CN111848376A - Glaucocalyx rabdosia root extract, extraction and separation method and application thereof - Google Patents

Glaucocalyx rabdosia root extract, extraction and separation method and application thereof Download PDF

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CN111848376A
CN111848376A CN202010967691.8A CN202010967691A CN111848376A CN 111848376 A CN111848376 A CN 111848376A CN 202010967691 A CN202010967691 A CN 202010967691A CN 111848376 A CN111848376 A CN 111848376A
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methanol
dichloromethane
volume ratio
rabdosia
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白乃生
王天义
张姗姗
郭森
岳文平
高兵
彭赛男
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Northwestern University
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Abstract

The invention provides a rabdosia glaucocalyx root extract, an extraction and separation method and application thereof, belongs to the technical field of plant extraction and separation, and comprises the following components: 5.61-5.93 mu g/g of 3-oxo-12-ene-28-ursolic acid, 4.75-5.37 mu g/g of ursolic acid, 4.82-5 mu g/g of oleanolic acid, 36.29-37.15 mu g/g of glaucocalyxin A, 3.2-3.52 mu g/g of rabdosia rubescens A, 2.72-2.86 mu g/g of sesamin, 11.32-11.76 mu g/g of glaucocalyxin B, 6.79-7.51 mu g/g of quercetin, 6.23-6.63 mu g/g of luteolin and 4.49-5.03 mu g/g of apigenin; the rabdosia japonica root extract can be used for preparing functional health products for reducing blood sugar, whitening skin, benefiting intelligence and strengthening brain, and has a good application prospect.

Description

Glaucocalyx rabdosia root extract, extraction and separation method and application thereof
Technical Field
The invention belongs to the technical field of plant extraction and separation, and particularly relates to a rabdosia japonica root extract, an extraction and separation method and application thereof.
Background
Isodon is a perennial herb of the family Labiatae (Labiatae) and the subfamily Ocimum (Ocimoideae). There is a very common growth situation of this genus around the world, up to over a hundred species, which is common in africa and in (sub) tropical regions of asia, with the most north distribution seen in days, korea and russia etc., although there is also less distribution in malaysia and islands nearby. In China, the plant of the genus is nearly hundred species, comprises more than twenty varieties, can grow in all places of China, and is most abundant in variety and quantity especially in the southwest region. Common varieties found in daily life include Rabdosia trichocarpa (Burm.f.) Hara), Rabdosia urophylla (Rabdosia excisa Hara), Rabdosia hosperma (Rabdosia henryi (Hemsl.) Hara), Rabdosia glaucocalyx (Rabdosia japonica var. glaucocalyx (Maxim.) Hara), and the like.
The Rabdosia plant has a very long planting history on the land of our ancestors, and the earliest record is traced to the 'saving and waster materia Medica' completed by the phoenix-tail weeds in the Ming dynasty. In the long history of Chinese herbal medicine, Rabdosia plants have been used for the treatment of various difficult and complicated diseases, and it is described that not less than 30 kinds of plants are used by people in daily life. The plant can be used for treating enteritis, jaundice hepatitis, pharyngolaryngitis, and red swelling and sore by topical application. The rabdosia has the effects of clearing heat, improving eyesight, detoxifying, inhibiting bacteria, eliminating inflammation, relaxing collaterals, promoting blood circulation, removing blood stasis, resisting tumors and the like, is always valued by medical personnel, is derived from the unique high-performance medicinal capacity of the rabdosia, and is deeply and carefully researched by scientific researchers in China and other countries for many years to separate and extract various compounds, and further discover components with excellent biological activity to perform corresponding pharmacological activity experiments.
Although there is a wide research on the active ingredients of the rabdosia plants, the chemical ingredients of the rabdosia plants are various and have significant bioactive functions, and the rabdosia plants are abundant in types and various in structures, and do not lack the characteristic compounds with novel structures, so further research is still needed.
Disclosure of Invention
The invention provides a rabdosia glaucocalyx root extract, an extraction and separation method and application thereof.
The first purpose of the invention is to provide a rabdosia glaucocalyx root extract, which comprises the following components: 5.61-5.93 mu g/g of 3-oxo-12-ene-28-ursolic acid, 4.75-5.37 mu g/g of ursolic acid, 4.82-5 mu g/g of oleanolic acid, 36.29-37.15 mu g/g of glaucocalyxin A, 3.2-3.52 mu g/g of rabdosia rubescens A, 2.72-2.86 mu g/g of sesamin, 11.32-11.76 mu g/g of glaucocalyxin B, 6.79-7.51 mu g/g of quercetin, 6.23-6.63 mu g/g of luteolin and 4.49-5.03 mu g/g of apigenin.
