Application of high gold line orchid extract in preparation of medicine or health care product for treating or relieving pain

Abstract

The application discloses application of high gold line orchid extract in preparation of medicines or health products for treating or relieving pain, wherein the high gold line orchid extract is at least one of high gold line orchid alcohol extract, high gold line orchid polysaccharide or monomer compound of macfarlanea glycoside. Experimental results show that the high gold line orchid alcohol extract, the high gold line orchid polysaccharide and the monomer compound of macfarlanea glycoside all have good in-vivo analgesic activity, can significantly inhibit acute or chronic pain caused by chemical stimulation and thermal stimulation, can effectively reduce the writhing frequency of mice induced by intraperitoneal injection of acetic acid, prolong the pain reaction time of mice, and improve the pain threshold of mice in a hot tail-flick test. The above results show that the high gold line orchid extract can be used for clinical drug treatment of various pains, especially inflammatory pain, or be prepared into health products for relieving pain.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of a high-quality Anoectochilus roxburghii extract in the preparation of drugs or health products for treating or relieving pain. Background Technology

[0002] Pain is a sensation produced when the body is subjected to various noxious stimuli, often associated with many diseases and traumas. With the increasing involvement of the military in war operations and the execution of diverse emergency missions, the use of strong analgesics with few side effects is crucial for ensuring the safety of wounded personnel, reducing their suffering, and preventing severe stress responses. Currently, the most commonly used method of analgesia in clinical practice is pharmacological analgesia, with nonsteroidal anti-inflammatory drugs (NSAIDs) and opioids being the most frequently used. While both have achieved good clinical efficacy, they also have varying degrees of adverse reactions. Therefore, the search for and development of novel, safe, and effective analgesics has always been an important task in pain management.

[0003] "In the north there is Cordyceps sinensis, in the south there is Anoectochilus roxburghii." Anoectochilus roxburghii is a characteristic traditional Chinese medicine from Fujian and Taiwan, derived from the plant *Anoectochilus roxburghii*, belonging to the genus *Anoectochilus* of the Orchidaceae family. The whole plant is used medicinally, possessing effects such as clearing heat and cooling blood, dispelling wind and dampness, and detoxifying. Anoectochilus roxburghii mainly contains lactones (such as anoectochiloside), polysaccharides, and flavonoids, and is widely used to treat hypertension, diabetes, hepatitis, and tumors (Zhang Hongyan, Pan Xin. Research progress on chemical constituents and pharmacological activities of Anoectochilus roxburghii. Strait Pharmaceutical Journal, 2009, 21, 82-84), with excellent efficacy. Therefore, Anoectochilus roxburghii has long been known in folk medicine as the "King of Herbs" and "Golden Herb." As a plant used for both medicinal and edible purposes, the demand for Anoectochilus roxburghii is extremely high, and wild resources are nearing depletion, leading to the emergence of many counterfeit and adulterated products in the market. In particular, some closely related species with plant morphology similar to Anoectochilus roxburghii, such as Anoectochilus zebrina, Anoectochilus xingrenensis, and Anoectochilus nandanensis, are often used interchangeably with Anoectochilus roxburghii in folk medicine, which seriously affects its clinical efficacy and safety.

