CA3117196C - Process for extracting compounds from dendrobium nobile lindl. and application thereof - Google Patents

Abstract

The present invention provides a process for extracting compounds from Dendrobium nobile Lindl., comprising: extracting the Dendrobium nobile Lindl. with ethyl acetate, passing the ethyl acetate extract liquid through a pressure-reduced column, a reversed-phase column, a sephadex column, purifying by semi-preparative HPLC, to obtain compounds represented by Formula I and Formula II. The process provides compounds represented by Formula I and Formula II, which can inhibit the activity of a-glucosidase and the production of NO, and exhibit an ami-inflammatmy activity, demonstrating that the compounds can be used to prepare a food product and drug for treating diabetes mellitus and inflammation.

Description

PROCESS FOR EXTRACTING COMPOUNDS FROM DENDROBIUM NOBILE LINDL.
AND APPLICATION THEREOF
[0001] This application claims the priority to Chinese Patent Application No.
202010159498.1, titled "PROCESS FOR EXTRACTING COMPOUNDS FROM DENDROBIUM NOBILE
LINDE AND APPLICATION THEREOF", filed on March 11, 2020 with the China National Intellectual Property Administration.
FIELD

[0002] The present disclosure belongs to the technical field of biotechnology, and specifically related to a process for extracting compounds from Dendrobium nobile Lindl.
and application thereof.
BACKGROUND

[0003] In recent years, with the improvement of people's living standards, the prevalence of diabetes mellitus is increasing year by year. According to data released by the International Diabetes Federation (IDF) in 2017, about 425 million people (20-79 years old) in the world suffer from diabetes mellitus, of which 114 million are in China, ranking first in the world. According to this trend, by 2045, there will be 629 million people with diabetes mellitus in the world. In addition, the incidence of diabetes is as high as 12%, and it is tend to occur more and more in young people. Diabetes mellitus (DM) is a metabolic disorder caused by many factors and etiologies, which is characterized by chronic hyperglycemia. Its onset is closely related to diet, genetics, environmental factors and dysfunction in the immune system. Diabetes mellitus is mainly divided into three types, namely type I, type II and gestational diabetes mellitus, in which type H patients account for more than 90% of the total number of patients with diabetes mellitus.
The precise molecular mechanism of its occurrence and development has not been yet fully understood. In recent years, many clinical experimental evidences have disclosed that the pathogenesis of type II diabetes mellitus is related to insulin resistance caused by obesity and inflammation.
- -Date Recue/Date Received 2022-11-17

[0004] Inflammation is a series of immune responses of the body and cells to harmful external physical, chemical and biological irritation. The common manifestations of these responses include fever, redness, pain, and dysfunction. Generally speaking, inflammation is the defensive response of tissues and organs to injury or infection, which is beneficial to the body, but a long-term inflammatory response may induce the body to produce excessive and abnormal responses, and even induce many diseases, such as diabetes mellitus and atherosclerosis, alzheimer's disease, and cancer. At present, steroid hormones and non-steroids (aspirin, diclofenac, ibuprofen, etc.) are often used clinically to treat inflammation, but after be taken, it will produce a series of toxic side effects in the human body, such as liver and gastrointestinal tract damage and cardiovascular system damage. In addition, plant-derived drugs have slow and mild effects, strong durability, low toxic and side effects, and are not easily tolerated by the body. Therefore, it is necessary to find substances with hypoglycemic and anti-inflammatory activities from plants to provide lead compounds for the development of new drugs for diabetes mellitus and inflammation.
SUMMARY

[0005] An object of the present disclosure is to provide a process for extracting compounds from Dendrobium nobile Lindl. and application thereof. According to the process in the present disclosure, the compounds of Formula I and II are extracted from the stems of the medicinal plant Dendrobium nobile Lindl., which can effectively inhibit the activity of enzymes key to blood sugar levels. When using LPS-induced RAW264.7 to produce NO as a model for evaluation of anti-inflammatory activity, the compounds of Formula I and II can inhibit the production of NO, thereby exhibiting anti-inflammatory activity.