The second purpose of the invention is to provide the extraction and separation method of the rabdosia japonica root extract, which comprises the following steps:
s1: the preparation method comprises the following steps of (1) placing rabdosia glaucocalyx powder in an ethanol solution with the volume concentration of 90% for leakage extraction for multiple times, combining filtrates, and concentrating to obtain an extract;
s2: re-suspending the extract in water, adding ethyl acetate solution, extracting for multiple times, mixing ethyl acetate layer solutions, and concentrating to obtain ethyl acetate extract;
s3: separating the ethyl acetate extract in S2 by silica gel packed column, and performing gradient elution with dichloromethane-methanol solution at volume ratio of 100:1-0:1 to obtain nine fractions;
nine of the fractions were: eluting fraction Fr1 when the volume ratio of dichloromethane-methanol solution is 100:1-80: 1; eluting fraction Fr2 when the volume ratio of dichloromethane-methanol solution is 80:1-70: 1; eluting fraction Fr3 when the volume ratio of dichloromethane-methanol solution is 70:1-55: 1; eluting fraction Fr4 when the volume ratio of dichloromethane-methanol solution is 55:1-45: 1; eluting fraction Fr5 when the volume ratio of dichloromethane-methanol solution is 45:1-35: 1; eluting fraction Fr6 when the volume ratio of dichloromethane-methanol solution is 35:1-25: 1; eluting fraction Fr7 when the volume ratio of dichloromethane-methanol solution is 25:1-15: 1; eluting fraction Fr8 when the volume ratio of dichloromethane-methanol solution is 15:1-7: 1; eluting fraction Fr9 when the volume ratio of dichloromethane-methanol solution is 7:1-0: 1;
s4: performing column separation on the nine fractions obtained in S3, eluting, and respectively preparing quercetin, sesamin, apigenin, glaucocalyxin B, glaucocalyxin A, luteolin, ursolic acid, rubescensin A, 3-oxo-12-ene-28-ursolic acid, and oleanolic acid.
Preferably, the quercetin and the sesamin are obtained by separating the following steps:
the fraction Fr4 was subjected to column chromatography using 100-200 mesh silica gel as packing, wet-packed, eluted with dichloromethane and methanol as solvents in a gradient of 60:1 to 0:1, and combined to give 5 fractions: obtaining Fr41 when the volume ratio of dichloromethane to methanol is 60:1-30: 1; when the volume ratio of the dichloromethane to the methanol is 30:1-20:1, Fr42 is obtained; obtaining Fr43 when the volume ratio of dichloromethane to methanol is 20:1-15: 1; obtaining Fr44 when the volume ratio of dichloromethane to methanol is 15:1-10: 1; obtaining Fr45 when the volume ratio of dichloromethane to methanol is 10:1-5: 1;
fr43 was separated by reverse phase column CHP with a gradient of water-methanol solution as eluent and a gradient of 100: 0-0: 100, when the volume concentration of the methanol is 60 percent, obtaining the quercetin; fr44 was separated by reverse phase column CHP with a gradient of water-methanol solution as eluent and a gradient of 100: 0-0: 100, and obtaining the sesamin when the volume concentration of the methanol is 50: 1.
Preferably, the apigenin and the glaucocalyxin B are obtained by the following steps:
fraction Fr5 was chromatographed on 200-300 mesh silica gel column, wet loaded, eluting with dichloromethane and methanol solvent as eluent, in a gradient of 50:1 to 0:1, and combined to give 4 fractions: obtaining Fr51 when the volume ratio of dichloromethane to methanol is 50:1-40: 1; obtaining Fr52 when the volume ratio of dichloromethane to methanol is 40:1-25: 1; obtaining Fr53 when the volume ratio of dichloromethane to methanol is 25:1-12: 1; obtaining Fr54 when the volume ratio of dichloromethane to methanol is 12:1-0: 1;
repeatedly performing column chromatography on Fr53 with 300-400-mesh silica gel, eluting with ethyl acetate-petroleum ether as solvent with gradient of 15:1-0:1, obtaining apigenin when the volume ratio of ethyl acetate to petroleum ether is 10:1, and performing column chromatography on the mixture of ethyl acetate: when the volume ratio of the petroleum ether is 3:1, the glaucocalyxin B is obtained.
Preferably, the glaucocalyxin A and luteolin are obtained by the following steps:
the fraction Fr6 was chromatographed on 100-200 mesh silica gel using a wet loading in dichloromethane and methanol with a gradient of 40:1 to 0:1, and the 4 fractions were combined: when the volume ratio of the dichloromethane to the methanol is 40:1-30:1, Fr61 is obtained; when the volume ratio of the dichloromethane to the methanol is 30:1-20:1, Fr62 is obtained; when the volume ratio of the dichloromethane to the methanol is 20:1-10:1, Fr63 is obtained; when the volume ratio of the dichloromethane to the methanol is 10:1-5:1, Fr64 is obtained;
fr63 is separated by reversed phase column CHP, the elution gradient is 80% -20% of water-methanol solution, when the volume concentration of methanol is 50%, the glaucocalyxin A is obtained, when the volume concentration of methanol is 10%, the luteolin is obtained.
Preferably, the ursolic acid and the oridonin are obtained by separation through the following steps:
the fraction Fr7 was subjected to column chromatography on 200-300 mesh silica gel, wet loaded with dichloromethane and methanol as solvents with a gradient of 40:1 to 0:1, and combined to give 5 fractions: when the volume ratio of the dichloromethane to the methanol is 40:1-30:1, Fr71 is obtained; when the volume ratio of the dichloromethane to the methanol is 30:1-25:1, Fr72 is obtained; when the volume ratio of the dichloromethane to the methanol is 25:1-20:1, Fr73 is obtained; when the volume ratio of the dichloromethane to the methanol is 20:1-10:1, Fr74 is obtained; when the volume ratio of the dichloromethane to the methanol is 10:1-4:1, Fr75 is obtained;
separating Fr74 with Sephadex LH-20 reversed phase column, wet loading, eluting with water-methanol solution 80-20%, obtaining ursolic acid when the volume concentration of methanol is 60%, and obtaining rubescensine A when the volume concentration of methanol is 20%.