[0004] Based on previous high-performance liquid chromatography (HPLC) methods, these easily confused varieties were thought to contain kinsenoside (3-(R)-3-β-d-Glucopyranosyloxybutanolide, the main component of *Anoectochilus roxburghii*). However, through optimized detection methods, it was found that the main component in some adulterants and counterfeits is gooderoside (3-(S)-3-β-d-Glucopyranosyloxybutanolide, gooderoside A), i.e., the kinsenoside isomer (Wu YB, Peng MC, Zhang C, Wu JG, Ye BZ, Yi J, Wu JZ, Zheng CJ. Quantitative determination of multi-class bioactive constituents for quality assessment of ten *Anoectochilus*, four *Goodyera* and one *Ludisia* species in China. Chinese Herbal Medicines, 2020, 4, 430-439). Kinsenoside and gooderoside are epimers, differing only in the configuration of the lactone hydroxyl group, and their polarities are extremely similar. Different main chemical components result in different medicinal effects, and the rationality of using easily confused varieties with Anoectochilus elatus in medicine will inevitably be questioned. In 2018, during the investigation of Anoectochilus elatus in Yunnan and the tracking of its production areas, a plant species of the genus Anoectochilus with a very similar plant morphology to Anoectochilus elatus (also used as Anoectochilus elatus in folk medicine) was discovered and identified as Anoectochilus elatus Lindl. Although the plant morphology is similar to Anoectochilus elatus, the flowers are larger and inverted; the front and base of the lip of the flower are nearly parallel, and the lip lobes are triangular, which can be clearly distinguished from Anoectochilus elatus. At present, tissue culture of Anoectochilus elatus has been successfully achieved, solving the resource problem. Anoectochilus elatus was discovered for the first time in China and is a new record species in China (Zheng Lixiang, Tian Huaizhen, Wu Jinzhong, Xu Baoling, Zheng Chengjian, Huang Zehao, Wu Yanbin. A new record species of Chinese orchids-Anoectochilus elatus. Modern Chinese Medicine, 2018, 20(1):14-16). Previous studies have shown that the main chemical component of Anoectochilus roxburghii is piperidine, rather than roxburghii glycoside.Furthermore, some scholars have found that although kinsenoside has various biological activities such as hepatoprotection, hypoglycemia, and hypolipidemia, its isomer, kinsenoside, has no activity (Xiang M, Liu T, Tan W, Ren H, Li H, Liu J, Cao H, Cheng Q, Liu X, Zhu H, Tuo Y, Wang J, Zhang Y. Effects of kinsenoside, a potential immunosuppressive drug for autoimmune hepatitis, on dendritic cells / CD8+T cells communication in mice. Hepatology. 2016, 64(6):2135-2150). To date, there are no reports on the pharmacological activities of kinsenoside and extracts of *Anoectochilus roxburghii*. There are only reports of using *Anoectochilus elatus* for treating insect and snake bites in India (Sherif NA, Kumar TS, Rao MV. In vitro regeneration by callus culture of *Anoectochilus elatus* Lindley, an endangered terrestrial jewelorchid. In Vitro Cellular & Developmental Biology-Plant, 2016, 52(1):72-80). However, there are no reports to date on the use of *Anoectochilus elatus*, its extracts, and the monomeric compound styracifoliin for analgesia. Summary of the Invention

[0005] The purpose of this invention is to provide an application of high-quality Anoectochilus roxburghii extract in the preparation of medicines or health products for treating or relieving pain.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides the use of a high-quality Anoectochilus roxburghii extract in the preparation of a medicine or health product for treating or relieving pain, wherein the high-quality Anoectochilus roxburghii extract is at least one of a high-quality Anoectochilus roxburghii alcohol extract, a high-quality Anoectochilus roxburghii polysaccharide, or a monosodium glutamate monomer compound.

[0008] The structure of the monomeric compound gooderoside A is shown below:

[0009]

[0010] The preparation method of the high-quality Anoectochilus roxburghii alcohol extract includes the following steps:

[0011] The high-quality Anoectochilus roxburghii was extracted by reflux with an aqueous ethanol solution of 40-95% (the ratio of ethanol to Anoectochilus roxburghii was 30:1). The extract was concentrated under reduced pressure to obtain an alcohol extract without alcohol odor.

[0012] The content of variegated glycosides in the high-quality Anoectochilus roxburghii alcohol extract is >50%.

[0013] The preparation method of the high-quality Anoectochilus roxburghii polysaccharide includes the following steps:

[0014] (1) Extraction: After the medicinal residue of *Anoectochilus roxburghii* is extracted with ethanol and water, it is dried until there is no alcohol taste. It is then boiled with water for extraction 2-3 times. The amount of solvent used each time is about 8-10 times the amount of raw medicinal material, and the extraction time is 1-2 hours each time. The extracts are combined.

