Uses of Aqueous Extract of *Rhizoctonia solani* in the Preparation of Drugs with Analgesic Effects

Abstract

This invention provides the use of an aqueous extract of *Derris elliptica* (Roxb.) Benth. in the preparation of a medicament with analgesic effects. This invention also provides a pharmaceutical composition with analgesic effects containing the aqueous extract of *Derris elliptica* (Roxb.) Benth. The oral administration of this *Derris elliptica* aqueous extract, extracted from a plant, is natural, environmentally friendly, economical, and readily available. It is used for analgesia and pain relief, exhibiting clear efficacy, safety, stability, and resistance to thermal decomposition. It also provides a long-lasting analgesic effect, offering a new clinical option.

Description

Technical Field

[0001] This invention relates to the use of water extract of *Rhizoctonia solani* in the preparation of drugs with analgesic effects, and belongs to the pharmaceutical field. Background Technology

[0002] Derris elliptica (Roxb.) Benth., a plant belonging to the genus Derris in the legume family, is distributed in India, the Indochina Peninsula, and the Malay Peninsula in Asia. It is also cultivated in Guangdong and Guangxi provinces of my country. In tropical regions, it is grown as a raw material for insecticides. The fresh roots of Derris elliptica and other plants in the genus Derris are crushed in water, and the resulting latex is used for fishing—a practice that has been practiced for centuries.

[0003] There are numerous reports on the use of *Derris fusiforme* in pesticides. For example, Wu Xinzhou's master's thesis, "Study on the Bioactivity and Chemical Constituents of the Aboveground Parts of *Derris fusiforme*", published in Guangxi Normal University in 2012, describes the separation of chemical components from the aboveground parts of *Derris fusiforme* to identify bioactive lead compounds, providing a theoretical basis and scientific evidence for the creation of new plant-derived pesticides and the development and utilization of this plant. The petroleum ether and ethyl acetate extracts of the aboveground parts of *Derris fusiforme* showed significant toxic activity against Aedes albopictus larvae, adult houseflies, and juvenile golden apple snails. The chemical components of these two extracts were separated. Through repeated separation using various methods such as silica gel column chromatography, gel chromatography, macroporous resin chromatography, and thin-layer chromatography, 18 compounds were isolated. Patent application number: CN201310745632.6, invention title: Pesticide Containing Extracts of *Annona squamosa* and *Derris fusiforme*, its Preparation Method and Uses. This patent relates to the field of pesticides, specifically to a pesticide containing extracts of *Annona squamosa* and *Derris fusiforme*, its preparation method and uses. This invention provides a pesticide containing extracts of custard apple and robininus peduncle, the active ingredients of which are extracted from the following raw materials in parts by weight: 1 part custard apple and 1-8 parts robininus peduncle. This pesticide containing extracts of custard apple and robininus peduncle exhibits a synergistic effect, showing significant control efficacy against lepidopteran pests. While providing rapid contact killing, it also improves the plant's immune system and significantly enhances plant vitality.

[0004] Research on rotenone has a long history, but it has mainly focused on its water-insoluble component, rotenone. Rotenone is an organic compound with the molecular formula C64. 23 H 22O6. Found in the roots of leguminous plants (Derris genus) in tropical and subtropical Asia, and also in some traditional Chinese medicines such as sweet potato seeds, bitter sandalwood, and *Spatholobus suberectus* roots. It is almost insoluble in water, but soluble in ethanol, acetone, carbon tetrachloride, chloroform, ether, and many other organic solvents. It decomposes upon exposure to light and air. Rotenone has been extensively reported for its use as a fish poison, insecticide, and in biosynthesis. Rotenone accumulates most abundantly in the roots, with higher concentrations in coarse roots than in fibrous roots, and higher in the xylem than in the phloem. Quantitative determination of rotenone content by high-performance liquid chromatography showed that *Derris hairyana* had the highest rotenone content (14.12%), higher than 13 other *Derris* species including *Derris hairyana*, *Derris macrocarpa*, and *Derris truncatula*. CN201010554904.0, Invention Title: Method for Increasing Rotenone Content in Rotenone Stems. This patent describes a method of treating 2-3 year old Rotenone stems with 50-200 mg / L naphthaleneacetic acid, followed by sand burial for 20-30 days. Using this method, the secondary metabolic system of Rotenone stems can be regulated, increasing the rotenone content by 10-20 times, thus significantly improving the rotenone content in Rotenone stems.

[0005] There are also reports of rotenone being used in pharmaceuticals, such as: Application No. 201710924467.9, Invention Title: Use of Rotenone in the Preparation of Drugs for Treating Acute Kidney Injury, The use of rotenone in the preparation of drugs for treating acute kidney injury, After treatment with a small dose (250ppm) of rotenone, the kidney damage in mice was significantly improved, and the serum urea nitrogen (BUN) and creatinine (Cr) levels were significantly reduced. Zhao Chunjing et al., Anti-inflammatory and Analgesic Effects of Rotenone Liniment, Journal of the Third Military Medical University, April 1998, disclosed that rotenone has a significant anti-inflammatory effect on inflammation induced by croton oil, formaldehyde, and carrageenan, and has a significant analgesic effect on pain induced by heat stimulation and formaldehyde in mice. Due to the high toxicity of rotenone, accidental ingestion can lead to poisoning. It has an irritant effect on the eyes and skin. Acute poisoning can cause nausea, vomiting, stomach pain, diarrhea, convulsions, and tremors. It has a damaging effect on the liver and kidneys. Moreover, rotenone is flammable, toxic, and irritating. It can produce highly toxic phosgene when in contact with open flames or hot objects.