Preferably, the 3-oxo-12-en-28-ursolic acid and oleanolic acid are isolated by the following steps:
the fraction Fr8 was subjected to column chromatography on 100-200 mesh silica gel, stirred over silica gel with the elution solvent dichloromethane in a gradient of 30:1 to 0:1, and combined to give 4 fractions: when the volume ratio of the dichloromethane to the methanol is 30:1-25:1, Fr81 is obtained; when the volume ratio of the dichloromethane to the methanol is 25:1-15:1, Fr82 is obtained; when the volume ratio of the dichloromethane to the methanol is 15:1-8:1, Fr83 is obtained; when the volume ratio of the dichloromethane to the methanol is 8:1-0:1, Fr84 is obtained;
separating Fr83 with Sephadex LH-20 reversed phase column, wet loading, eluting with water-methanol solution 100-20%, and obtaining 3-oxo-12-ene-28-ursolic acid when the volume concentration of methanol is 70%; when the volume concentration of the methanol is 40 percent, the oleanolic acid is obtained.
The third purpose of the invention is to provide the application of the rabdosia japonica root extract in preparing whitening products.
The fourth purpose of the invention is to provide the application of the rabdosia japonica root extract in preparing the intelligence-improving and brain-strengthening health care product.
The fifth purpose of the invention is to provide the application of the rabdosia japonica root extract in preparing the hypoglycemic medicament.
Compared with the prior art, the invention has the beneficial effects that:
the rabdosia glaucocalyx root extract has good tyrosinase inhibitory activity and acetylcholinesterase inhibitory activity, can be used for preparing functional health products for reducing blood sugar, whitening, benefiting intelligence, strengthening brain and the like, has a good application prospect, and obviously expands the application value of the rabdosia glaucocalyx root.
Detailed Description
Several embodiments of the present invention are described in detail below, but it should be understood that the scope of the present invention is not limited to the embodiments.
A method for extracting and separating a rabdosia japonica root extract comprises the following steps:
s1, crushing 8.173kg of dried rabdosia japonica roots into powder, performing leakage extraction by using an ethanol solution with the volume concentration of 90%, fully immersing a sample in the solvent, pouring the solvent while stirring the sample, soaking for 48 hours, stirring during soaking to prevent the sample from settling and aggregating, promoting the sample and the solvent to be fully mixed, then starting to permeate, respectively collecting primary filtrate and primary filter residue, taking the primary filtrate as an extracting solution, performing leakage extraction twice on the primary filter residue according to the steps, respectively collecting secondary filtrate and the secondary extracting solution, combining the three extracting solutions, and performing reduced pressure concentration at 45 ℃ and under the condition of 0.10Mpa to obtain an extract;
s2, re-suspending the extract in S1 in water, adding ethyl acetate with the volume 2 times that of the extract water solution for extraction, uniformly mixing, standing, layering, retaining the ethyl acetate layer solution, repeatedly extracting for 2 times, combining the ethyl acetate layer solutions obtained by 3 times of extraction, and performing rotary evaporation to obtain an ethyl acetate extract;
wherein, the conditions of reduced pressure concentration are as follows: 45 deg.C and 0.10 MPa.
S3, separating the ethyl acetate extract in the S2 by a silica gel packed column, and then performing gradient elution by using dichloromethane-methanol solution (100:1-0:1) to obtain nine fractions: fr1 (dichloromethane-methanol solution (100:1-80:1), Fr2 (dichloromethane-methanol solution (80:1-70:1), Fr3 (dichloromethane-methanol solution (70:1-55:1), Fr4 (dichloromethane-methanol solution (55:1-45:1), Fr5 (dichloromethane-methanol solution (45:1-35:1), Fr6 (dichloromethane-methanol solution 35:1-25:1), Fr7 (dichloromethane-methanol solution (25:1-15:1), Fr8 (dichloromethane-methanol solution (15:1-7:1), Fr9 (dichloromethane-methanol solution (7:1-0: 1);
fr4 using 100-200 mesh silica gel as packing material for column chromatography, wet loading, eluting with dichloromethane and methanol as solvent with gradient of 60:1-0:1, combining to obtain 5 parts: fr41 (dichloromethane: methanol volume ratio of 60:1-30:1), Fr42 (dichloromethane: methanol volume ratio of 30:1-20:1), Fr43 (dichloromethane: methanol volume ratio of 20:1-15:1), Fr44 (dichloromethane: methanol volume ratio of 15:1-10:1), Fr45 (dichloromethane: methanol volume ratio of 10:1-5: 1); separating Fr43 (dichloromethane: methanol volume ratio of 20:1-15:1) with reversed phase column CHP, wherein the elution gradient is water-methanol solution (100% -0%), and when the methanol volume concentration is 60%, obtaining compound 8; and (3) performing separation on Fr44 (dichloromethane: methanol volume ratio of 15:1-10:1) by using a reverse phase column CHP, wherein the elution gradient is water-methanol solution (100% -0%), and when the volume concentration of methanol is 50%, obtaining the compound 6.
Fr5 was chromatographed on 200-mesh 300-mesh silica gel using a wet loading gradient of 50:1 to 0:1 eluting with dichloromethane and methanol as eluent to give 4 fractions: fr51 (dichloromethane: methanol volume ratio is 50:1-40:1), Fr52 (dichloromethane: methanol volume ratio is 40:1-25:1), Fr53 (dichloromethane: methanol volume ratio is 25:1-12:1), Fr54 (dichloromethane: methanol volume ratio is 12:1-0: 1); and (2) performing repeated column chromatography on Fr53 (dichloromethane: methanol volume ratio is 25:1-12:1) by using 300-400-mesh silica gel, eluting by using ethyl acetate-petroleum ether as a solvent, and obtaining a compound 10 when the volume ratio of the ethyl acetate to the petroleum ether is 10:1 and a compound 7 when the volume ratio of the ethyl acetate to the petroleum ether is 3:1 in a gradient (15:1-0: 1).