[0006] The present disclosure provides a process for extracting compounds from Dendrobium nobile Lindl., comprising the following steps:

[0007] A) pulverizing Dendrobium nobile Lindl., leaching with 1-3 times volume of ethanol aqueous solution for 2-5 times, filtering the obtained leaching solution, and combining and concentrating the filtrate to obtain extract A;

[0008] B) preparing a suspension from the extract A and water, extracting with petroleum ether, 16838106.2 Date Recue/Date Received 2021-05-04 ethyl acetate and normal butanol in sequence, screening three extract liquids, concentrating ethyl acetate extract liquid to prepare extract B;

[0009] C) passing the extract B through a pressure-reduced column, performing gradient elution with a mixed eluent of petroleum ether and ethyl acetate, washing the column with acetone to collect fractions, concentrating and combining each of the obtained fractions, to finally obtain 16 fractions recorded as Fr.1 - Fr.16;
100101 D) passing fraction Fr.13 through a reversed-phase column, performing gradient elution with 30-100% methanol aqueous solution to obtain 14 fractions recorded as Fr.13-1 - Fr.13-14; and 100111 E) passing fraction Fr.13-7 through a sephadex column, eluting with a mixed liquid of methanol and chloroform to obtain 18 fractions recorded as Fr.13-7-1 - Fr.13-7-18, combining fractions Fr.13-7-15 - Fr.13-7-17, and purifying by semi-preparative HPLC to obtain a compound represented by Formula I at tR = 13.5 min and a compound represented by Formula II at tR = 16.4 min;

OH HO OH
OH

H3CO, NN-HO NH
Formula I Formula II.
100131 Preferably, in step B), a volume ratio of the extract A to the water is 1 : (0.5-2);
100141 a volume ratio of the extract A to the petroleum ether is 1 : (0.5-2);
a volume ratio of the extract A to the ethyl acetate is 1 : (0.5-2); a volume ratio of the extract A
to the normal butanol is 1 : (0.5-2).
100151 Preferably, in step C), the gradient elution with the mixed eluent of petroleum ether and ethyl acetate is specifically by uniformly reducing a volume ratio of petroleum ether to ethyl acetate from 20 : 1 to 0 : 1 within 80-120 hours, with a total time of the gradient elution of 90-110 hours 16838106.2 Date Recue/Date Received 2021-05-04 preferably, and more preferably 100 hours.
[0016] Preferably, in step C), each of the obtained fractions is concentrated under reduced pressure, and detected by spotting on a thin layer chromatography plate, and similar fractions are combined according to color visualization.
.. 100171 Preferably, in step D), the gradient elution is specifically by eluting with methanol aqueous solution having a mass concentration of 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%
in sequence, with an equal elution time for each of elution gradients and a total elution time of 24-48 hours, more preferably 32-36 hours.
[0018] Preferably, in step E), a volume ratio of the methanol to the chloroform is 1 : (0.5-2).
.. [0019] The present disclosure provides use of a compound represented by Formula I and a compound represented by Formula II extracted by the process described above in the preparation of a medicament for preventing and treating diabetes mellitus and relieving inflammation.
[0020] Preferably, the medicament for preventing and treating diabetes mellitus is an oi-glucosidase activity inhibitor; and the medicament for relieving inflammation is an inhibitor against NO production.
100211 The present disclosure provides a pharmaceutical preparation, comprising a compound represented by Formula I or a compound represented by Formula II extracted by the process described above and a pharmaceutically acceptable adjuvant.
[0022] Preferably, a dosage form of the pharmaceutical preparation is a tablet, a capsule, a pill, a granule, a decoction, an ointment, a distillate formula, an oral liquid, a dropping pill or asyrup.
[0023] The present disclosure provides a process for extracting compounds from Dendrobium nobile Lindl., comprising the following steps: A) pulverizing Dendrobium nobile Lindl., leaching with 1-3 times volume of ethanol aqueous solution for 2-5 times, filtering the obtained leaching solution, and combining and concentrating the filtrate to obtain extract A; B) preparing a suspension from the extract A and water, extracting with petroleum ether, ethyl acetate and normal butanol in sequence, and concentrating ethyl acetate extract liquid to prepare extract B;
C) passing the extract B through a pressure-reduced column, performing gradient elution with a mixed eluent of petroleum ether and ethyl acetate, and then washing the column with acetone to collect fractions, 16838106.2 Date Recue/Date Received 2021-05-04 concentrating and combining each of the obtained fractions, to finally obtain 16 fractions recorded as Fri - Fr.16; D) passing fraction Fr.13 through a reversed-phase column, performing gradient elution with 30-100% methanol aqueous solution to obtain 14 fractions recorded as Fr.13-1 - Fr.13-14; E) passing Fr.13-7 through a sephadex column, eluting with a mixed liquid of methanol and chloroform to obtain 18 fractions recorded as Fr.13-7-1 - Fr.13-7-18, combining fractions Fr.13-7-- Fr.13-7-17, and purifying by semi-preparative HPLC to obtain a compound represented by Formula I at tR = 13.5 min and a compound represented by Formula II at tR =
16.4 min. The researches of the present disclosure have shown that the compounds represented by Foimula I and II can inhibit the activity of a-glucosidase. When using LPS-induced RAW264.7 to produce NO

