CN110204589B - Active ingredients, extraction method and application in the preparation of neuroprotective drugs - Google Patents
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Abstract
本发明涉及青葙子有效成分，其特征在于，包括齐墩果酸型三萜皂苷类化合物和/或苯乙腈苷类化合物，齐墩果酸型三萜皂苷类化合物的结构式如下所示：；苯乙腈苷类化合物的结构式如下所示：
。本发明还提供了该青葙子有效成分的制备方法。本发明经试验发现：青葙子有效成分对神经细胞NSC34氧化损伤具有很好保护作用，可用作制备神经保护药物或者保健品。The present invention relates to the active ingredient of A. chinensis, which is characterized in that it comprises oleanolic acid-type triterpenoid saponins and/or phenylacetonitrile glycosides, and the structural formula of the oleanolic acid-type triterpenoid saponins is as follows: 
; The structural formula of phenylacetonitrile glycosides is as follows: . The present invention also provides a preparation method of the active ingredient of the Fructus Arugulae. According to the experiment, the invention finds that the active components of Fructus chinensis have a good protective effect on the oxidative damage of nerve cells NSC34, and can be used for preparing neuroprotective drugs or health care products.
Description
Technical Field
The invention belongs to the technical field of pharmacy, and particularly relates to an effective component of feather cockscomb seeds (oleanolic acid type triterpenoid saponins and/or benzyl cyanide glycosides compounds), an extraction method and application thereof in preparing neuroprotective drugs or health care products.
Background
Neurodegenerative diseases are characterized by progressive damage to nerve cells and loss of neurons, which worsen over time, leading to impaired motor or cognitive function, and belong to a class of progressively developing disabling, severely fatal complex diseases. Common neurodegenerative diseases include Alzheimer's Disease (AD), Amyotrophic Lateral Sclerosis (ALS), Parkinson's Disease (PD), Huntington's Disease (HD), and spinocerebellar ataxia (SCA). Although the pathology of neurodegenerative diseases remains unclear, many reports have revealed that oxidative stress plays a critical role, leading to the massive production of Reactive Oxygen Species (ROS) and DNA oxidation. In neurodegenerative disease patients, excess reactive oxygen species are often produced due to aging and mitochondrial dysfunction, which further damage proteins and DNA, and ROS are released in large quantities after apoptosis damages other cells, thereby triggering the cytotoxic domino effect, and ROS cause neurotransmission disorders and gradually increase the risk of neurodegenerative diseases. The accumulated evidence suggests that traditional Chinese medicines with neuroprotective effects can reduce the risk of suffering from neurodegenerative diseases by inhibiting oxidative stress.
The semen Celosiae (Celosiae seed) is semen Celosiae (Celosia) of genus Celosia (Amaranthaceae)Ce losia argenteaL.) dried mature seeds, a pharmacopoeia collection variety. Feather cockscomb seed is bitter in taste and slightly cold in nature, enters liver meridian, has the main functions of improving eyesight, removing nebula, clearing liver-fire, and nourishing brain and marrow recorded in Ben Cao gang mu. The stems, leaves and seedlings of the feather cockscomb can be eaten, and the feather cockscomb is an edible wild vegetable with good quality. Modern pharmacological research shows that the feather cockscomb seed has the functions of resisting oxidation, protecting liver, resisting inflammation, reducing blood sugar, resisting tumor and infection, treating cataract, mitosis and the like. The main chemical components of the feather cockscomb seed extract comprise saponin, cyclic peptide, alkaloid, phenols and the like, the feather cockscomb seed resource is distributed in most regions of China, and is also widely distributed in other tropical and subtropical countries or regions. Has important significance for developing high-efficiency and low-price nerve protection medicines or health care products and solving the problems of difficult and expensive medical care. At present, no report about the application of feather cockscomb seeds in nerve protection is available.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an effective component of feather cockscomb seeds consisting of oleanolic acid type triterpenoid saponins and/or phenylacetonitrile glycosides, an extraction method and application thereof in preparing neuroprotective medicaments.