Fr6 was separated by 100-pass 200 mesh silica gel column chromatography, wet loading in dichloromethane and methanol with a gradient of 40:1 to 0:1, and combining to give 4 fractions: fr61 (dichloromethane: methanol volume ratio is 40:1-30:1), Fr62 (dichloromethane: methanol volume ratio is 30:1-20:1), Fr63 (dichloromethane: methanol volume ratio is 20:1-10:1), Fr64 (dichloromethane: methanol volume ratio is 10:1-5: 1); fr63 (dichloromethane: methanol volume ratio of 20:1-10:1) is separated by a reversed phase column CHP, the elution gradient is water-methanol solution (80% -20%), when the methanol volume concentration is 50%, the compound 4 is obtained, and when the methanol volume concentration is 10%, the compound 9 is obtained.
Fr7 was separated by column chromatography on 200-300 mesh silica gel with wet loading in dichloromethane and methanol at a gradient of 40:1 to 0:1, and the 5 fractions were combined: fr71 (dichloromethane: methanol volume ratio is 40:1-30:1), Fr72 (dichloromethane: methanol volume ratio is 30:1-25:1), Fr73 (dichloromethane: methanol volume ratio is 25:1-20:1), Fr74 (dichloromethane: methanol volume ratio is 20:1-10:1), Fr75 (dichloromethane: methanol volume ratio is 10:1-4: 1); separating Fr74 (dichloromethane: methanol volume ratio is 20:1-10:1) with Sephadex LH-20 reversed phase column, wet loading, eluting with water-methanol solution (80% -20%), and obtaining compound 2 when methanol volume concentration is 60%; when the volume concentration of the methanol is 20 percent, the compound 5 is obtained.
Fr8 was subjected to column chromatography using 100-200 mesh silica gel, which was stirred with dichloromethane and eluted with a gradient of 30:1 to 0:1, to give 4 fractions: fr81 (dichloromethane: methanol volume ratio of 30:1-25:1), Fr82 (dichloromethane: methanol volume ratio of 25:1-15:1), Fr83 (dichloromethane: methanol volume ratio of 15:1-8:1), Fr84 (dichloromethane: methanol volume ratio of 8:1-0: 1); separating Fr83 (dichloromethane: methanol volume ratio is 15:1-8:1) with Sephadex LH-20 reversed phase column, wet loading, eluting with water-methanol solution (100% -20%), and obtaining compound 1 when the volume concentration of methanol is 70%; when the volume concentration of the methanol is 40 percent, obtaining a compound 3; in conclusion, 10 rabdosia japonica root extracts are obtained by the co-separation of (1) to (10).
In order to identify which substance the 10 rabdosia glaucocalyx root extracts separated in the above extraction method are, the chemical identification method, the HPLC and the NMR method are used for identification, and the spectrogram data is as follows:
chemical combinationAn object 1:13C-NMR(400MHz,CD3OD),36.82(C-1),33.83(C-2),206.14(C-3),46.65(C-4),54.82(C-5),18.09(C-6),31.78(C-7),39.23(C-8),45.65(C-9),36.18(C-10),22.94(C-11),38.14(C-12),140.91(C-13),43.89(C-14),28.46(C-15),22.17(C-16),49.84(C-17),53.53(C-18),39.79(C-19),37.03(C-20),29.89(C-21),35.83(C-22),25.14(C-23),22.65(C-24),16.42(C-25),17.09(C-26),18.98(C-27),183.30(C-28),16.65(C-29),17.81(C-30)。
compound 2:13C-NMR(400MHz,CD3OD),40.89(C-1),22.83(C-2),76.58(C-3),41.65(C-4),52.42(C-5),18.09(C-6),33.98(C-7),40.30(C-8),47.65(C-9),36.96(C-10),23.94(C-11),124.14(C-12),138.91(C-13),41.89(C-14),28.43(C-15),22.17(C-16),47.84(C-17),54.53(C-18),31.79(C-19),28.03(C-20),28.89(C-21),36.83(C-22),23.14(C-23),17.65(C-24),17.42(C-25),15.09(C-26),24.98(C-27),178.30(C-28),21.65(C-29),23.81(C-30)。
compound 3:13C-NMR(400MHz,CD3OD),36.89(C-1),27.83(C-2),76.14(C-3),38.65(C-4),54.42(C-5),19.09(C-6),31.98(C-7),39.30(C-8),49.65(C-9),36.81(C-10),21.94(C-11),123.14(C-12),146.91(C-13),43.89(C-14),28.39(C-15),29.17(C-16),45.84(C-17),41.53(C-18),47.79(C-19),31.03(C-20),35.89(C-21),33.83(C-22),26.14(C-23),15.65(C-24),14.42(C-25),16.09(C-26),28.98(C-27),179.30(C-28),34.65(C-29),21.81(C-30)。
compound 4:13C-NMR(400MHz,CD3OD),36.82(C-1),32.83(C-2),216.12(C-3),45.65(C-4),50.42(C-5),27.09(C-6),72.98(C-7),59.30(C-8),51.65(C-9),36.81(C-10),18.94(C-11),30.04(C-12),44.91(C-13),73.83(C-14),206.39(C-15),146.17(C-16),115.84(C-17),27.53(C-18),19.79(C-19),17.03(C-20)。