10 as a model for evaluation of anti-inflammatory activity, the compounds represented by Formula I
and II can inhibit the production of NO, thereby exhibiting anti-inflammatory activity. This shows that the compound can be used to prepare a food product and/or drug for treating and/or preventing diabetes mellitus and inflammatory.

[0024] Hereinafter, in order to more clearly describe the technical solutions in the embodiments of the present disclosure or the existing technology, a brief introduction will be made to the drawings that need to be used in the description of the embodiments or the existing technology. It is obvious that the drawings described below are only some examples of the present disclosure and that for those skilled in the art, other drawings may also be derived from them without inventive effort.
[0025] FIG.1 is a 1H NMR spectrum of a compound represented by Formula I in Example 1 of the present disclosure;
[0026] FIG.2 is a 13C NMR+DEPT135 spectrum of a compound represented by Formula I in Example 1 of the present disclosure;
[0027] FIG.3 is a HSQC spectrum of a compound represented by Formula Tin Example 1 of the present disclosure;
[0028] FIG.4 is a 1H NMR spectrum of a compound represented by Formula II in Example 1 of the present disclosure;
16838106.2 Date Recue/Date Received 2021-05-04 [0029] FIG.5 is a "C NMIR +DEPT135 spectrum of a compound represented by Formula II in Example 1 of the present disclosure; and [0030] FIG.6 is a HSQC spectrum of a compound represented by Formula II in Example 1 of the present disclosure.
DETAILED DESCRIPTION
[0031] The present disclosure provides a process for extracting compounds from Dendrobium nobile Lindl., comprising the following steps:
[0032] A) pulverizing Dendrobium nobile Lindl., leaching with 1-3 times volume of ethanol aqueous solution for 2-5 times, filtering the obtained leaching solution, and combining and concentrating the filtrate to obtain extract A;
[0033] B) preparing a suspension from the extract A and water, extracting with petroleum ether, ethyl acetate and normal butanol in sequence, screening three extract liquids, concentrating ethyl acetate extract liquid to prepare extract B;
[0034] C) passing the extract B through a pressure-reduced column, performing gradient elution with a mixed eluent of petroleum ether and ethyl acetate, and then washing the column with acetone to collect fractions, concentrating and combining each of the obtained fractions, to finally obtain 16 fractions recorded as Fr.1 - Fr.16;
[0035] D) passing fraction Fr.13 through a reversed-phase column, performing gradient elution with 30-100% methanol aqueous solution to obtain 14 fractions recorded as Fr.13-1 - Fr.13-14; and [0036] E) passing Fr.13-7 through a sephadex column, eluting with a mixed liquid of methanol and chlorofoim to obtain 18 fractions recorded as Fr.13-7-1 - Fr.13-7-18, combining fractions Fr.13-7-15 - Fr.13-7-17, and purifying by semi-preparative HPLC to obtain a compound represented by Formula I at tR = 13.5 min and a compound represented by Formula II at tR = 16.4 min;
[0037]
16838106.2 Date Recue/Date Received 2021-05-04 OH HO OH
O