[0006] There are currently no reports on the use of dermal aralia water extract for pain relief or analgesia. Summary of the Invention

[0007] The technical solution of this invention provides a new use for the aqueous extract of *Rhizoctonia solani*. Another technical method of this invention provides a pharmaceutical composition with analgesic effects.

[0008] This invention provides the use of an aqueous extract of Derris elliptica (Roxb.) Benth. in the preparation of a drug with analgesic effects.

[0009] The aforementioned *Derris elliptica* comes from the roots or stems of *Derris elliptica* (Roxb.) Benth., a plant belonging to the genus *Derris* of the legume family.

[0010] The water extract of *Dendrobium nobile* is either a decoction extract or an ultrasound-assisted water extract.

[0011] The drug is an oral preparation made by adding pharmaceutically acceptable excipients or auxiliary ingredients to a water extract of *Derris fusiforme* as the active ingredient.

[0012] The oral preparations mentioned above are tablets, capsules, granules, pills, and oral liquids.

[0013] The conditions for ultrasonic-assisted water extraction are: ultrasonic frequency 40±2kHz and power 720-900W.

[0014] More preferably, the method for ultrasound-assisted water extraction is as follows:

[0015] a. Take the dried roots and stems of the hairy fish plant and crush them into coarse powder;

[0016] b. Ultrasonic-assisted water extraction was used, with the following parameters: a medium ultrasonic frequency of 40±2kHz; a power of 900W; a processing time of 2 hours; and a temperature controlled below 20℃. After extraction, the mixture was left to stand overnight.

[0017] c. Centrifugation: Centrifuge at 3000 rpm for 15 minutes, take the supernatant and filter to obtain the water extract of *Gynostemma pentaphyllum* of this invention.

[0018] The water decoction extraction method is as follows:

[0019] a. Take the dried roots and stems of the hairy fish plant and crush them into coarse powder;

[0020] b. Soak the ingredients in 25 to 50 times their weight of distilled water and stir continuously on a magnetic stirrer for 60 to 90 minutes; heat the water at 80 to 100°C and continue decocting for 60 to 90 minutes; after decocting, cool and centrifuge to obtain the water extract of *Gnaphalium affine* of this invention.

[0021] The centrifuge was set to centrifuge at 3000 rpm for 15 minutes.

[0022] The isocratic elution HPLC chromatogram of the ultrasound-assisted water extract contains four characteristic peaks, with the retention time, peak height, and peak area of ​​each peak being:

[0023] The retention time of the second peak was 2.312 minutes, the peak height was 625.503 mAU, and the peak area was 5910.828 mAU.s.

[0024] The retention time of the third peak was 2.467 minutes, the peak height was 1058.993 mAU, and the peak area was 11470.414 mAU.s.

[0025] The retention time of the fourth peak was 2.808 minutes, the peak height was 268.572 mAU, and the peak area was 4485.034 mAU.s.

[0026] The retention time of the ninth peak was 6.956 minutes, the peak height was 166.365 mAU, and the peak area was 1773.271 mAU.s.

[0027] The isocratic elution HPLC chromatogram of the water decoction extract contains four characteristic peaks, with the following retention times, peak heights, and peak areas:

[0028] The retention time of the first peak was 1.644 minutes, the peak height was 475.435 mAU, and the peak area was 3218.328 mAU.s;

[0029] The retention time of the second peak was 2.318 minutes, the peak height was 981.870 mAU, and the peak area was 8771.686 mAU.s;

[0030] The retention time of the third peak was 2.480 minutes, the peak height was 1172.479 mAU, and the peak area was 13846.608 mAU.s;

[0031] The retention time of the twelfth peak was 6.954 minutes, the peak height was 350.738 mAU, and the peak area was 3820.238 mAU.s;

[0032] The relative retention time fluctuated within ±0.05 minutes; the peak height fluctuated within ±5%; and the peak area fluctuated within ±10%.

[0033] The chromatographic conditions were as follows: column: reversed-phase C18, size 250 mm x 4.6 mm, packed material particle size 5 μm; in isocratic elution mode, the mobile phase consisted of HPLC-grade distilled water (A) and HPLC-grade methanol (B) in a volume ratio of 20% A and 80% B, the flow rate was set to 1 mL / min, the detection wavelength was 214 nm, and the injection volume was 20 μL.

[0034] The gradient elution HPLC chromatogram of the ultrasound-assisted water extract contains four characteristic peaks, with the following retention times, peak heights, and peak areas:

[0035] The second peak: retention time 3.310 minutes, peak height 675.796 mAU, peak area 5514.894 mAU.s;

[0036] The third peak: retention time 3.515 minutes, peak height 112.733 mAU, peak area 1092.375 mAU.s;

[0037] The thirteenth peak: retention time 37.678 minutes, peak height 321.401 mAU, peak area 15557.904 mAU.s;

[0038] The fourteenth peak: retention time 40.904 minutes, peak height 82.399 mAU, peak area 2728.250 mAU.s;

[0039] The gradient elution HPLC chromatogram of the water decoction extract contains four characteristic peaks, with the following retention times, peak heights, and peak areas:

[0040] First peak: retention time 2.880 minutes, peak height 623.523 mAU, peak area 4239.315 mAU.s;

[0041] The second peak: retention time 3.037 minutes, peak height 120.289 mAU, peak area 696.857 mAU.s;

[0042] The twelfth peak: retention time 41.963 minutes, peak height 527.401 mAU, peak area 11362.166 mAU.s;

[0043] The thirteenth peak: retention time 43.833 minutes, peak height 127.460 mAU, peak area 2269.173 mAU.s;

[0044] The relative retention time fluctuated within ±0.05 minutes; the peak height fluctuated within ±5%; and the peak area fluctuated within ±10%.

