the quassop injection.

5. The preparation method according to claim 4, characterized in that, The preparation process of the *Paspalum notatum* extract is as follows: Step 1: Extract the branches and leaves of the bitter tree by reflux with 70% ethanol three times, each time for 1.5 hours, and combine the extracts; Step 2: Concentrate the extract under reduced pressure at -20~-30 MPa for 1-2 hours, pass it through a macroporous resin column AB-8, first elute impurities with 5% ethanol, then elute quassin with 80% ethanol; after concentrating the quassin solution, freeze-dry it at -30~-40℃ for 2 hours to obtain a brownish-yellow powder with a purity ≥90%.

6. The use of the *Quercus mongolica* injection prepared by the method of claim 4 or 5 in the preparation of heat-clearing and detoxifying drugs / anti-inflammatory drugs.

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