* 0 HO NH
Formula I Formula H.
100381 Dendrobium nobile Lindl. is a plant belonging to orchidaceae dendrobium Sw., also known as Harba Dendrolii Nobilis, Dendrobium chrysanthum, Dendrobium chrysanthum Wall, etc..
It is precious traditional Chinese medicine in China, always called as "extremely precious herb", and often used as medicine with its fresh or dry stems. Dendrobium nobile Lindl. is mainly distributed in the subtropical regions, such as Guizhou, Yunnan, and Guangxi located in south of the Changjiang River in China. In the present disclosure, it is preferred to use the dry stems of the Dendrobium nobile Lindl. as raw material to extracte.
100391 In the present disclosure, the dry stems of Dendrobium nobile Lindl.
are pulverized, and leached with ethanol aqueous solution; the obtained leaching solution is filtered and then combined and concentrated to obtain extract A.
100401 In the present disclosure, the dry stems of Dendrobium nobile Lindl.
are preferably pulverized to a particle size of 0.1-1 cm, and more preferably to 0.5-0.6 cm;
a volume ratio of ethanol to water in the ethanol aqueous solution is preferably (15-20) : 1, and more preferably (18-19) :1; a volume of the ethanol aqueous solution is 1-3 times the volume of the pulverized Dendrobium nobile Lindl., and preferably 2 times the volume; the leaching is preferably carried out for 2-5 times, and more preferably 3-4 times; the leaching solution is concentrated preferably at a concentration ratio of (3-5) :1, and more preferably 4 : 1.
100411 In the present disclosure, after the extract A is obtained, the extract A and water are prepared into a suspension, and then the suspension is extracted with petroleum ether, ethyl acetate and normal butanol in sequence. The extraction is stopped when each extract layer is extracted until the extract liquid becomes colorless. The ethyl acetate extract liquid is concentrated to prepare extract B.
16838106.2 Date Recue/Date Received 2021-05-04 100421 The polarity of petroleum ether, ethyl acetate and normal butanol increases in sequence.
The normal butanol extract liquid may contain some highly polar glycoside compounds. In a specific experiment, the fraction with medium polarity, that is, the ethyl acetate extract liquid is selected. In the present disclosure, a volume ratio of the extract A to the water preferably is 1: (0.5-2), and more preferably 1 : (1-1.5); a volume ratio of the extract A to the petroleum ether is 1 : (0.5-2), and more preferably 1: (1-1.5). Specifically, in embodiments of the present disclosure, a volume ratio of extract A: water : petroleum ether is 1 : 1 : 1. A volume ratio of the extract A to the ethyl acetate is preferably 1 : (0.5-2), and more preferably 1 : (1-1.5).
Specifically, in embodiments of the present disclosure, a volume ratio of extract A: water: ethyl acetate is 1: 1 : 1. A volume ratio of the extract A to the normal butanol is 1 : (0.5-2), and more preferably 1 (1-1.5). Specifically, in embodiments of the present disclosure, a volume ratio of extract A: water:
normal butanol is 1 :
1 : 1.
[0043] In the present disclosure, the ethyl acetate extract liquid is concentrated preferably at a concentration ratio of (2-5) :1, and more preferably (3-4) : 1.
[0044] In the present disclosure, after the extract B is obtained, the extract B is passed through a pressure-reduced column, and subjected to gradient elution with a mixed eluent of petroleum ether and ethyl acetate. Then the column is washed with acetone, to collect the fractions. Each of the obtained fractions is concentrated, and combined to finally obtain 16 fractions recorded as Fr.1 -Fr.16.
[0045] In the present disclosure, the pressure-reduced column is silica gel column chromatography. The silica gel column is silica gel H with a particle size of 100-200 meshes. A
volume ratio of petroleum ether to ethyl acetate in their mixed solution is (20-0) : 1. Specifically, in embodiments of the present disclosure, the gradient elution is specifically carried out as follows.
[0046] The volume ratio of petroleum ether to ethyl acetate in the mixed solution is initially 20:
1, and with the progress of the gradient elution, the volume ratio is uniformly reduced until it decreased to 0 : 1. The gradient elution is performed preferably at a total time of 80-120 hours, more preferably 90-110 hours, and most preferably 100 hours. The gradient elution is performed preferably at a temperature of room temperature, i.e. 20-35 C, and more preferably 25-30 C. A
volume of the eluate used in each gradient preferably is 3-4 L.
16838106.2 Date Recue/Date Received 2021-05-04 100471 After the elution is completed, the column is washed with acetone, to collect fractions preferably once each 500 mL. Each of the obtained fractions is detected by spotting it on a thin layer chromatography (TLC) plate. Similar fractions are combined to obtain 16 fractions, recorded as Fr.1 - Fr.16.
100481 In the present disclosure, each of the obtained fractions is detected by spotting on a thin layer chromatography (TLC) plate. The fractions with the same or similar main points observed by the naked eye are combined together to obtain a total of 16 fractions.
100491 After analysis by high performance liquid chromatography, it is showed that the characteristic peak of Fr.13 is relatively obvious. Fr.13 is passed through a reversed-phase column .. (in which the filler is C18), and subjected to gradient elution with 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% of methanol aqueous solutions in sequence, with 100 mL collected once. For each gradient, 1.5 L of the mixed solution is used. Each of the obtained fractions is detected by spotting it on a thin layer chromatography (TLC) plate. The fractions with the same or similar main points observed by the naked eye are combined together to obtain a total of 14 fractions, recorded as Fr.13-1 - Fr,13-14.
[0050] In the present disclosure, in the gradient elution, the elution time for each gradient is equal;
the total elution time is preferably 24-48 hours, and more preferably 32-36 hours.
100511 The fraction Fr.13-7 is passed through a Sephadex LH-20 column, and eluted with a mixed solution of methanol and chloroform at room temperature at a rate of 2-3 seconds per drop, with each 5 mL collected in one tube. Each of the obtained fractions is concentrated under reduced pressure, and detected by spotting it on a thin layer chromatography (TLC) plate. The similar fractions are combined together to obtain 18 fractions, recorded as Fr.13-7-1 -Fr.13-7-18.
[0052] In the present disclosure, a volume ratio of the methanol and the chloroform preferably is 1 : (0.5-2), and more preferablyl (1-1.5).
[0053] Fr.13-7-15 - Fr.13-7-17 are similar in spots. They are combined and purified by semi-preparative HPLC (a C18 chromatography column, eluted with 35% methanol-water), to obtain the compound represented by Formula I (tR = 13.5 min) and the compound of Formula II (tR = 16.4 min).
16838106.2 Date Recue/Date Received 2021-05-04 [0054]
OH HO OH
3' 4' 0 2çOH