[0045] The chromatographic conditions were as follows: column: reversed-phase AQ-C18, size 250mm x 4.6mm, packed material particle size 5μm; mobile phase: HPLC grade methanol (A) and HPLC grade distilled water (B); gradient elution mode, volume ratio: 0–20 min, 40% A → 60% A; 20–30 min, 60% A; 30–45 min, 60% A → 80% A; 45–60 min, 80% A; flow rate: 1 mL / min; detection wavelength: 296 nm; injection volume: 10 μL.

[0046] The present invention provides a pharmaceutical composition with analgesic effect, which contains an aqueous extract of Derris elliptica (Roxb.) Benth.

[0047] The pharmaceutical composition is prepared from the aqueous extract of Derris elliptica (Roxb.) Benth., with the addition of pharmaceutically acceptable excipients or auxiliary ingredients, into a commonly used oral formulation.

[0048] This invention relates to an oral administration of the aqueous extract of Derris elliptica (Roxb.) Benth. for analgesia and pain relief. The extract is effective, safe, stable, and does not easily decompose when heated. It is extracted from a plant, making it natural, environmentally friendly, economical, easy to obtain, and convenient to extract, thus providing a new option for clinical use. Attached Figure Description

[0049] Figure 1 Chromatogram of ultrasonic-assisted aqueous extract of *Derris fusiforme* (30 g / L) eluted with high performance liquid chromatography (HPLC);

[0050] Figure 2 Chromatogram of 20 g / L isocratic elution high performance liquid chromatography (HPLC) of decoction of *Rhizoctonia solani*.

[0051] Figure 3 Chromatogram of ultrasonic-assisted aqueous extract of *Derris fusiforme* (30 g / L gradient elution) by high-performance liquid chromatography;

[0052] Figure 4 Chromatogram of 20 g / L water decoction of *Rhizophora stylosa* using high performance liquid chromatography with gradient elution;

[0053] Figure 5 Chromatogram of rotenone eluted by high performance liquid chromatography;

[0054] Figure 6 Comparative test of ultrasound-assisted supernatant and sedimentation of *Derris fusiforme* supernatant and sedimentation solution: Pain threshold (seconds) in each group of mice under hot plate test before drug administration (* indicates comparison with the model group, * means P < 0.05, ** means P < 0.05).

[0055] <0.01, *** means P<0.001 (the same applies below);

[0056] Figure 7 Comparative test of ultrasound-assisted supernatant and sediment of *Derris fusiforme*, pain threshold (seconds) in each group of mice under hot plate test 2 hours after drug administration;

[0057] Figure 8 : Comparative experiment of ultrasound-assisted supernatant and sedimentation of *Derris fusiforme*: Number of writhing movements in mice in each group during a 30-minute observation period 2 hours after drug administration;

[0058] Figure 9 Ultrasound-assisted comparison of supernatant and sediment of *Derris fusiforme*: Time (seconds) at which mice in each group began to writhe 2 hours after drug administration;

[0059] Figure 10Pain threshold (seconds) in mice under hot plate test before administration of ultrasound-assisted aqueous extract in each group;

[0060] Figure 11 Pain threshold (seconds) in mice under hot plate test 2 hours after administration of ultrasound-assisted aqueous extract;

[0061] Figure 12 The number of writhing movements in mice in each group during a 30-minute observation period 2 hours after administration of ultrasound-assisted aqueous extract;

[0062] Figure 13 Time (in seconds) for mice in each group to begin writhing 2 hours after administration of ultrasound-assisted aqueous extract;

[0063] Figure 14 Pain threshold (seconds) in each group of mice under hot plate test before administration of decoction;

[0064] Figure 15 Pain threshold (seconds) in mice under hot plate test 2 hours after administration of decoction;

[0065] Figure 16 The number of writhing movements in mice in each group during a 30-minute observation period 2 hours after administration of the decoction;

[0066] Figure 17 Time (in seconds) for mice in each group to begin writhing 2 hours after administration of the decoction. Detailed Implementation

[0067] Example 1: Preparation of the ultrasonic-assisted water extract of *Derris fusiforme* according to the present invention

[0068] The dried roots and stems of the hairy vine plant are cleaned under high pressure with purified water, dried in an environment, cut into suitable sizes, processed by a pulverizer, and passed through a 100-mesh sieve to obtain coarse powder.

[0069] 0.5g of the extract was precisely weighed on an analytical balance and added to a 250ml sterile flask. It was then dissolved in 50ml of distilled water to prepare a 10g / L suspension. Next, an ultrasound-assisted water extraction method was used, with parameters including a moderate ultrasonic frequency of 40±2kHz, a power of 900W, a processing time of 2 hours, and a temperature controlled below 20℃. The following day, the suspension, which had been soaked overnight at 22℃, was centrifuged at 3000rpm for 15 minutes. The supernatant was then filtered to remove residual particles, yielding a clear 10g / L (based on the original medicinal material) water extract. This supernatant is the *Rottosporum tobira* water extract of this invention.