[0015] (2) Alcohol precipitation: Concentrate the above extract to 1 / 4 of the original volume, let it cool, slowly add 95% ethanol to adjust the ethanol concentration in the extract to 80%, place it in a 4°C refrigerator overnight, filter by suction, and wash the precipitate repeatedly with anhydrous ethanol and acetone until the filtrate is pale yellow.

[0016] (3) Purification: Dissolve the above precipitate in water at a ratio of 8 mg / ml and centrifuge. Take the supernatant and wrap it in a 3500 KD ready-to-use dialysis bag. Place the dialysis bag in running water overnight. Then wash the dialysis bag with double-distilled water 2-4 times, 1 hour each time. Collect the aqueous solution in the dialysis bag, concentrate under reduced pressure, and dry to obtain high-gold-thread polysaccharide.

[0017] The content of high-quality Anoectochilus roxburghii polysaccharide is greater than or equal to 90%.

[0018] The high-gold-thread polysaccharide is mainly composed of mannose, rhamnose, glucose, galactose, xylose and arabinose, with a molar ratio of 1:0.14:5.19:0.75:0.13:0.46.

[0019] The preparation method of the variegated glycoside monomer compound includes the following steps:

[0020] (1) Extraction: The extract of high-quality agastache rugosa alcohol is fully suspended in water (the ratio of extract to water is 10:3), sonicated for 1 to 60 minutes, and centrifuged to collect the supernatant; the precipitate is repeatedly suspended in water, sonicated for 1 to 60 minutes, and the supernatant is collected by filtration; repeat at least 3 times and combine the supernatants.

[0021] (2) Drying: The combined supernatant was freeze-dried to obtain the monosodium glutamate monomer compound.

[0022] The pain mentioned in the medications or health products for treating or relieving pain specifically refers to inflammatory pain.

[0023] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0024] This invention uses acetic acid-induced writhing, hot tail flicking, and formalin-licked paw models in mice (Zheng CJ, Huang BK, Han T, Zhang QY, Zhang H, Rahman K, Qin LP. Antinociceptive activities of the liposoluble fraction from Vitex negundo seeds. Pharmaceutical Biology, 2010, 48, 651-658) to observe the analgesic effect of oral administration of Vitex negundo extract. Experimental results show that the high-quality *Anoectochilus roxburghii* ethanol extract, high-quality *Anoectochilus roxburghii* polysaccharide, and *Anoectochilus roxburghii* glycoside monomer compound of this invention all have good in vivo analgesic activity, significantly inhibiting acute or chronic pain induced by chemical and thermal stimuli. They can effectively reduce the number of writhing movements induced by intraperitoneal injection of acetic acid in mice and prolong the pain response time in mice; they also increase the pain threshold in mice during the thermal tail-flicking test; and the ethanol extract and *Anoectochilus roxburghii* glycoside monomer compound can significantly reduce the phase II paw-licking time induced by formalin injection in mice. This effect cannot be antagonized by naloxone, indicating that it has no morphine receptor agonist-like addictive side effects. These results suggest that the high-quality *Anoectochilus roxburghii* extract (including the ethanol extract, polysaccharide, and glycoside monomer compound) can be used clinically for the treatment of various types of pain, especially inflammatory pain, or prepared into health products for pain relief.

[0025] This invention provides a new source for seeking new drugs and health products for treating or relieving pain. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the chemical structure of the monomer compound gooderoside A.

[0027] Figure 2 This is an HPLC chromatogram of the alcohol extract of *Anoectochilus roxburghii*.