H3C0 3 7 = 1 5. 4 3 it Formula I Formula II.
[0055] The present disclosure further provides use of a compound represented by Formula I or a compound represented by Formula II in the preparation of a medicament for preventing and treating diabetes mellitus and relieving inflammation. Preferably, the medicament for preventing and treating diabetes mellitus is an a-glucosidase activity inhibitor, and the diabetes mellitus is type II
diabetes mellitus. The compounds represented by Formula I and Formula II in the present disclosure inhibit the activity of a-glucosidase, reduce the decomposition of oligosaccharides in the digestive tract, and delay the absorption of glucose by the intestine, thereby reducing the risk of postprandial glycemia, and achieving the effect of lowering blood sugar levels.
[0056] In the present disclosure, the medicament for relieving inflammation is an inhibitor against NO production. The compound represented by Formula I or the compound represented by Formula II in the present disclosure can inhibit the production of NO, exhibit anti-inflammatory activity, and relieve symptoms of redness, swelling, fever, pain in body, etc.
[0057] The present disclosure further provides a pharmaceutical preparation, comprising a compound represented by Formula I or a compound represented by Formula II
extracted by the process described above and a pharmaceutically acceptable adjuvants.
[0058] Preferably, a dosage form of the pharmaceutical preparation is an oral preparation, and more preferably a tablet, a capsule, a pill, a granule, a decoction, an ointment, a distillate formula, an oral liquid, a dropping pill or a syrup.
[0059] More preferably, the capsule is a hard capsule or a soft capsule. More preferably, the tablet is an oral tablet or a buccal tablet. More preferably, the oral tablet refers to a tablet for oral 16838106.2 Date Recue/Date Received 2021-05-04 administration. The medicaments in most of these tablets are absorbed by gastrointestinal tract to exert their effects, while the medicaments in some of the tablets act locally in the gastrointestinal tract. In some embodiments provided by the present disclosure, the oral tablet is an ordinary compressed tablet, a dispersible tablet, an effervescent tablet, a chewable tablet, a coated tablet or a sustained-release tablet.
[0060] The pharmaceutically acceptable adjuvant includes one selected from fruit powders, an edible essence, a sweetener, a sour agent, a filler, a lubricant, a preservative, a suspending agent, a food coloring, a diluent, an emulsifier, a disintegrating agent, a plasticizer or a mixture of two or more of them.
100611 The present disclosure provides a process for extracting compounds from Dendrobium nobile Lindl., comprising the following steps: A) pulverizing Dendrobium nobile Lindl., leaching with 1-3 times volume of ethanol aqueous solution for 2-5 times, filtering the obtained leaching solution, and combining and concentrating the filtrate to obtain extract A; B) preparing a suspension from the extract A and water, extracting with petroleum ether, ethyl acetate and normal butanol in sequence, and concentrating ethyl acetate extract liquid to prepare extract B;
C) passing the extract B through a pressure-reduced column, performing gradient elution with a mixed eluent of petroleum ether and ethyl acetate, and then washing the column with acetone to collect fractions, concentrating and combining each of the obtained fractions, to finally obtain 16 fractions recorded as Fr.1 - Fr.16; D) passing fraction Fr.13 through a reversed-phase column, performing gradient elution with 30-100% methanol aqueous solution to obtain 14 fractions recorded as Fr.13-1 - Fr.13-14; E) passing fraction Fr.13-7 through a sephadex column, eluting with a mixed liquid of methanol and chloroform to obtain 18 fractions recorded as Fr.13-7-1 - Fr.13-7-18, combining fractions Fr.13-7-15 - Fr.13-7-17, and purifying by semi-preparative HPLC to obtain a compound represented by Formula I at tR = 13.5 min and a compound represented by Formula II at tR = 16.4 min. The researches of the present disclosure have shown that the compounds represented by Formula I and II can inhibit the activity of a-glucosidase. When using LPS-induced RAW264.7 to produce NO as a model for evaluation of anti-inflammatory activity, the compounds represented by Formula I and II can inhibit the production of NO, thereby exhibiting anti-inflammatory activity.
This shows that the compound can be used to prepare a food product and/or drug for treating and/or 16838106.2