[0070] Example 2: Preparation of the decoction extract of *Derris fusiforme* according to the present invention

[0071] The cleaned *Dendrobium nobile* plant material was pulverized and sieved through a 100-mesh sieve. 2g of the pulverized material was then precisely weighed using an analytical balance and added to 100ml of distilled water to prepare a 20g / L suspension. This suspension was placed on a magnetic stirrer and stirred continuously for 60 minutes to promote component dissolution. Next, a heating mantle was used, with a condenser connected to recover steam, and the water temperature was controlled at 80-100℃ for another 60 minutes. After decoction, the suspension was cooled to room temperature, centrifuged at 3000rpm for 15 minutes, and then filtered to remove insoluble impurities, ensuring the purity of the decoction. Finally, the resulting decoction was refrigerated for storage.

[0072] Example 3: Quality Detection of the Ultrasonic-Assisted Water Extract of the present invention, *Derris fusiforme*

[0073] The supernatant from the preparation of *Rotenone spp.* in Example 1, along with purchased rotenone monomer (glpbio, 5g*bottle), was dissolved in methanol. A high-performance liquid chromatography (HPLC) system was used, equipped with a reversed-phase C18 column (250mm length, 5μm particle size) with an inner diameter of 4.6mm. In isocratic elution mode, the mobile phase consisted of HPLC-grade distilled water (A) and HPLC-grade methanol (B), with a volume ratio of 20% A and 80% B. The flow rate was set to 1mL / min, and detection was performed at 214nm using a UV detector. The injection volume was 20μL, and the sample was pre-filtered through a 0.22μm filter. The HPLC system was pre-run until the baseline stabilized before analysis. All operations were performed at room temperature (approximately 25°C).

[0074] Test results

[0075] This analysis used an Elite HPLC 3200 system for sample testing. The chromatographic column was a reversed-phase C18, 250 mm x 4.6 mm, with a packed particle size of 5 μm. In isocratic elution mode, the mobile phase consisted of HPLC-grade distilled water (A) and HPLC-grade methanol (B), with a volume ratio of 20% A and 80% B, and a flow rate of 1 mL / min. Detection was performed at 214 nm using a UV detector. The injection volume was 20 μL, and the sample was pre-filtered through a 0.22 μm filter. The HPLC system was pre-run until the baseline stabilized before analysis. All operations were performed at room temperature (approximately 25 °C).

[0076] The resulting chromatogram showed good separation, with symmetrical peak shapes for each component (see...). Figure 1Under the experimental conditions, in the isocratic elution high-performance liquid chromatography (HPLC) analysis of the ultrasonic-assisted aqueous extract of *Rottosporum tobira*, we observed four main chromatographic peaks. The specific parameters of each peak are as follows: the retention time of the second peak was 2.312 min, peak height: 625.503 mAU, and peak area: 5910.828 mAU·s; the retention time of the third peak was 2.467 min, peak height: 1058.993 mAU, and peak area: 11470.414 mAU·s; the retention time of the fourth peak was 2.808 min, peak height: 268.572 mAU, and peak area: 4485.034 mAU·s; and the retention time of the ninth peak was 6.956 min, peak height: 166.365 mAU, and peak area: 1773.271 mAU·s. Although these four peaks were not completely separated, we could still infer the possible components by their retention times and peak areas. To further resolve and confirm the specific compounds represented by these peaks, it may be necessary to optimize chromatographic conditions (such as changing the composition of the mobile phase, column temperature, or using a more selective column), or employ other supplementary separation techniques, such as mass spectrometry (MS). Current HPLC analysis suggests that the sample may contain four distinct components that cannot be completely separated under the current chromatographic conditions. Future work will focus on improving resolution to more accurately quantify and identify these components.

[0077] Isocratic elution high-performance liquid chromatography (HPLC) chromatogram of the decoction extract of *Rhizophora stylosa* (see [link]). Figure 2 The sample contains four characteristic peaks, with the following retention times, peak heights, and peak areas: the first peak has a retention time of 1.644 minutes, a peak height of 475.435 mAU, and a peak area of ​​3218.328 mAU.s; the second peak has a retention time of 2.318 minutes, a peak height of 981.870 mAU, and a peak area of ​​8771.686 mAU.s; the third peak has a retention time of 2.480 minutes, a peak height of 1172.479 mAU, and a peak area of ​​13846.608 mAU.s; and the twelfth peak has a retention time of 6.954 minutes, a peak height of 350.738 mAU, and a peak area of ​​3820.238 mAU.s.

[0078] HPLC analysis results of rotenone (see...) Figure 5 It was detected at 6.259 minutes, forming a region with a height of 3992.059 mAU and an area of ​​66353.048 mAU.s.

[0079] In summary, the peak time of the ultrasonic-assisted aqueous extract of *Rotenone oleracea* is earlier than that of the rotenone monomer, proving that the ultrasonic-assisted aqueous extract of *Rotenone oleracea* contains active components other than rotenone.

[0080] Alternatively, change the chromatographic column, gradient elution mode, and wavelength, and separate the various peaks.

[0081] The analysis was performed using an Elite HPLC 3200 system. The chromatographic column was a reverse-phase AQ-C18, 250 mm x 4.6 mm, with a packed particle size of 5 μm. The mobile phase consisted of HPLC-grade methanol (A) and HPLC-grade distilled water (B), in gradient elution mode with the following volume ratios: 0–20 min, 40% A → 60% A; 20–30 min, 60% A; 30–45 min, 60% A → 80% A; 45–60 min, 80% A). The flow rate was set to 1 mL / min, and the detection wavelength was 296 nm. The injection volume was 10 μL. Samples were pre-filtered through a 0.45 μm filter. The HPLC system was pre-run until the baseline stabilized before analysis. All operations were performed at room temperature (approximately 25 °C).