compound 5:13C-NMR(400MHz,CD3OD),71.89(C-1),29.83(C-2),37.14(C-3),33.65(C-4),60.42(C-5),72.09(C-6),96.98(C-7),62.30(C-8),55.65(C-9),41.81(C-10),20.94(C-11),30.14(C-12),42.91(C-13),73.89(C-14),208.39(C-15),152.17(C-16),118.84(C-17),21.53(C-18),31.79(C-19),62.03(C-20)。
compound 6:13C-NMR(400MHz,DMSO),164.47(C-2),103.39(C-3),181.42(C-4),148.38(C-5),128.85(C-6),153.52(C-7),92.04(C-8),145.70(C-9),103.64(C-10),121.55(C-1′),115.20(C-2′),144.79(C-3′),148.21(C-4′),115.23(C-5′),120.55(C-6′)。
compound 7:13C-NMR(400MHz,CD3OD),36.82(C-1),32.83(C-2),215.27(C-3),48.64(C-4),50.42(C-5),29.09(C-6),72.17(C-7),62.30(C-8),54.65(C-9),36.81(C-10),18.41(C-11),33.04(C-12),41.82(C-13),74.79(C-14),104.46(C-15),144.30(C-16),115.96(C-17),27.53(C-18),19.79(C-19),116.70(C-20),20.54(C-21),171.29(-OAc)。
compound 8:13C-NMR(400MHz,DMSO),146.47(C-2),135.39(C-3),175.42(C-4),160.38(C-5),97.85(C-6),163.52(C-7),92.94(C-8),155.70(C-9),102.64(C-10),121.55(C-1′),115.24(C-2′),144.68(C-3′),147.21(C-4′),115.23(C-5′),121.55(C-6′)。
compound 9:13C-NMR(400MHz,DMSO),163.9(C-2),103.9(C-3),181.6(C-4),157.3(C-5),98.9(C-6),164.2(C-7),93.9(C-8),161.5(C-9),103.7(C-10),119.1(C-1′),113.4(C-2′),145.8(C-3′),149.7(C-4′),115.9(C-5′),124.4(C-6′)。
compound 10:13C-NMR(400MHz,DMSO),163.2(C-2),102.3(C-3),181.2(C-4),156.8(C-5),98.4(C-6),163.7(C-7),93.7(C-8),160.9(C-9),110.0(C-10),120.9(C-1′),128.2(C-2′),115.8(C-3′),161.2(C-4′),115.6(C-5′),128.1(C-6′)。
the above data indicate that these 10 compounds are:
compound 1: 3-oxo-12-en-28-ursolic acid; compound 2: ursolic acid; compound 3: oleanolic acid; compound 4: glaucocalyxin A (4); compound 5: oridonin; compound 6: sesamin (6); compound 7: glaucocalyxin B (7); compound 8: quercetin; compound 9: luteolin (9); compound 10: apigenin (10);
the structural formula is as follows:
Figure BDA0002682936050000101
Figure BDA0002682936050000111
in order to determine the content of the compounds 1-10, the following provides an analysis method of the rabdosia glaucocalyx root extract, comprising the following steps:
(1) accurately weighing 1.0mg of the above compound standard substance with an analytical balance to 0.001g, dissolving with 0.2mL of methanol, diluting to a constant volume of 1.0mL in a volumetric flask, and freezing for storage. Respectively taking 10 mu L, 50 mu L, 100 mu L, 150 mu L and 200 mu L of the standard solutions to a 1.0mL volumetric flask, fixing the volume with methanol, uniformly mixing to prepare a series of standard solutions with the concentrations of 10mg/L, 50mg/L, 100mg/L, 150mg/L and 200mg/L, and storing in a refrigerator at 4 ℃ for later use.
(2) Respectively detecting 10 standard substance solutions with the concentration of 50mg/L by using a high performance liquid chromatograph, selecting different mobile phase gradient elution methods, continuously adjusting chromatographic conditions according to a detection structure, and determining optimal chromatographic conditions and absorption wavelengths, wherein the optimal chromatographic conditions are as follows: a chromatographic column: phenomenex Luna C-18(250 mm. times.4.6 mm, 5 μm), mobile phase: acetonitrile-water (containing 0.2% phosphoric acid) system, flow rate: 1mL/min, column temperature: 30 ℃, sample introduction: 15 μ L, detection wavelength: 210nm, 230nm and 260 nm.
Analyzing the series of standard solutions of the prepared compounds by using a high performance liquid chromatograph, wherein the sample amount is 15 mu L, respectively recording the peak area of each compound, and drawing a standard curve by taking the concentration of the standard solution as a horizontal coordinate and the peak area as a vertical coordinate. The results show that the peak area and the concentration of each compound are in good linear relation when the concentration is in the range of 0.2-1000 mu g/mL, and R2All values are greater than 0.99. Wherein, the high performance liquid chromatography conditions are as shown in table 1:
TABLE 1 high Performance liquid chromatography conditions
Figure BDA0002682936050000121
A chromatographic column: phenomenex Luna C-18(250 mm. times.4.6 mm, 5 μm)
Column temperature: 30 deg.C
Mobile phase: a (0.2% phosphoric acid in water) -B (acetonitrile)
Flow rate: 1mL/min
Sample introduction amount: 15 μ L
Detection wavelength: 210nm, 230nm and 260 nm.