- 11 -Date Recue/Date Received 2021-05-04 preventing diabetes mellitus and inflammatory.
100621 Hereafter, in order to further describe the present disclosure, the process for extracting compounds from Dendrobium nobile Lindl. and application thereof provided by the present disclosure will be described in detail in conjunction with examples, but it should not be construed as limiting the protection scope of the present disclosure.
Example 1 100631 Step 1: The dry stems of Dendrobium nobile Lindl. (13 kg) were pulverized, and then leached with 2 times the volume of ethanol aqueous solution for 3 times. The obtained leaching solution was filtered, and then combined and concentrated to prepare extract A.
100641 Step 2: A suspension was prepared from extract A and water at a volume ratio of 1 : 1, and extracted with petroleum ether (extract A : water : petroleum ether = 1 :
1 : 1), ethyl acetate (extract A: water :ethyl acetate = 1 : 1: 1), normal butanol ( extract A:
water :normal butanol = 1:
1 : 1) in sequence. The extraction was stopped when each extraction layer was extracted until the extract liquid became colorless. The ethyl acetate extract liquid was concentrated to prepare extract B.
100651 Step 3: The extract B was passed through a pressure-reduced column, and subjected to gradient elution with a mixed solution of petroleum ether and ethyl acetate (20: 1 ¨> : 1, V/V).
Finally, the column was washed with acetone, with each 500 mL collected in one bottle. Each of the obtained fractions was detected by spotting it on a thin layer chromatography (TLC) plate.
Similar fractions were combined to obtain 16 fractions, recorded as Fr.1 -Fr.16.
100661 Step 4: Analysis by high performance liquid chromatography showed that the characteristic peak of Fr.13 was relatively obvious. Fr.13 was passed through a reversed-phase column, and subjected to gradient elution with 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%
of methanol aqueous solution, with 100 mL collected in one bottle. For each gradient, 1.5 L of the mixed solution was used. Each of the obtained fractions was detected by spotting it on a thin layer chromatography (TLC) plate. The similar fractions were combined together to obtain 14 fractions, recorded as Fr.13-1 - Fr.13-14.
16838106.2