[0082] In gradient elution high-performance liquid chromatography (HPLC) analysis of the ultrasonic-assisted aqueous extract of *Rhizoctonia solani*, we observed four main chromatographic peaks (see...). Figure 3 The specific parameters for each peak are as follows:

[0083] The second peak: retention time 3.310 minutes, peak height 675.796 mAU, peak area 5514.894 mAU.s;

[0084] The third peak: retention time 3.515 minutes, peak height 112.733 mAU, peak area 1092.375 mAU.s;

[0085] The thirteenth peak: retention time 37.678 minutes, peak height 321.401 mAU, peak area 15557.904 mAU.s;

[0086] The fourteenth peak: retention time 40.904 minutes, peak height 82.399 mAU, peak area 2728.250 mAU.s;

[0087] Future work will focus on improving the separation rate in order to more accurately quantify and identify these components.

[0088] Gradient elution high-performance liquid chromatography (HPLC) chromatogram of rotenone decoction extract (see...) Figure 4 It contains four characteristic peaks, and the retention time, peak height, and peak area of ​​the characteristic peaks are as follows:

[0089] First peak: retention time 2.880 minutes, peak height 623.523 mAU, peak area 4239.315 mAU.s;

[0090] The second peak: retention time 3.037 minutes, peak height 120.289 mAU, peak area 696.857 mAU.s;

[0091] The twelfth peak: retention time 41.963 minutes, peak height 527.401 mAU, peak area 11362.166 mAU.s;

[0092] The thirteenth peak: retention time 43.833 minutes, peak height 127.460 mAU, peak area 2269.173 mAU.s;

[0093] The relative retention time of the above spectra fluctuated within ±0.05 minutes; the peak height fluctuated within ±5%; and the peak area fluctuated within ±10%.

[0094] Separation can be achieved by changing the chromatographic column and detection conditions.

[0095] The following pharmacodynamic experiments demonstrate the beneficial effects of this invention.

[0096] Experimental Example 1: Experiment on the analgesic effect of the drug of the present invention

[0097] I. Experimental Methods:

[0098] Experimental group: The precipitate after centrifugation in Example 1 was mixed with 50 ml of water, which was the same volume as the initial suspension preparation, to prepare a suspension, referred to as the sediment.

[0099] 1. Hot plate test for comparing supernatant and sediment with ultrasound assistance.

[0100] One hundred and twenty healthy female Kunming mice (18-22g) were divided into four groups (n=30). To ensure the accuracy and animal welfare of the oral gavage drug administration experiment, stainless steel gavage needles of appropriate size and thoroughly cleaned and sterilized were used before the experiment. When restraining the mice, trained researchers used a gentle and stable technique, gently pinching the skin on their necks with their left hand to stabilize their tails, ensuring the mice remained stable, their limbs were not compressed, and they could breathe freely. The laboratory environment was controlled under suitable temperature and soft lighting conditions to reduce stress in the mice. During drug administration, the operator carefully inserted the gavage needle along the dorsal side of the mouth into the esophagus, avoiding accidental aspiration into the trachea, and slowly pushed the drug in, then carefully removed the gavage needle. The mice's response was monitored throughout the process; if adverse reactions such as respiratory distress or convulsions occurred, emergency procedures were immediately implemented and humane treatment was provided. Mice were administered 0.9% saline (Saline, 10 mL / kg, normal control group), 0.9% saline (Saline, 10 mL / kg, model control group), *Derris elliptica* (8 g / L, supernatant of experimental group (prepared in Example 1)), and *Derris elliptica* (8 g / L, sediment of experimental group) by gavage, respectively. Before the experiment, mice were allowed to acclimatize at room temperature (22°C) for 30 minutes and on an unheated hot plate for 15 minutes. The hot plate was cleaned with 0.5% acetic acid solution to minimize odor interference between mice. During the formal experiment, the hot plate temperature was adjusted to 55 ± 0.5°C. Pain screening was first performed: the time it took for a mouse to lick its hind paw, lift its hind paw, or jump for the first time was recorded as the heat pain latency (seconds), i.e., the heat pain threshold. Mice with a threshold less than 5 seconds or greater than 30 seconds were excluded, and sufficient mice were ensured in each group. Two hours after the last administration, the heat pain threshold of each group was measured again. If the mice do not respond, the test duration should be no more than 60 seconds to prevent burns to the hind paws. The responses of the control group mice after gavage should also be observed and recorded. The pain inhibition rate % is calculated as follows: (average baseline pain threshold after administration - average average pain threshold before administration) / average baseline pain threshold before administration × 100%

[0101] 2. Acetic acid-induced writhing test comparing supernatant and sediment with ultrasound assistance.

[0102] Forty healthy female Kunming mice (18-22g) were divided into four groups (n=10). Each animal was acclimatized to room temperature (22℃) for 30 minutes before gavage administration. The mice were administered the following medications by gavage: 0.9% saline (Saline, 10mL / kg, normal control group), 0.9% saline (Saline, 10mL / kg, model control group), *Derris elliptica* (8g / L, experimental group supernatant (prepared in Example 1)), and *Derris elliptica* (8g / L, experimental group sediment). Before each experiment, the 0.5% acetic acid solution was confirmed to be freshly prepared. Furthermore, prior to the formal experiments, additional mice were used to verify the activity of the solution, ensuring that it induced a typical writhing response within a short time (less than 3 minutes), thus guaranteeing the scientific rigor and scientific validity of the experiment. Two hours after the last administration, intraperitoneal injection was performed according to standardized procedures: a sterile 1 ml syringe and a needle of appropriate specification were used, operated by trained technicians; mice were gently restrained in a stress-reduced environment with their heads down; the injection site was located at the junction of the left groin and abdominal cavity, with the needle inserted slowly at a 45-degree angle, the depth precisely controlled; the fluid was injected at a moderate rate, and the needle was smoothly withdrawn after injection; if bleeding or subcutaneous hematoma was observed at the injection site, the relevant mice were culled according to animal welfare standards. After injection, each mouse was placed in a separate cage and numbered, and observers recorded the number of writhing movements within 30 minutes after administration and the time of the first writhing movement under blinded conditions. The pain inhibition rate was calculated as follows: Pain inhibition rate % = [1 - Mean of treatment group / Mean of control group] × 100%.