[0028] Figure 3 These are HPLC chromatograms of the monosaccharide composition of monosaccharide reference standards and high-quality Anoectochilus roxburghii polysaccharide (A. Monosaccharide reference standards: 1. Mannose; 2. Rhamnose; 3. Glucuronic acid; 4. Galacturonic acid; 5. Glucose; 6. Galactose; 7. Xylose; 8. Arabinose; B. High-quality Anoectochilus roxburghii polysaccharide: 1. Mannose; 2. Rhamnose; 5. Glucose; 6. Galactose; 7. Xylose; 8. Arabinose).

[0029] Figure 4The effect of *Anoectochilus roxburghii* extract on the number of writhing movements induced by acetic acid in mice was compared with the blank control group. *p<0.05, **p<0.01 (CON, blank group; COX, indomethacin; PS, polysaccharide; EE, alcohol extract; GA, variegated glycoside; L / M / H, low / medium / high).

[0030] Figure 5 The effect of *Anoectochilus roxburghii* extract on the latency of acetic acid-induced writhing in mice was compared with the blank control group. *p<0.05, **p<0.01 (CON, blank group; COX, indomethacin; PS, polysaccharide; EE, alcohol extract; GA, variegated glycoside; L / M / H, low / medium / high).

[0031] Figure 6 The effect of high-quality *Anoectochilus roxburghii* extract on the pain threshold of hot-tail-flick mice was compared with the blank control group. *p<0.05, **p<0.01 (CON, blank group; COX, indomethacin; PS, polysaccharide; EE, alcohol extract; GA, variegated glycoside; L / M / H, low / medium / high).

[0032] Figure 7 This is a schematic diagram showing the effect of *Anoectochilus roxburghii* extract on the phase I paw licking time induced by formalin in mice. The results are compared with the blank control group. *p<0.05, **p<0.01 (CON, blank group; MOR, morphine; ANT, naloxone; PS, polysaccharide; EE, alcohol extract; GA, variegata glycoside; L / M / H, low / medium / high).

[0033] Figure 8 This is a schematic diagram showing the effect of *Anoectochilus roxburghii* extract on the period of formalin-induced phase II paw licking in mice. The results are compared with the blank control group. *p<0.05, **p<0.01 (CON, blank group; MOR, morphine; ANT, naloxone; PS, polysaccharide; EE, alcohol extract; GA, variegata glycoside; L / M / H, low / medium / high). Detailed Implementation

[0034] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1

[0036] Preparation of high-quality Anoectochilus roxburghii extract

[0037] 1) Preparation of high-quality Anoectochilus roxburghii alcohol extract:

[0038] 100g of *Anoectochilus roxburghii* herb (tissue culture seedlings obtained through the applicant's laboratory) was extracted with 3L of 95% ethanol aqueous solution under conventional reflux conditions (ethanol to *Anoectochilus roxburghii* material ratio 30:1). The extract was concentrated under reduced pressure to a non-alcoholic extract, yielding 20g of *Anoectochilus roxburghii* ethanol extract. Liquid chromatography analysis showed that the content of variegated glycoside (a *Anoectochilus roxburghii* glycoside isomeric) in this ethanol extract was 59%. Figure 2 As shown, Figure 2 This is an HPLC chromatogram of the high-quality Anoectochilus roxburghii alcohol extract. As can be seen from the figure, the high-quality Anoectochilus roxburghii alcohol extract has relatively few peaks, indicating few impurities, with roxburghii glycosides being the main component.

[0039] 2) Preparation of high-quality *Anoectochilus roxburghii* polysaccharide:

[0040] (1) Extraction: After the medicinal residue of *Anoectochilus roxburghii* is extracted with ethanol and water, it is dried until there is no alcohol taste. It is then boiled with water for extraction 2-3 times. The amount of solvent used each time is about 8-10 times the amount of raw medicinal material, and the extraction time is 1-2 hours each time. The extracts are combined.

[0041] (2) Alcohol precipitation: Concentrate the above extract to 1 / 4 of the original volume, let it cool, slowly add 95% ethanol to adjust the ethanol concentration in the extract to 80%, place it in a 4°C refrigerator overnight, filter by suction, and wash the precipitate repeatedly with anhydrous ethanol and acetone until the filtrate is pale yellow.