- 12 -Date Recue/Date Received 2021-05-04 100671 Step 5: Fr.13-7 was better in spot. Fr.13-7 was passed through a sephadex column, and eluted with 500 mL of a mixed solution with methanol : chloroform = 1 : 1, with 6 mL collected in one tube. After spotting on the plate, the similar fractions were combined together to obtain 18 fractions, recorded as Fr.13-7-1 - Fr.13-7-18.
100681 Step 6: Fr.13-7-15 - Fr.13-7-17 were similar in spot. They were combined together, and then purified by semi-preparative HPLC (a C18 chromatography column, eluted with 35%
methanol-water), to obtain compounds of Formula I and II (tR = 13.5 min and tR
= 16.4 min).
100691 The identification spectra of the compounds having the structures represented by Formula land Formula II are shown in spectrum FIGs.1-6.
[0070] The identification data of the structures represented by Formula I and Formula II was as follows: low resolution mass spectrometry miz was 352.4354 [M + Na]; the molecular formula was C18H19N05; the data in NMR(500 MHz) and 1.3C NMR(125 MHz) are shown in Table 1.
[0071] Table 1 The NMR data of the compounds of Formula I and II (the solvent was deuterated methanol) I II
position Oc, type off(J in Hz) Oc, type c5H(J in Hz) 1 128.2 128.5 2 111.5 7.12, brs,1H 113.9 7.36, brs,1H
3 149.9 148.6 4 149.3 148.5 5 116.5 6.79, d,1H (8.07) 115.8 6.73, d,1H(8.2) 6 123.2 7.02, brd,1H 124.9 6.93,brd,1H (8.2) (8.16) 7 142.0 7.43,d,1H (15.65) 138.4 6.61,d,1H (12.7) 8 118.7 6.40,d,1H (15.58) 121.6 5.81,d,1H(12.7) 9 169.2 170.3 132.1 132.0 2' 116.9 6.67,brs 116.8 6.64, brs 3' 146.3 146.3 4' 144.8 144.8 5' 116.4 6.69, d,1H (8.0) 116.4 6.66, d,1H(8.2) 6' 121.0 6.55, brd,1H (8.0) 121.0 6.49, brd,1H(8.2) 7' 36.1 2.70, t,2H (7.34) 35.8 2.64,t,2H(7.51) 8' 42.5 3.46, t,2H (7.40) 42.4 3.39,t,2H(7.53) 3- OCH3 56.4 3.88,s,3H 56.4 3.83,s,3H
16838106.2

- 13 -Date Recue/Date Received 2021-05-04 [0072] Inhibition effects of the compounds of Formula I and II against a-glucosidase [0073] The test sample solution was prepared as follows. 450 [IL of the prepared 2 U/ml a-glucosidase solution (diluted with a PBS solution with pH = 6.8) was taken into an EP tube. The test compound was dissolved in DMSO (with a concentration of 2 mM), and then 45 I:IL of the solution was added to the EP tube and shaken well. Four uniformly mixed test solutions each with a volume of 110 p.L were taken into a 96-well plate separately. (the negative control group and blank control group were prepared as follows: 4500_, of the prepared 2 U/ml a-glucosidase solution was taken into an EP tube, and then to the EP tube 45 III, of DMSO solution was added, and shaken well; four uniformly mixed test solutions each with a volume of 110 [IL were taken into a 96-well plate separately.) [0074] After the 96-well plate was placed at 37 C for 15 minutes, each group was added with 40 [IL of (2.5 mmol/L of 4-nitropheny1-13-D-glucopyranoside) PNPG solution; (the blank control group was added with 401.1L (0.1 mol/L) of PBS solution).
[0075] After the 96-well plate was placed at 37 C for 15 minutes, the OD
absorbance in each well was measured by an enzyme reader set at a wavelength of 405 nm.
[0076] The inhibitory activity of the compound against a-glycosidase was calculated using the following formula.
[0077] Inhibition rate = (ODDmso - ODsample) / (ODDMSO - ODPBS) X 100%
[0078] After the compounds were diluted to several gradients in a two-fold serial dilution way, inhibition rates of the samples with different concentrations against a-glucosidase were detected by the same detection method, and their IC50 values were calculated by GraphPad Prism 7 software.
[0079] The results are shown in Table 2. The compounds have a certain inhibitory activity against a-glucosidase, and are better than the positive control acarbose.
[0080] Table 2 Inhibitory activity of the compounds against ct-glucosidase Test samples IC50 SD (j.1M) Geni stein (Positive control) 8.54 0.69 16838106.2