[0103] 3. Ultrasonic-assisted hot plate experiment of water extraction solution in each group

[0104] Seventy healthy female Kunming mice (18-22g) were randomly divided into seven groups (n=10). Each group was administered the following medications by gavage: 0.9% saline (Saline, 10mL / kg, normal control group), 0.9% saline (Saline, 10mL / kg, model control group), *Derris elliptica* (10, 20, 30, 40g / L, experimental group (prepared in Example 1)), or tramadol (Tramadol, 40mg / kg, positive control group). The experimental methods were the same as in Example 1. Two hours after the last administration, the pain threshold of each mouse was measured. The response of the normal control group after gavage was observed, while the other groups underwent a hot plate test, and the thermal pain threshold was recorded to calculate the pain inhibition rate.

[0105] 4. Ultrasonic-assisted acetic acid writhing test in each group

[0106] Seventy healthy female Kunming mice (18-22g) were divided into seven groups and administered the following medications by gavage: 0.9% saline (Saline, 10mL / kg, normal control group), 0.9% saline (Saline, 10mL / kg, model control group), Derris elliptica (10, 20, 30, 40g / L, experimental group (prepared in Example 1)), and Tramadol (Tramadol, 40mg / kg, positive control group). The experimental method was the same as in Example 2. Two hours after the last administration, the number of writhing movements within 30 minutes after administration and the time of the first writhing movement were recorded under blinded conditions, and the pain inhibition rate was calculated.

[0107] 5. Hot plate experiment with water decoction

[0108] Forty healthy female Kunming mice (18-22g) were divided into four groups and administered the following medications by gavage: 0.9% saline (Saline, 10mL / kg, normal control group), 0.9% saline (Saline, 10mL / kg, model control group), and *Derris elliptica* (20, 40g / L, decoction experimental group, prepared in Example 2). The experimental methods were the same as in Example 1. Two hours after the last administration, the pain threshold of each mouse was measured. The response of mice in the normal control group was observed after gavage, while the other groups underwent a hot plate test, and the thermal pain threshold was recorded to calculate the pain inhibition rate.

[0109] 6. Acetic acid writhing test in water decoction

[0110] Forty healthy female Kunming mice (18-22g each) were divided into four groups and administered the following medications by gavage: 0.9% saline (Saline, 10mL / kg, normal control group), 0.9% saline (Saline, 10mL / kg, model control group), and *Derris elliptica* (20, 40g / L, decoction experimental group, prepared in Example 2). The experimental methods were the same as in Example 2. Two hours after the last administration, the response of the mice in the normal control group was observed. Under blinded conditions, the number of writhing movements within 30 minutes after administration and the time of the first writhing movement were recorded, and the pain inhibition rate was calculated.

[0111] 7. Statistical methods

[0112] All experimental data are expressed as mean ± standard deviation (mean ± SD). Graphpad Prism 8.0 software was used for plotting, and SPSS 26.0 software was used for data processing. One-way ANOVA or repeated measures ANOVA was used to compare the means between groups, depending on the data type. P < 0.05 was considered statistically significant.

[0113] II. Experimental Results

[0114] 1. Two hours after gavage administration of the supernatant of the ultrasonic-assisted aqueous extract of *Derris fusiforme*, the pain threshold in mice was significantly prolonged.

[0115] No deaths occurred in the normal control group after gavage. The pain thresholds of the other groups were as follows: Figure 6 , Figure 7 As shown, before administration, compared with the model group, the pain threshold of mice in each administration group was within 5–30 seconds, with no significant difference among the groups (P>0.05); 2 hours after the last administration, compared with the model group, the pain threshold of mice in each group of *Dendrobium nobile* was significantly prolonged (P<0.05). Gavage administration of 8 g / L of *Dendrobium nobile* supernatant for 2 hours significantly prolonged the pain threshold of mice, and the pain inhibition rate of the supernatant (34.47%) was superior to that of the sediment (26.45%).

[0116] 2. The supernatant of the ultrasonic-assisted aqueous extract of *Derris fusiforme* significantly reduced the number of acetic acid-induced writhing movements and prolonged the time to the onset of writhing in mice 2 hours after administration.

[0117] like Figure 8 , Figure 9 As shown, compared with the model control group, mice in the normal control group showed no writhing response after injection of physiological saline (P<0.001), and the time to the first writhing was longer than the observation time. The number of writhing episodes in both the supernatant and sediment of *Dendrobium nobile* was significantly reduced (P<0.05), and the time to the onset of writhing was significantly prolonged (P<0.05). In the 8 g / L group, both the supernatant and sediment of *Dendrobium nobile* significantly reduced the number of acetic acid writhing episodes and prolonged the time to the onset of writhing in mice 2 hours after administration. This demonstrates that the ultrasonic-assisted aqueous extract of *Dendrobium nobile* has an analgesic effect.

[0118] 3. The ultrasonic-assisted aqueous extract of *Dendrobium nobile* significantly prolonged the pain threshold in mice 2 hours after administration.