[0042] (3) Purification: Dissolve the above precipitate in water at a ratio of 8 mg / ml and centrifuge. Take the supernatant and wrap it in a 3500 KD ready-to-use dialysis bag. Place the dialysis bag in running water overnight. Then wash the dialysis bag with double-distilled water 2-4 times, 1 hour each time. Collect the aqueous solution in the dialysis bag, concentrate under reduced pressure, and dry to obtain high-gold-thread polysaccharide.

[0043] The polysaccharide content of *Anoectochilus roxburghii* was determined to be 90% by the phenol-sulfuric acid method. High-performance liquid chromatography (HPLC) analysis revealed that the monomer composition mainly consisted of mannose, rhamnose, glucose, galactose, xylose, and arabinose, with molar ratios of 1:0.14:5.19:0.75:0.13:0.46. Figure 3 As shown, Figure 3 The figures show the HPLC chromatograms of the monosaccharide composition of the monosaccharide reference standard and high-quality *Anoectochilus roxburghii* polysaccharide (A. Monosaccharide reference standard: 1. Mannose; 2. Rhamnose; 3. Glucuronic acid; 4. Galacturonic acid; 5. Glucose; 6. Galactose; 7. Xylose; 8. Arabinose; B. High-quality *Anoectochilus roxburghii* polysaccharide: 1. Mannose; 2. Rhamnose; 5. Glucose; 6. Galactose; 7. Xylose; 8. Arabinose). As can be seen from the figures, the monosaccharide composition of high-quality *Anoectochilus roxburghii* polysaccharide mainly consists of glucose, mannose, and galactose, with only small amounts of arabinose, rhamnose, and xylose, in a molar ratio of 5.19:1:0.75:0.46:0.14:0.13.

[0044] 3) Preparation of monoclonal compounds of variegated glycosides:

[0045] (1) Extraction: The extract of high-quality agastache rugosa alcohol is fully suspended in water (the ratio of extract to water is 10:3), sonicated for 30 min, and centrifuged to obtain the supernatant; the precipitate is repeatedly suspended in water, sonicated for 30 min, and the supernatant is filtered; this process is repeated 3 times and the supernatants are combined.

[0046] (2) Drying: The combined supernatant was freeze-dried at below 0°C to obtain the monoclonal compound of piperazine, with a content >95% as determined by HPLC-ELSD. 1 H-NMR, 13 C-NMR, DEPT 1 H- 1 Spectroscopic data from H COSY, HSQC, HMBC, and NOESY (Zhang Y, Xia Y, Lai Y, Tang F, Luo Z, Xue Y, Yao G, Zhang Y, Zhang J. Efficient synthesis of kinsenoside and goodyeroside a by a chemo-enzymatic approach. Molecules, 2014 Oct 22; 19(10): 16950-8) were used to identify the chemical structure of the kinsenoside monomer compound as follows: Figure 1 As shown, Figure 1 This is a schematic diagram of the chemical structure of the monomer compound gooderoside A.

[0047] Example 2

[0048] Analgesic pharmacodynamics of Anoectochilus roxburghii extract

[0049] 2.1. Acetic acid-induced writhing test in mice

[0050] 2.1.1 Modeling and Grouping

[0051] Eighty healthy ICR mice were randomly divided into eight groups of ten mice each: a blank control group, a positive control drug (indomethacin) group, three groups of high-, medium-, and low-dose Anoectochilus roxburghii polysaccharide, and three groups of high-, medium-, and low-dose Anoectochilus roxburghii alcohol extract.

[0052] 2.1.2 Dosage and Administration

[0053] Mice in each group were administered 0.1 mL / 10 g of the drug by gavage daily. The positive control group was administered 5 mg / kg indomethacin solution. The experimental groups of high, medium, and low doses were administered 25 mg / kg, 50 mg / kg, and 100 mg / kg by gavage, respectively. The blank control group was administered an equal volume of physiological saline by gavage. The treatment lasted for 7 days.