- 14 -Date Recue/Date Received 2021-05-04 Acarbose (Positive control) 701.45+0.52 Compound I 22.98+1.46 Compound II 46.42+1.12 [0081] Evaluation of anti-inflammatory activity of the compounds of Formula I
and II
[0082] RAW264.7 (mouse mononuclear macrophage leukemia cells) were selected out. 100 !IL
of these cells were seeded in a 96-well flat-bottom cell culture plate at a concentration of 5x 104 cells/mL, and cultured under the condition of 37 C, 5% CO2 and a humidity above 90%. After 24 .. h, the cells were added with 50 [IL of the prepared test compound solution, and continued to be cultured under this condition. After 1 h, 50 pt of the prepared LPS (a final concentration of 500 ng/mL) solution was added. After 24 h, 100 j.iL of the supernatant from each well was added to another 96-well plate, and then 100 .1_, (40 mg/mL) of the Griess reagent was added to each well, and they are mixed well using the cross method. The absorbance in each well was measured by an .. enzyme reader at a wavelength of 540 nm and recorded. NO inhibition rates were calculated according to the following formula. The control group was indomethacin. The negative control group was DMSO. The test compounds were diluted to 5 concentration gradients in a two-fold serial dilution way. Plotting was carried out to calculate IC50 values of the test compounds with the concentrations of the test compounds on abscissa and their inhibition rates on ordinate.
[0083] Inhibition rate (%) = (C2-C1) / (C2-Co) x 100%
100841 In the formula, Co, Ci, and C2 were the absorbance measured at 540 nm of the blank controlled group (without LPS added), the experimental group, and the negative (with LPS added) control group, respectively. The inhibition rates at each of concentrations were calculated and a curve of compound concentrations __ inhibition rate was plotted. Half maximal inhibitory concentrations (IC50 value) of the compounds against LPS-induced NO production by RAW264.7 were calculated. The results are shown in Table 3. The compounds can effectively inhibit the amount of NO produced by RAW264.7 cells, exhibit a certain anti-inflammatory activity, and have significantly better effects than the positive control indomethacin.
[0085] Table 3 Inhibitory effects of the compounds against the production of NO from RAW264.7 16838106.2

- 15 -Date Recue/Date Received 2021-05-04 Test samples IC50 SD (p.M) Indomethacin (Positive control) 52.30 2.11 Compound! 11.07 0.61 Compound II 10.43 1.17 100861 The above description is only the preferred embodiments of the present disclosure. It should be noted that for those skilled in the art, various improvements and modifications may be made without departing from the principle of the present disclosure, and these improvements and modifications should fall within the scope of protection of the present disclosure.
16838106.2

- 16 -Date Recue/Date Received 2021-05-04

Claims (6)

1. A process for extracting compounds from Dendrobium nobile Lindl., comprising the following steps:
A) pulverizing Dendrobium nobile Lindl., leaching with 1-3 times volume of ethanol aqueous solution for 2-5 times, filtering the obtained leaching solution, and combining and concentrating the filtrate to obtain extract A;
B) preparing a suspension from the extact A and water, extracting with petroleum ether, ethyl acetate and normal butanol in sequence, screening three extract liquids, concentrating ethyl acetate extract liquid to prepare extract B;
C) passing the extract B through a pressure-reduced column, performing gradient elution with a mixed eluent of petroleum ether and ethyl acetate, washing the column with acetone to collect fractions, concentrating and combining each of the obtained fractions, to finally obtain 16 fractions recorded as Fr.1 - Fr.16;
D) passing fraction Fr.13 through a reversed-phase column, performing gradient elution with 30-100% methanol aqueous solution to obtain 14 fractions recorded as Fr.13-1 -Fr.13-14; and E) passing fraction Fr.13-7 through a sephadex column, eluting with a mixed liquid of methanol and chloroform to obtain 18 fractions recorded as Fr.13-7-1 - Fr.13-7-18, combining fractions Fr.13-7-15 - Fr.13-7-17, and purifying by semi-preparative HPLC to obtain a compound represented by Formula I at tR = 13.5 min and a compound represented by Formula II at IR = 16.4 min;
OH HO OH
OH

HO NH
Formula I Formula II.

301351971.1 Date Recue/Date Received 2023-07-10

2. The process according to claim 1, wherein in step B), a volume ratio of the extract A to the water is 1 : (0.5-2);
a volume ratio of the extract A to the petroleum ether is 1 : (0.5-2);
a volume ratio of the extract A to the ethyl acetate is 1 : (0.5-2); and a volume ratio of the extract A to the normal butanol is 1 : (0.5-2).

3. The process according to claim 1, wherein in step C), the gradient elution with the mixed eluent of petroleum ether and ethyl acetate is specifically by unifolinly reducing a volume ratio of petroleum ether to ethyl acetate from 20 : 1 to 0 : 1 within 80-120 hours.

4. The process according to claim 1, wherein in step C), each of the obtained fractions is concentrated under reduced pressure, and detected by spotting on a thin layer chromatography plate, and similar fractions are combined according to color visualization.

5. The process according to claim 1, wherein in step D), the gradient elution is specifically by eluting with methanol aqueous solution having a mass concentration of 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% in sequence, with an equal elution time for each of elution gradients and a total elution time of 24-48 hours.

6. The process according to claim 1, wherein in step E) a volume ratio of the methanol to the chloroform is 1 : (0.5-2).

301351971.1 Date Recue/Date Received 2023-07-10