[0119] No deaths occurred in the normal control group after gavage. The pain thresholds of the other groups were as follows: Figure 10 , Figure 11 As shown, before administration, compared with the model group, the pain threshold of mice in each administration group was within 5-30 seconds, with no significant difference among the groups (P>0.05); 2 hours after the last administration, compared with the model group, the pain threshold of mice in each group of *Dendrobium nobile* was significantly prolonged (P<0.05). The ultrasound-assisted aqueous extract of *Dendrobium nobile* significantly prolonged the pain threshold of mice 2 hours after administration, with the pain inhibition rate of the 20 g / L group (35.55%) being closest to that of the tramadol group (40.11%).

[0120] 4. Two hours after administration of the ultrasonic-assisted aqueous extract of *Rottosporum tobira*, all groups showed a significant reduction in the number of acetic acid-induced writhing movements and a prolonged time to the onset of writhing in mice.

[0121] like Figure 12 , Figure 13As shown, compared with the model control group, the normal control group mice showed no writhing response after injection of physiological saline (P<0.001), and the time for the first writhing was longer than the observation time. The number of writhing episodes in all groups of *Dendrobium nobile* was significantly reduced (P<0.05), and the time to the start of writhing was significantly prolonged in all groups (P<0.05). There was no statistically significant difference in the number of writhing episodes in the tramadol groups (P>0.05), but the writhing time was prolonged (P<0.05). Two hours after administration, the ultrasonic-assisted aqueous extract of *Dendrobium nobile* significantly reduced the number of acetic acid writhing episodes and prolonged the time to the start of writhing in mice. The 10 g / L group showed the best effect, with a pain inhibition rate of 65.26%, which was superior to the tramadol group (23.56%).

[0122] 5. Two hours after oral administration of the decoction of *Dendrobium nobile*, the pain threshold in mice was significantly prolonged.

[0123] No deaths occurred in the normal control group after gavage. The pain thresholds of the other groups were as follows: Figure 14 , 15 As shown, before administration, compared with the model group, the pain threshold of mice in each administration group was within 5-30 seconds, with no significant difference among the groups (P>0.05); 2 hours after the last administration, compared with the model group, the pain threshold of mice in each group of *Dendrobium nobile* was significantly prolonged (P<0.05). Gavage administration of 40 g / L of *Dendrobium nobile* supernatant for 2 hours significantly prolonged the pain threshold of mice, and the pain inhibition rate at 40 g / L (99.43%) was better than that at 20 g / L (90.92%), and both pain inhibition rates were superior to those of the ultrasound-assisted extraction method.

[0124] 6. Two hours after oral administration of the decoction of *Dendrobium nobile*, the number of writhing movements in mice induced by acetic acid was significantly reduced, and the time to the onset of writhing was prolonged.

[0125] like Figure 16 , Figure 17 As shown, compared with the model control group, the normal control group mice showed no writhing response after injection of physiological saline (P<0.001), and the time to the first writhing was longer than the observation time. The number of writhing episodes in all groups of *Dendrobium nobile* was significantly reduced (P<0.05), and the 40 g / L group significantly prolonged the time to the start of writhing (P<0.05). Two hours after administration, the aqueous decoction of *Dendrobium nobile* significantly reduced the number of acetic acid-induced writhing episodes and prolonged the time to the start of writhing in mice. The pain inhibition rates in the 20 g / L and 40 g / L groups were 57.00% and 56.28%, respectively.

[0126] 7. Statistical table of experimental results for each group of *Derris fusiforme*

[0127] Table 1: Comparison of supernatant and sediment from ultrasound-assisted aqueous extract of *Derris fusiforme* (prepared in Example 1) in mouse hot plate test and acetic acid writhing test.

[0128]

[0129] Table 2: Statistical data of mouse hot plate test and acetic acid writhing test of ultrasonic-assisted aqueous extract of *Derris fusiforme* (prepared in Example 1)

[0130]

[0131] Table 3: Statistical data of mouse hot plate test and acetic acid writhing test using the aqueous decoction of *Rotala rotundifolia* (prepared in Example 2).

[0132]

[0133] Analysis of the pain inhibition rate showed that the pain inhibition rate of the ultrasound-assisted aqueous extract was lower than that of the decoction, indicating that the decoction had a more significant advantage in analgesia.

[0134] III. Summary

[0135] 1. Water extracts of *Derris fusiforme* contain water-soluble analgesic substances.

[0136] Rotenone topical application has a good analgesic effect. Rotenone is insoluble in water. This experiment investigated the analgesic effect of the aqueous extract of *Rotenoptera litura* (hair vine) by examining components excluding rotenone. In the hot plate test, *Rotenoptera litura* at various dosages prolonged the pain threshold in mice. In the acetic acid writhing test, various dosages prolonged the time to writhing and reduced the number of writhing movements. High-performance liquid chromatography (HPLC) analysis confirmed that the components in the aqueous extract were not rotenone. This verifies the analgesic effect of the water-soluble compounds in *Rotenoptera litura*.

[0137] 2. Oral analgesic activity of water extract of *Rhizoctonia solani*

[0138] Rotenone topical application has been reported for its analgesic effects, but no other routes of administration have been reported, nor has the analgesic effect of the whole plant of *Rotenoptera hainanensis* been reported. This experiment verifies the oral analgesic effect of *Rotenoptera hainanensis* water extract, which is innovative and has good application prospects.

