阅读说明：本技术 新甾体皂苷类化合物及其制备方法和在制备治疗2型糖尿病药物中的应用 (New steroid saponin compound, preparation method thereof and application thereof in preparing medicament for treating type 2 diabetes ) 是由 丛悦 郭敬功 刘*** 王书云 李钦 于 2020-07-03 设计创作，主要内容包括：本发明属于医药技术领域,涉及从韭菜子中提取新甾体皂苷类化合物,尤其是韭菜子苷A(Alliumin A)、韭菜子苷B(Alliumin B)、韭菜子苷C(Alliumin C)、韭菜子苷D(Alliumin D)、韭菜子苷E(Alliumin E)；其结构通式如下所示,<Image he="216" wi="556" file="100004_DEST_PATH_IMAGE001.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>经动物实验发现,该甾体皂苷类化合物可以显著降低小鼠空腹血糖、糖化血清蛋白(GSP)、甘油三酯(TG)水平,可用于制备预防和治疗2型糖尿病的药物、保健品或食品等。本发明化合物制备方法简便,溶剂可回收利用,提取量大,可工业化生产并且韭菜子来源广泛,取材便利。(The invention belongs to the technical field of medicines, and relates to a new steroid saponin compound extracted from semen allii tuberosi, in particular to allin A (alliumin A), allin B (alliumin B), allin C (alliumin C), allin D (alliumin D) and allin E (alliumin E); the general formula of the structure is shown as follows, animal experiments show that the steroid saponin compound can obviously reduce fasting blood glucose and saccharification of miceSerum Protein (GSP) and Triglyceride (TG) levels, and can be used for preparing medicine, health product or food for preventing and treating type 2 diabetes. The compound of the invention has simple preparation method, recyclable solvent, large extraction amount, wide source of the semen allii tuberosi and convenient material taking, and can be industrially produced.)

1. The novel steroid saponin compound is characterized in that the structural general formula is shown as follows,

and/or

Wherein R is H or OH, R₂Is H or OH, R₃Is H or OH, R₄Is CH₃Or OH, R₅Is H, OH or CH₃，

R₁Is composed of

R₆Is composed of

Wherein GLC is glucose, RHA is rhamnose, and XYL is xylose.

2. The novel steroidal saponins compound according to claim 1, comprising chikungum A, chikungum B, chikungum C, chikungum D and chikungum E, wherein the specific structural formula is as follows:

and chive seed glycoside B

3. The process for preparing novel steroidal saponins compounds according to claim 2, comprises the following steps:

1) extracting pulverized semen Allii Tuberosi, parched semen Allii Tuberosi or salt-processed semen Allii Tuberosi with 30-40% ethanol under reflux for 1-2 hr for 2-3 times, mixing extractive solutions, and recovering ethanol under reduced pressure to obtain water extractive solution;

2) adsorbing the water extract with macroporous adsorbent resin, sequentially gradient eluting with water, 30% ethanol and 60% ethanol, respectively collecting 30% ethanol eluate and 60% ethanol eluate, and recovering solvent under reduced pressure to obtain component A and component B;

3) purifying the component A by ODS open column, gradient eluting with 30% methanol, 50% methanol, 70% methanol, and 100% methanol, collecting 50% methanol eluate and 70% methanol eluate, mixing, and recovering solvent under reduced pressure to obtain component A1;

4) purifying the component A1 with ODS open column, gradient eluting with 35% methanol, 55% methanol, and 75% methanol, respectively collecting 55% methanol eluate and 75% methanol eluate, and recovering solvent under reduced pressure to obtain components A1-1 and A1-2;

5) subjecting component A1-1 to preparative high performance liquid chromatography, and isocratic eluting with methanol-water mixture solution with flow rate of 6ml/min, wavelength of 203nm, and concentration of 63% to obtain eleuside A and eleuside B;

6) subjecting the component A1-2 to semi-preparative high performance liquid chromatography, and isocratic eluting with 21% acetonitrile water mixture at flow rate of 6ml/min and wavelength of 203nm to obtain leek seed glycoside E and leek seed glycoside C;

7) purifying the component B by an ODS open column, performing gradient elution by using 40% methanol, 50% methanol and 70% methanol, collecting 50% methanol eluent and 70% methanol eluent, merging, recovering the solvent under reduced pressure, detecting and merging by TLC (thin layer chromatography) to obtain four components, and sequentially marking the components as components B1-B4 from large to small according to the polarity of the obtained components; and (3) subjecting the component B2 to semi-preparative high performance liquid chromatography, and performing isocratic elution by using 23% acetonitrile water mixed solution with the flow rate of 6ml/min and the wavelength of 203nm to obtain the chikusetoside D.

4. The method for preparing novel steroidal saponin compounds according to claim 3, wherein in step 2), the model of the macroporous adsorbent resin is HPD100, HPD600, HPD400, D101, AB-8, ADS-17 or DM 130.