[0054] 2.1.3 Observation Indicators

[0055] Thirty minutes after the last administration, each group received an intraperitoneal injection of 0.2 mL of 0.6% acetic acid solution per animal. The number of animals exhibiting writhing response (abdomen concavity, body and hind legs extension, buttock elevation, and crawling), the time of the first writhing occurrence, and the number of writhing episodes in mice within 20 minutes were observed.

[0056] 2.2. Mouse hot tail flick test

[0057] 2.2.1 Modeling and Grouping

[0058] Eighty healthy ICR mice were randomly divided into eight groups of ten mice each: a blank control group, a positive control drug (indomethacin) group, three groups of high-, medium-, and low-dose Anoectochilus roxburghii polysaccharide, and three groups of high-, medium-, and low-dose Anoectochilus roxburghii alcohol extract.

[0059] 2.2.2 Dosage and Administration

[0060] Mice in each group were administered 0.1 mL / 10 g of the drug daily by gavage. The positive control group was administered 5 mg / kg indomethacin solution. The experimental groups of high, medium, and low doses were administered 25 mg / kg, 50 mg / kg, and 100 mg / kg by gavage, respectively. The blank control group was administered an equal volume of physiological saline by gavage.

[0061] 2.2.3 Observation Indicators

[0062] The lower tail of mice was vertically immersed in a 55°C constant temperature water bath. The latency period when the tail retracted out of the water was used as the pain threshold. Two measurements were taken 5 minutes apart before drug administration, and the average value was used as the baseline pain threshold. After drug administration, measurements were taken on both sides, and the average value was used as the post-drug pain threshold. The rate of increase in pain threshold on the day of drug administration was measured.

[0063] 2.3. Formalin-induced foot-licking test in mice

[0064] 2.3.1 Modeling and Grouping

[0065] 140 healthy ICR mice were randomly divided into 14 groups of 10 mice each: blank control group, positive control group, positive control group + antagonist group, three groups each of high-dose, high-dose, and low-dose monoclonal compounds of *Anoectochilus roxburghii* extract, high-dose, and low-dose monoclonal compounds of *Anoectochilus roxburghii* polysaccharide, and high-dose monoclonal compounds of *Anoectochilus roxburghii* monoclonal compounds + antagonist group.

[0066] 2.3.2 Dosage and Administration

[0067] The positive control group received a tail vein injection of 5 mg / kg morphine, the positive control + antagonist group received a 5 mg / kg + 0.4 mg / kg naloxone injection, the experimental groups of high, medium and low doses were administered by gavage of 25 mg / kg, 50 mg / kg and 100 mg / kg respectively, the blank group was administered by gavage of an equal volume of physiological saline, and the high dose + naloxone group was administered by gavage of 100 mg / kg of naloxone + 0.4 mg / kg naloxone via tail vein injection. The administration volume was 1 ml / 100g per day for a total of 7 days.

[0068] 2.3.3 Observation Indicators

[0069] Thirty minutes after the last administration, 30 μL of 2.5% formalin solution (0.92% formaldehyde) was injected subcutaneously into the right hind paw and immediately placed in a transparent container. The time of licking (biting) the paw was observed and recorded in two different stages: 0–5 min (phase I response) and 15–30 min (phase II response). This time was used as an indicator of the intensity of the animal's pain response.

[0070] 2.4 Experimental Results

[0071] SPSS 22.0 was used to perform a two-sample t-test statistical analysis on the two groups of data. A p-value < 0.05 was considered statistically significant.