[0139] 3. Long-acting oral analgesic activity of roe drupe aqueous extract

[0140] This experiment verified the oral analgesic effect of the aqueous extract of *Dendrobium nobile*. It exerted an analgesic effect 2 hours after administration in all dose groups. In the ultrasound-assisted acetic acid writhing test of the aqueous extract, its efficacy was superior to that of tramadol, the positive control drug, and it could significantly improve the pain threshold rate, showing good development prospects.

[0141] Experimental Example 2: Acute toxicity test of the ultrasonic-assisted aqueous extract of the present invention, *Derris fusiforme*.

[0142] Four healthy Kunming mice (18-22g each time) were administered the extract via gavage at a concentration gradient ten times that of the previously described treatment. Mice were observed for mortality within 14 days; if mortality occurred, the concentration was reduced. This preliminary investigation aimed to explore the acute toxicity of the ultrasonic-assisted aqueous extract of *Rotenone oleracea* in mice. No acute deaths were observed in any experimental group. One mouse died within 14 days in the 100g / L group, equivalent to a body weight ratio of 1000mg / kg, administered according to body weight. The safety profile of this extract is higher than that of rotenone (LD50: 2.8mg / kg), and this extraction method successfully reduced the drug's toxicity.

Claims

1. Use of an aqueous extract of Derris elliptica (Roxb.) Benth. in the preparation of a medicament with analgesic effects; wherein the aqueous extract of Derris elliptica is a decoction extract; The water decoction extraction method is as follows: a. Take the dried roots and stems of the hairy fish plant and crush them into coarse powder; b. Soak the fish in 25 to 50 times its weight of distilled water and stir continuously on a magnetic stirrer for 60 to 90 minutes; heat the water at 80 to 100°C and continue to decoct for 60 to 90 minutes; after decoction, cool and centrifuge to obtain the water extract of the present invention.

2. The use according to claim 1, characterized in that: The aforementioned Derris elliptica is derived from the roots or stems of Derris elliptica (Roxb.) Benth., a plant belonging to the genus Derris in the family Leguminosae.

3. The use according to claim 1 or 2, characterized in that: The drug is an oral preparation made from water extract of *Derris fusiforme* as the active ingredient, with the addition of pharmaceutically acceptable excipients or auxiliary ingredients.

4. The use according to claim 3, characterized in that: The oral preparations mentioned are tablets, capsules, granules, pills, and oral liquids.

5. The use according to claim 1, characterized in that: The isocratic elution HPLC chromatogram of the water decoction extract contains four characteristic peaks, with the following retention times, peak heights, and peak areas: First peak: Retention time: 1.644 minutes, peak height: 475.435 mAU, peak area: 3218.328 mAU.s; Second peak: Retention time: 2.318 minutes, peak height: 981.870 mAU, peak area: 8771.686 mAU.s; The third peak: retention time: 2.480 minutes, peak height: 1172.479 mAU, peak area: 13846.608 mAU.s; Twelfth peak: Retention time: 6.954 minutes, peak height: 350.738 mAU, peak area: 3820.238 mAU.s; The relative retention time fluctuated within ±0.05 minutes; the peak height fluctuated within ±5%; and the peak area fluctuated within ±10%. The chromatographic conditions were as follows: column: reversed-phase C18, size 250 mm x 4.6 mm, packed material particle size 5 μm; isocratic elution mode, mobile phase consisted of HPLC-grade distilled water (A) and HPLC-grade methanol (B), volume ratio 20% A and 80% B, flow rate set at 1 mL / min, detection wavelength: 214 nm; injection volume 20 μL. The gradient elution HPLC chromatogram of the water decoction extract contains four characteristic peaks, with the following retention times, peak heights, and peak areas: First peak: retention time 2.880 minutes, peak height 623.523 mAU, peak area 4239.315 mAU.s; The second peak had a retention time of 3.037 minutes, a peak height of 120.289 mAU, and a peak area of ​​696.857 mAU.s. The twelfth peak: retention time 41.963 minutes, peak height 527.401 mAU, peak area 11362.166 mAU.s; The thirteenth peak: retention time 43.833 minutes, peak height 127.460 mAU, peak area 2269.173 mAU.s; The relative retention time fluctuated within ±0.05 minutes; the peak height fluctuated within ±5%; and the peak area fluctuated within ±10%. The chromatographic conditions were as follows: column: reversed-phase AQ-C18, size 250 mm x 4.6 mm, packed material particle size 5 μm; mobile phase consisted of HPLC grade methanol (A) and HPLC grade distilled water (B), gradient elution mode, volume ratio: 0~20 min, 40%A→60%A; 20~30 min, 60%A; 30~45 min, 60%A→80%A; 45~60 min, 80%A); flow rate set at 1 mL / min; detection wavelength: 296 nm; injection volume 10 μL.

6. A pharmaceutical composition having analgesic effects, characterized in that: It is prepared from the aqueous extract of Derris elliptica (Roxb.) Benth.; The aforementioned *Derris fusiforme* water extract is a decoction extract; The water decoction extraction method is as follows: a. Take the dried roots and stems of the hairy fish plant and crush them into coarse powder; b. Soak the fish in 25 to 50 times its weight of distilled water and stir continuously on a magnetic stirrer for 60 to 90 minutes; heat the water at 80 to 100°C and continue to decoct for 60 to 90 minutes; after decoction, cool and centrifuge to obtain the water extract of the present invention.

7. The pharmaceutical composition with analgesic effect according to claim 6, characterized in that: It is prepared into a commonly used oral formulation by adding pharmaceutically acceptable excipients or auxiliary ingredients to the water extract of Derris elliptica (Roxb.) Benth.

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