5. Use of the novel steroidal saponins compounds according to claim 1 or 2 in the preparation of medicaments for the prevention and treatment of type 2 diabetes.

Technical Field

The invention belongs to the technical field of pharmacy, and particularly relates to five novel steroid saponin compounds, a preparation method thereof and application thereof in preparing a medicament for treating type 2 diabetes.

Background

Type 2 diabetes is a complex chronic metabolic disorder, the cause of which is mainly pancreatic beta-cell dysfunction caused by insulin resistance and insulin deficiency; it features the disorder of metabolism of sugar, lipid and protein, and the clinical symptoms of blood sugar rise, polydipsia, diuresis, polyphagia, emaciation and fatigue. Type 2 diabetes mellitus can cause damage to various organs, thereby causing various complications, and has become a major health problem worldwide. The increasing incidence of type 2 diabetes is a global public health concern in the 21 st century, and environmental factors and genetic factors have led to an accelerated prevalence of diabetes. At present, diet and exercise are combined with drug therapy to be a common strategy for treating diabetes, and the clinical drug therapy mainly comprises oral hypoglycemic drugs and insulin injection. However, most of the current hypoglycemic drugs have toxic and side effects on human bodies, and long-term insulin injection can also enhance insulin resistance, so that the search for new drugs or health care products for treating or preventing type 2 diabetes is urgent.

Semen Allii tuberosi (Semen Allii Tuberose) is a dried mature seed of a perennial plant allium tuberosi (allium tuberosum Rottl. exSpreng.) of allium in liliaceae, is a traditional Chinese medicine with homology of medicine and food, has the effects of tonifying liver and kidney, strengthening spleen and refreshing, promoting qi circulation and regulating blood, relaxing bowel and the like, is used for liver and kidney deficiency, waist and knee soreness, impotence and spermatorrhea and the like, and is collected in the Chinese pharmacopoeia. Research shows that the chemical components of the semen allii tuberosi mainly comprise alkaloids, sulfides, flavonoids and other compounds. Modern pharmacological research proves that the semen allii tuberosi has the effects of warming kidney, tonifying yang, enhancing immunity, resisting oxidation, resisting aging, inhibiting bacteria and the like. The semen allii tuberosi is a traditional Chinese medicine with homology of medicine and food, and is an accessory product produced by the semen allii tuberosi, and the semen allii tuberosi has wide planting area and sufficient resources. However, at present, reports of extracting a large amount of steroid saponins from semen allii tuberosi are few, and no new steroid saponin compound extracted from semen allii tuberosi and relevant reports of the compound on treatment of type 2 diabetes mice are available.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a novel steroid saponin compound and a preparation method thereof, and tests show that: the steroid saponin compounds can be used for preparing medicine, food or health product for preventing and treating type 2 diabetes.

In order to achieve the purpose, the invention adopts the following technical scheme:

the structural general formula of the novel steroid saponin compound is shown as follows,

wherein R is H or OH, R₂Is H or OH, R₃Is H or OH, R₄Is CH₃Or OH, R₅Is H, OH or CH₃，

R₁Is composed of

R₆Is composed of

Wherein GLC is glucose, RHA is rhamnose, and XYL is xylose.

Preferably, the novel steroidal saponins compounds include leek seed glycoside a (alliumin a), leek seed glycoside b (alliumin b), leek seed glycoside c (alliumin c), leek seed glycoside d (alliumin d) and leek seed glycoside e (alliumin e), and the specific structural formula is shown as follows:

and chive seed glycoside B

The invention provides a preparation method of the novel steroid saponin compound, which comprises the following steps:

1) extracting pulverized semen Allii Tuberosi, parched semen Allii Tuberosi or salt-processed semen Allii Tuberosi with 30-40% ethanol under reflux for 1-2 hr for 2-3 times, mixing extractive solutions, and recovering ethanol under reduced pressure to obtain water extractive solution;

2) adsorbing the water extract with macroporous adsorbent resin, gradient eluting with water (water eluate is discarded), 30% ethanol and 60% ethanol sequentially, collecting 30% ethanol eluate and 60% ethanol eluate respectively, and recovering solvent under reduced pressure to obtain component A and component B respectively;

3) purifying the component A by ODS open column, gradient eluting with 30% methanol, 50% methanol, 70% methanol, and 100% methanol, collecting 50% methanol eluate and 70% methanol eluate, mixing, and recovering solvent under reduced pressure to obtain component A1;

4) purifying the component A1 with ODS open column, gradient eluting with 35% methanol, 55% methanol, and 75% methanol, respectively collecting 55% methanol eluate and 75% methanol eluate, and recovering solvent under reduced pressure to obtain components A1-1 and A1-2;

5) subjecting component A1-1 to preparative high performance liquid chromatography, and isocratic eluting with methanol-water mixture solution with flow rate of 6ml/min, wavelength of 203nm, and concentration of 63% to obtain eleuside A and eleuside B;

6) subjecting the component A1-2 to semi-preparative high performance liquid chromatography, and isocratic eluting with 21% acetonitrile water mixture at flow rate of 6ml/min and wavelength of 203nm to obtain leek seed glycoside E and leek seed glycoside C;

7) purifying the component B by an ODS open column, performing gradient elution by using 40% methanol, 50% methanol and 70% methanol, collecting 50% methanol eluent and 70% methanol eluent, merging, recovering the solvent under reduced pressure, detecting and merging by TLC (thin layer chromatography) to obtain four components, and sequentially marking the components as components B1-B4 from large to small according to the polarity of the obtained components; and (3) subjecting the component B2 to semi-preparative high performance liquid chromatography, and performing isocratic elution by using 23% acetonitrile water mixed solution with the flow rate of 6ml/min and the wavelength of 203nm to obtain the chikusetoside D.