The standard curves and their parameters for each compound are shown in table 2:
TABLE 2 Standard Curve formula of Rabdosia glaucocalyx extract
Figure BDA0002682936050000122
Figure BDA0002682936050000131
(3) Accurately weighing dried powder of rabdosia japonica root 5.0g, adding 30mL of methanol as an extracting solution, performing ultrasonic treatment for 1h, and filtering; concentrating the filtrate under vacuum, diluting to 5mL, filtering the supernatant with 0.45 μm microporous membrane, collecting 1mL, injecting into HPLC sample bottle, and storing at 4 deg.C.
Precisely sucking 15 μ L of each reference solution and test solution, detecting with high performance liquid chromatograph, finding out the corresponding peak of each reference compound according to the detection result, recording the peak area, substituting the peak area into the standard curve, and calculating the content of each compound respectively, wherein the result is shown in Table 3.
TABLE 3 content of 1-10 compounds in the root of Isodon japonicus
Figure BDA0002682936050000132
To verify the efficacy of the rabdosia glaucocalyx root extract of the present invention, the following experiments were performed:
(1) tyrosinase activity inhibition assay
The determination method comprises the following steps: accurately weighing and preparing 1mg/mL of L-tyrosine standard solution, adding 1mg of tyrosinase into 5mLPBS to prepare 100U/mL of tyrosinase solution, and storing at 4 ℃ for later use.
Preparing the medicines into 1.0mg/mL solution to be detected by using a methanol solution, and adding 80 mu L of 0.1mol/L phosphate buffer, 50 mu L of solvent and 50 mu L of tyrosinase into 1 and 2 rows of wells in a 96-well plate to serve as a blank group; 3. 130 mu L of 0.1mol/L phosphate buffer and 50 mu L of solvent are added into the 4 rows of wells to be used as a blank background group; 5. 80 mu L of 0.1mol/L phosphate buffer solution, 50 mu L of sample to be detected and 50 mu L of tyrosinase are added into the 6 rows of holes to be used as experimental groups; 7. 130 mu L of 0.1mol/L phosphate buffer solution and 50 mu L of samples to be detected are added into 8 rows of holes to be used as experiment background groups; finally, 20. mu.L of substrate (L-tyrosine) was added to each well to trigger the reaction, and the 96-well plate was incubated at 37 ℃ for 30min, followed by measurement of absorbance at 475nm using a microplate reader, and kojic acid as a positive control, with the results as shown in Table 4:
inhibition rate [ < 1- (C) >Experiment of-DBackground of the experiment)/(ABlank space-BBlank background)]×100％
(2) Acetylcholinesterase activity inhibition assay
The determination method comprises the following steps: the frozen acetylcholine esterase powder was dissolved in 0.01mol/L phosphate buffer to prepare 5U/mL enzyme solution, and then 15mM ACTI solution and 2mM DTNB solution were prepared.
Preparing the extract into a solution with the concentration of 1.0mg/mL by using a methanol solution, and adding 140 mu L of 0.1mol/L phosphate buffer solution, 20 mu L of a sample to be detected and 20 mu L of acetylcholinesterase into 1 and 2 rows of wells in a 96-well plate to serve as a sample group; 3. 160 mu L of 0.1mol/L phosphate buffer solution and 20 mu L of samples to be detected are added into the 4 rows of holes to be used as control groups; 5. a blank group was prepared by adding 160. mu.L of 0.1mol/L phosphate buffer (pH 7.4) and 20. mu.L acetylcholinesterase to each well of 6 rows.
After incubation at 4 ℃ for 20min, 10. mu.L of 15mM ACTI and 10. mu.L of 2mM DTNB were added to each well, and after incubation at 37 ℃ for 20min, the absorbance at 412nm was measured using a microplate reader. Huperzine a was used as a positive control, and the results are shown in table 4:
inhibition ratio [ < 1- (OD)% ]Sample (I)-ODControl of)/ODBlank space]×100％
(3) Alpha-glucosidase activity inhibition assay
The determination method comprises the following steps: accurately weighing PNPG glucoside, fully dissolving the PNPG glucoside by using 0.1mol/L phosphate buffer solution, and preparing 2.5mmol/L substrate PNPG glucoside solution after constant volume; dissolving the frozen alpha-glucosidase powder by 0.01mol/L phosphate buffer solution to prepare 2U/mL enzyme solution, and then preparing 0.2mol/L sodium carbonate solution.
Preparing the extract in the embodiment 1 into a solution to be detected of 1.0mg/mL by using a methanol solution, and adding 20 mu L of 0.1mol/L phosphate buffer solution, 20 mu L of a sample to be detected and 20 mu L of alpha-glucosidase into 1 and 2 rows of wells in a 96-well plate to serve as a sample group; 3. adding 40 mu L of 0.1mol/L phosphate buffer solution and 20 mu L of sample to be detected into the 4 rows of holes to be used as a control group; 5. 40. mu.L of 0.1mol/L phosphate buffer and 20. mu.L of alpha-glucosidase were added to the 6 wells as a blank.