[0072] 2.4.1 Effects of *Anoectochilus roxburghii* extract on the number of writhing movements and latency period induced by acetic acid in mice

[0073] like Figure 4 As shown, Figure 4 The effect of *Anoectochilus roxburghii* extract on acetic acid-induced writhing frequency in mice was investigated, with *p<0.05 and **p<0.01 compared to the blank control group (CON, blank group; COX, indomethacin; PS, polysaccharide; EE, ethanol extract; GA, variegataside; L / M / H, low / medium / high). The figure shows that after 7 days of administration, the *Anoectochilus roxburghii* ethanol extract, *Anoectochilus roxburghii* polysaccharide, and variegataside monomeric compound groups all dose-dependently inhibited the writhing frequency induced by intraperitoneal injection of acetic acid in mice, showing significant differences compared to the model group (p<0.05). The writhing frequency in the high-dose groups of *Anoectochilus roxburghii* ethanol extract, *Anoectochilus roxburghii* polysaccharide, and variegataside monomeric compound were 5.4±2.8, 4.4±2.8, and 4.2±2.9, respectively, all comparable to the writhing frequency (4.1±1.9) in the positive control group (indomethacin). The time to first writhing (latency) was significantly prolonged in all treatment groups, showing a significant difference from the model group (p<0.05). Figure 5 As shown, Figure 5The effect of high-dose Anoectochilus roxburghii extract on the latency of acetic acid-induced writhing in mice was investigated. The results were compared with the control group (*p<0.05, **p<0.01, CON, control group; COX, indomethacin; PS, polysaccharide; EE, alcohol extract; GA, variegata glycoside; L / M / H, low / medium / high). The figure shows that the latency time (in minutes) of the high-dose variegata glycoside monomer group was comparable to that of the positive control group, at 6.0±2.7 and 6.2±2.3 minutes, respectively.

[0074] 2.4.2 Effect of High-Quality Anoectochilus roxburghii Extract on Pain Threshold in Hot-Tail-Flick Mice

[0075] like Figure 6 As shown, Figure 6 This study investigated the effect of *Anoectochilus roxburghii* extract on the pain threshold of mice subjected to heat-induced tail whipping. The results were compared with the control group (*p<0.05, **p<0.01, CON: control group; COX: indomethacin; PS: polysaccharide; EE: ethanol extract; GA: variegataside; L / M / H: low / medium / high). The figure shows that the increase in pain threshold in mice was measured on the day of administration. The ethanol extract, high-dose polysaccharide, and medium-dose variegataside significantly increased the pain threshold compared to the control group (p<0.05). The increases in pain threshold in mice from the ethanol extract, polysaccharide, and variegataside were 20.0%, 16.1%, and 22.1%, respectively, compared to 30.9% in the positive control group.

[0076] 2.4.3 Effect of High-quality Anoectochilus roxburghii extract on formalin-induced paw-licking time in mice

[0077] like Figure 7 As shown, Figure 7 This is a schematic diagram illustrating the effect of *Anoectochilus roxburghii* extract on the paw-licking time in formalin-induced phase I mice. The comparison is between the control group and the control group; *p<0.05, **p<0.01 (CON, control group; MOR, morphine; ANT, naloxone; PS, polysaccharide; EE, alcohol extract; GA, chamomile glycoside; L / M / H, low / medium / high). The figure shows that in phase I, the paw-licking time (seconds) in the model group was 55.5±8.9. Except for the positive control morphine (24.4±6.4), only the high-dose chamomile glycoside monomer group (45.4±9.3) significantly reduced the paw-licking time, showing a significant difference from the model group (p<0.05). In phase II, as... Figure 8 As shown, Figure 8This is a schematic diagram illustrating the effect of *Anoectochilus roxburghii* extract on the paw-licking time in formalin-induced phase II mice. The comparison is between the model group and the blank control group. *p<0.05, **p<0.01 (CON, blank group; MOR, morphine; ANT, naloxone; PS, polysaccharide; EE, alcohol extract; GA, variegata glycoside; L / M / H, low / medium / high). The figure shows that the paw-licking time (seconds) in the model group was 138.9±23.7. The medium and high doses of the *Anoectochilus roxburghii* alcohol extract and the low, medium, and high doses of variegata glycoside effectively reduced the paw-licking time in a dose-dependent manner, showing a significant difference from the model group (p<0.05). Among these, except for the morphine group (36.9±15.1), the high dose group of variegata glycoside showed the best effect (80.1±31.1). Naloxone alone had no significant effect on the time mice spent licking their paws (140.2±25.3), but naloxone combined with morphine (143.3±29.1) significantly antagonized the analgesic activity of morphine. However, naloxone combined with a monomeric compound of cinnamyl glycoside (95.3±29.6) did not antagonize the analgesic activity of cinnamyl glycoside. This indicates that the monomeric compound of cinnamyl glycoside does not exert its analgesic effect through opioid receptors, suggesting that it may not have side effects such as morphine receptor agonist-like addiction and has good safety.