Further, in the step 2), the model of the macroporous adsorption resin is HPD100, HPD600, HPD400, D101, AB-8, ADS-17 or DM130, and the like.

The invention also provides application of the novel steroid saponin compound in preparation of medicines, functional foods, health-care products and the like for preventing and treating type 2 diabetes.

Compared with the prior art, the invention has the beneficial effects that:

1) the compound of the invention has stable structure, belongs to steroid saponin compounds, and the extraction raw material is from edible leek seeds, widely distributed in most areas of China and has abundant resources. The compound of the invention has mild extraction and preparation conditions, small technical difficulty, low production cost and small environmental pollution; meanwhile, the raw materials are rich and belong to natural renewable resources; the extraction and separation technology has small difficulty, and the solvent can be recycled, so that the ecological environment pollution can not be caused; the extraction amount is large, the industrial production can be realized, the sources of the semen allii tuberosi are wide, and the material drawing is convenient;

2) the test shows that: the novel steroid saponin compound has obvious treatment effect on the aspect of treating type 2 diabetes, and can be used for preparing medicaments, functional foods, health-care products and the like for preventing and treating type 2 diabetes.

Drawings

FIG. 1 is an HR-ESI-MS spectrum of chikusetin A;

FIG. 2 shows the preparation of chive seed glycoside A¹H NMR spectrum;

FIG. 3 shows the preparation of chive seed glycoside A¹³C NMR spectrum;

FIG. 4 is the HSQC spectrum of chive seed glycoside A;

FIG. 5 is an HMBC profile of chikusetin A;

FIG. 6 is the HSQC-TOCSY spectrum of chive seed glycoside A;

FIG. 7 is the NOESY spectrum of chikusetoside A;

FIG. 8 is an HR-ESI-MS spectrum of chikusetin B;

FIG. 9 shows the formula of chive seed glycoside B¹H NMR spectrum;

FIG. 10 shows the preparation of chive seed glycoside B¹³C NMR spectrum;

FIG. 11 is the HSQC spectrum of chive seed glycoside B;

FIG. 12 is an HMBC profile of chikusetin B;

FIG. 13 is the NOESY spectrum of chikusetoside B;

FIG. 14 is an HR-ESI-MS spectrum of chikusetin C;

FIG. 15 shows the formula of chive seed glycoside C¹H NMR spectrum;

FIG. 16 shows the formula of chive seed glycoside C¹³C NMR spectrum;

FIG. 17 is the HSQC spectrum of chironiside C;

FIG. 18 is an HMBC profile of chikusetin C;

FIG. 19 is the NOESY spectrum of leetin C;

FIG. 20 is an HR-ESI-MS spectrum of chikusetin D;

FIG. 21 shows the production of chive seed glycoside D¹H NMR spectrum;

FIG. 22 shows the production of chive seed glycoside D¹³C NMR spectrum;

FIG. 23 is an HSQC spectrum of chironiside D;

FIG. 24 is an HMBC profile of chikusetin D;

FIG. 25 is the HSQC-TOCSY spectrum of chikusetin D;

FIG. 26 is the NOESY spectrum of chikusetoside D;

FIG. 27 is an HR-ESI-MS spectrum of leek seed glycoside E;

FIG. 28 is a schematic representation of chive seed glycoside E¹H NMR spectrum;

FIG. 29 shows the formula of chive seed glycoside E¹³C NMR spectrum;

FIG. 30 is an HSQC spectrum of chironiside E;

FIG. 31 is an HMBC profile of chikusetin E;

FIG. 32 is the HSQC-TOCSY spectrum of chikusetin E;

FIG. 33 is the NOESY spectrum of leek seed glycoside E.

Detailed Description

The technical solution of the present invention is further described in detail with reference to the following examples, but the scope of the present invention is not limited thereto.

The extraction raw material of the novel steroidal saponin compound is dried mature seeds of Chinese chives (Allium tuberosum Rottll. ex Spreng.), which are purchased from Yuzhou medicinal material market in Henan province in 2017 in 9 months, identified by professor junyue in Henan university, and specimens are stored in specimen museum of the college of pharmacy in Henan university.

In the following examples, unless otherwise specified, methanol, ethanol, and acetonitrile all refer to volume percent concentrations.