Culturing at 37 deg.C for 15min, adding 20 μ L of 2.5mmol/L PNPG glycoside per well, culturing at 37 deg.C for 15min, adding 80 μ L of 0.2mol/L Na per well2CO3The reaction is stopped by the solution, and the light absorption value at the wavelength of 405nm is measured by a microplate reader. Acarbose was used as a positive control, and the results are given in table 4:
inhibition ratio [ < 1- (OD)% ]Sample (I)-ODControl of)/ODBlank space]×100％
TABLE 4 inhibitory Activity of the Compounds on the three enzymes
Figure BDA0002682936050000151
The results in table 4 show that apigenin, luteolin, sesamin and quercetin have tyrosinase inhibitory activity, tyrosinase has a unique catalytic function, is a key enzyme for controlling melanin generation of organisms, is closely related to aging and beauty treatment, and can be used for common pigmentation skin diseases.
Apigenin, luteolin, sesamin, quercetin and oleanolic acid have acetylcholinesterase inhibitory activity, the acetylcholinesterase is a key enzyme in biological nerve conduction, and the enzyme can degrade acetylcholine between cholinergic synapses, stop the excitation of neurotransmitters on postsynaptic membranes and ensure the normal transmission of nerve signals in organisms. The acetylcholinesterase participates in the development and maturation of cells, can promote the development and nerve regeneration of neurons, and has the effects of improving intelligence and strengthening brain, so the rabdosia glauca root extract has the effects of improving intelligence and strengthening brain.
Apigenin, luteolin, sesamin and quercetin have alpha-glucosidase inhibitory activity, glucosidase is an important member in a carbohydrate metabolism path in an organism, the alpha-glucosidase directly participates in a starch and glycogen metabolism path, and the chemical metabolism of sugar can be reduced by inhibiting the alpha-glucosidase, so that the effect of reducing blood sugar is achieved, and therefore the rabdosia japonica root extract has the effect of reducing blood sugar.
To verify the accuracy of the above experiments, the following repeatability and stability experiments were performed:
accurately weighing 5.0g of each standard substance powder, carrying out High Performance Liquid Chromatography (HPLC) detection analysis, wherein the sample amount is 15 mu L, recording the response value of the peak area of each reference substance compound, and calculating the content value of the reference substance compound through a standard, wherein the results are shown in Table 5. The RSD value of each compound is lower than 2.45, and the experimental analysis method has good repeatability.
Each stock solution of the standard substance of 1.0mg/mL is weighed and analyzed by HPLC at 0, 2, 4, 8, 12, 16, 24 and 48h respectively, the content of the compound of the standard substance is calculated by the peak area response value of each compound of the standard substance, the result is shown in Table 5, the RSD value is lower than 2.23, and the analytical method for the experiment completely has excellent stability.
TABLE 5 repeatability and stability tests
Figure BDA0002682936050000161
Figure BDA0002682936050000171
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims (10)

1. The rabdosia glaucocalyx root extract is characterized by comprising the following components: 5.61-5.93 mu g/g of 3-oxo-12-ene-28-ursolic acid, 4.75-5.37 mu g/g of ursolic acid, 4.82-5 mu g/g of oleanolic acid, 36.29-37.15 mu g/g of glaucocalyxin A, 3.2-3.52 mu g/g of rabdosia rubescens A, 2.72-2.86 mu g/g of sesamin, 11.32-11.76 mu g/g of glaucocalyxin B, 6.79-7.51 mu g/g of quercetin, 6.23-6.63 mu g/g of luteolin and 4.49-5.03 mu g/g of apigenin.
2. The method for extracting and separating the rabdosia glaucocalyx root extract according to claim 1, comprising the steps of:
s1: the preparation method comprises the following steps of (1) placing rabdosia glaucocalyx powder in an ethanol solution with the volume concentration of 90% for leakage extraction for multiple times, combining filtrates obtained multiple times, and concentrating to obtain an extract;
s2: re-suspending the extract in water, adding ethyl acetate solution, extracting for multiple times, mixing ethyl acetate layer solutions, and concentrating to obtain ethyl acetate extract;
s3: separating the ethyl acetate extract in S2 by silica gel packed column, and performing gradient elution with dichloromethane-methanol solution at volume ratio of 100-0:1 to obtain nine fractions;
nine of the fractions were: eluting fraction Fr1 when the volume ratio of dichloromethane-methanol solution is 100-80: 1; eluting fraction Fr2 when the volume ratio of dichloromethane-methanol solution is 80-70: 1; eluting fraction Fr3 when the volume ratio of dichloromethane-methanol solution is 70-55: 1; eluting fraction Fr4 when the volume ratio of dichloromethane-methanol solution is 55-45: 1; eluting fraction Fr5 when the volume ratio of dichloromethane-methanol solution is 45-35: 1; eluting fraction Fr6 when the volume ratio of dichloromethane-methanol solution is 35-25: 1; eluting fraction Fr7 when the volume ratio of dichloromethane-methanol solution is 25-15: 1; eluting fraction Fr8 when the volume ratio of dichloromethane-methanol solution is 15-7: 1; eluting fraction Fr9 when the volume ratio of dichloromethane-methanol solution is 7-0: 1;
s4: performing column separation on the nine fractions obtained in S3, eluting, and respectively preparing quercetin, sesamin, apigenin, glaucocalyxin B, glaucocalyxin A, luteolin, ursolic acid, rubescensin A, 3-oxo-12-ene-28-ursolic acid, and oleanolic acid.