[0078] Based on the above pharmacological results, the high-quality Anoectochilus roxburghii extract of the present invention (including high-quality Anoectochilus roxburghii alcohol extract, high-quality Anoectochilus roxburghii polysaccharide, and variegated glycoside monomer compound) has good in vivo analgesic activity and significantly inhibits acute or chronic pain caused by chemical and thermal stimuli. This suggests that the high-quality Anoectochilus roxburghii extract (including high-quality Anoectochilus roxburghii alcohol extract, high-quality Anoectochilus roxburghii polysaccharide, and variegated glycoside monomer compound) can be used clinically for the drug treatment of various types of pain, especially inflammatory pain, or prepared into health products for pain relief.

[0079] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. The application of *Anoectochilus roxburghii* extract in the preparation of medicines for treating or relieving pain, characterized in that, The high-quality Anoectochilus roxburghii extract is at least one of high-quality Anoectochilus roxburghii alcohol extract and high-quality Anoectochilus roxburghii polysaccharide; The preparation method of the high-quality Anoectochilus roxburghii alcohol extract includes the following steps: The high-quality Anoectochilus roxburghii was extracted by reflux with a 95% ethanol aqueous solution. The extract was then concentrated under reduced pressure to obtain an alcohol extract without alcohol odor. The preparation method of the high-quality Anoectochilus roxburghii polysaccharide includes the following steps: (1) Extraction: After the medicinal residue of *Anoectochilus roxburghii* is extracted with ethanol and water, it is dried until there is no alcohol smell. It is then boiled with water for extraction 2-3 times. The amount of solvent used each time is 8-10 times the amount of raw drug, and the extraction time is 1-2 hours each time. The extracts are combined. (2) Alcohol precipitation: Concentrate the above extract to 1 / 4 of the original volume, let it cool, slowly add 95% ethanol, adjust the ethanol concentration in the extract to 80%, place it in a 4°C refrigerator overnight, filter by suction, and wash the precipitate repeatedly with anhydrous ethanol and acetone until the filtrate is pale yellow. (3) Purification: Dissolve the above precipitate in water at a ratio of 8 mg / ml and centrifuge. Take the supernatant and wrap it in a 3500 KD ready-to-use dialysis bag. Place the dialysis bag in running water overnight. Then wash the dialysis bag with double-distilled water 2-4 times, 1 hour each time. Collect the aqueous solution in the dialysis bag, concentrate under reduced pressure, and dry to obtain high-gold-thread polysaccharide.

2. The use of the *Anoectochilus roxburghii* extract according to claim 1 in the preparation of a medicament for treating or relieving pain, characterized in that... The content of variegated glycosides in the high-quality Anoectochilus roxburghii alcohol extract is >50%.

3. The use of the *Anoectochilus roxburghii* extract according to claim 1 in the preparation of a medicament for treating or relieving pain, characterized in that... The high-golden thread orchid polysaccharide is mainly composed of mannose, rhamnose, glucose, galactose, xylose and arabinose, with a molar ratio of 1:0.14:5.19:0.75:0.13:0.46.

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