3. The method for extracting and separating rabdosia glaucocalyx root extract according to claim 2, wherein the quercetin and the sesamin are obtained by the following steps:
performing column chromatography on the fraction Fr4 by using 100-200 mesh silica gel as a packing material, performing wet column packing, performing elution by using a mixed solvent of dichloromethane and methanol with a gradient of 60-0:1, and combining to obtain 5 parts: when the volume ratio of the dichloromethane to the methanol is 60-30:1, Fr41 is obtained; when the volume ratio of the dichloromethane to the methanol is 30-20:1, Fr42 is obtained; when the volume ratio of the dichloromethane to the methanol is 20-15:1, Fr43 is obtained; when the volume ratio of the dichloromethane to the methanol is 15-10:1, Fr44 is obtained; when the volume ratio of the dichloromethane to the methanol is 10-5:1, Fr45 is obtained;
fr43 was separated by reverse phase column CHP with a gradient of water-methanol solution as eluent and a gradient of 100: 0 to 0: 100, when the volume concentration of the methanol is 60 percent, obtaining the quercetin; fr44 was separated by reverse phase column CHP with a gradient of water-methanol solution as eluent and a gradient of 100: 0 to 0: 100, and obtaining the sesamin when the volume concentration of the methanol is 50 percent.
4. The method for extracting and separating the rabdosia glaucocalyx root extract as claimed in claim 2, wherein the apigenin and the glaucocalyxin B are separated by the following steps:
fraction Fr5 was chromatographed on 200-300 mesh silica gel column, wet loaded, eluted with a mixed eluent of dichloromethane and methanol at a gradient of 50-0:1, and combined to give 4 fractions: when the volume ratio of dichloromethane to methanol is 50-40:1, Fr51 is obtained; when the volume ratio of dichloromethane to methanol is 40-25:1, Fr52 is obtained; when the volume ratio of the dichloromethane to the methanol is 25-12:1, Fr53 is obtained; when the volume ratio of dichloromethane to methanol is 12-0:1, Fr54 is obtained;
repeatedly performing column chromatography on Fr53 with 300-400 mesh silica gel, eluting with ethyl acetate-petroleum ether as solvent with gradient of 15-0:1, obtaining apigenin when the volume ratio of ethyl acetate to petroleum ether is 10:1, and performing column chromatography on the mixture of ethyl acetate: when the volume ratio of the petroleum ether is 3:1, the glaucocalyxin B is obtained.
5. The method for extracting and separating the rabdosia japonica root extract according to claim 2, wherein the glaucocalyxin A and the luteolin are obtained by the following steps:
the fraction Fr6 was chromatographed on 100-200 mesh silica gel using a wet loading in dichloromethane and methanol with a gradient of 40-0:1 to give 4 fractions: when the volume ratio of the dichloromethane to the methanol is 40-30:1, Fr61 is obtained; when the volume ratio of the dichloromethane to the methanol is 30-20:1, Fr62 is obtained; when the volume ratio of the dichloromethane to the methanol is 20-10:1, Fr63 is obtained; when the volume ratio of the dichloromethane to the methanol is 10-5:1, Fr64 is obtained;
fr63 is separated by reversed phase column CHP, the elution gradient is 80% -20% of water-methanol solution, when the volume concentration of methanol is 50%, glaucocalyxin A is obtained, and when the volume concentration of methanol is 10%, luteolin is obtained.
6. The method for extracting and separating the rabdosia japonica root extract according to claim 2, wherein the ursolic acid and the rubescensine A are obtained by the following steps:
subjecting fraction Fr7 to column chromatography with 200-300 mesh silica gel, loading by wet method, eluting with mixed solvent of dichloromethane and methanol at gradient of 40-0:1, and combining to obtain 5 fractions: when the volume ratio of the dichloromethane to the methanol is 40-30:1, Fr71 is obtained; when the volume ratio of the dichloromethane to the methanol is 30-25:1, Fr72 is obtained; when the volume ratio of the dichloromethane to the methanol is 25-20:1, Fr73 is obtained; when the volume ratio of the dichloromethane to the methanol is 20-10:1, Fr74 is obtained; when the volume ratio of the dichloromethane to the methanol is 10-4:1, Fr75 is obtained;
separating Fr74 with Sephadex LH-20 reversed phase column, wet loading, eluting with water-methanol solution 80-20%, obtaining ursolic acid when the volume concentration of methanol is 60%, and obtaining rubescensine A when the volume concentration of methanol is 20%.
7. The method for extracting and separating the rabdosia japonica root extract according to claim 2, wherein the 3-oxo-12-en-28-ursolic acid and oleanolic acid are obtained by the steps of:
subjecting fraction Fr8 to column chromatography with 100-200 mesh silica gel, mixing with dichloromethane, and gradient of 30-0:1, and combining to obtain 4 fractions: when the volume ratio of the dichloromethane to the methanol is 30-25:1, Fr81 is obtained; when the volume ratio of the dichloromethane to the methanol is 25-15:1, Fr82 is obtained; when the volume ratio of the dichloromethane to the methanol is 15-8:1, Fr83 is obtained; when the volume ratio of the dichloromethane to the methanol is 8-0:1, Fr84 is obtained;
separating Fr83 with Sephadex LH-20 reversed phase column, wet loading, eluting with water-methanol solution 100-20%, and obtaining 3-oxo-12-ene-28-ursolic acid when the volume concentration of methanol is 70%; when the volume concentration of the methanol is 40 percent, the oleanolic acid is obtained.
8. The use of rabdosia glaucocalyx root extract according to claim 1 for preparing a whitening product.
9. The use of rabdosia glaucocalyx root extract according to claim 1 for preparing a brain-strengthening and intelligence-improving health product.
10. The use of the rabdosia glaucocalyx root extract according to claim 1 for the preparation of a hypoglycemic medicament.
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