In order to achieve the purpose, the invention adopts the following technical scheme:
the effective component of the seeds of feather cockscomb comprises an oleanolic acid type triterpene saponin compound and/or a phenylacetonitrile glycoside compound, wherein the structural formula of the oleanolic acid type triterpene saponin compound is shown as follows:
the structural formula of the phenylacetonitrile glycoside compound is shown as follows:
the preparation method of the effective component of the feather cockscomb seeds comprises the following steps:
1) performing flash extraction on the crushed feather cockscomb seeds or fried feather cockscomb seeds with 20-50% ethanol, and concentrating the extracting solution to obtain extract; dispersing the extract in water, adsorbing with macroporous adsorbent resin, sequentially gradient-eluting with water, 20% ethanol and 60% ethanol, collecting 20% ethanol eluate, and recovering solvent under reduced pressure to obtain 20% ethanol extract A; collecting 60% ethanol eluate, and recovering solvent under reduced pressure to obtain 60% ethanol extract B;
2) performing thin-layer chromatography on the 20% ethanol extract A by using a silica gel column with the volume ratio of 8: 1, eluting with dichloromethane and methanol solvent, and recovering solvent to obtain fraction; eluting with 20% methanol-water by semi-preparative HPLC chromatography for 24min to obtain phenylacetonitrile glycosides;
3) subjecting the 60% ethanol extract B to MCIGELCHP20 open column, selecting methanol-water solvent system, sequentially and respectively gradient-eluting with 15% methanol, 40% methanol, 50% methanol, 60% methanol, and 100% methanol to obtain components: b-1, B-2, B-3, B-4 and B-5; and (3) carrying out thin-layer chromatography on the component B-5 by using a silica gel column, wherein the volume ratio is 5: 1, eluting with dichloromethane and methanol solvent to obtain the oleanolic acid type triterpenoid saponin compound.
The preparation method of the effective components of semen Celosiae comprises, in step 1), flash-extracting for 2-3 times, wherein 5-10 mL of 20-50% ethanol is added into 1 g of semen Celosiae or parched semen Celosiae, and each flash-extraction time is 3-10 min.
The invention provides application of the effective component of the feather cockscomb seed in preparing neuroprotective drugs or health products.
The invention also provides application of the feather cockscomb seed effective component in preparing medicines or health products for treating neurodegenerative diseases.
Compared with the prior art, the invention has the beneficial effects that:
1) the two compounds have stable structures, belong to oleanolic acid type triterpenoid saponin compounds and phenylacetonitrile glycoside compounds respectively, and the extraction raw material is from the feather cockscomb growing on the plain or hillside bank. The feather cockscomb is widely distributed in most areas of China, and resources are very rich. The invention has mild production conditions, few experimental steps, small technical difficulty, low production cost and little environmental pollution; meanwhile, the raw materials are rich and belong to natural renewable resources; the extraction and separation technology has small difficulty, the solvent can be recycled, and the separation filler can be repeatedly used without causing ecological environment pollution;
2) the test shows that: the celosia seeds effective component containing the oleanolic acid type triterpenoid saponin compound and/or the phenylacetonitrile glycoside compound has a good protection effect on a t-BHP induced nerve cell NSC34 damage model, shows no cytotoxicity, improves cell activity, reduces the intracellular ROS level and reduces the apoptosis rate.
Drawings
FIG. 1 shows oleanolic acid type triterpene saponin compound 113C NMR spectrum;
FIG. 2 shows oleanolic acid type triterpene saponin compound 11H NMR spectrum;
FIG. 3 is an HSQC spectrum of oleanolic acid type triterpenoid saponin compound 1;
FIG. 4 is an HMBC spectrum of oleanolic acid type triterpene saponin compound 1;
FIG. 5 is a NOESY spectrum of oleanolic acid type triterpene saponin type compound 1;
FIG. 6 is an HR-TOF-MS spectrum of oleanolic acid type triterpenoid saponin compound 1;
FIG. 7 shows phenylacetonitrile glycoside compound 213C NMR spectrum;
FIG. 8 shows phenylacetonitrile glycoside compound 21H NMR spectrum;
FIG. 9 is HSQC spectrum of phenylacetonitrile glycoside compound 2;
FIG. 10 is an HMBC spectrum of phenylacetonitrile glycoside compound 2;
FIG. 11 is an HR-TOF-MS spectrum of phenylacetonitrile glycoside compound 2;
FIG. 12 is an infrared spectrum of phenylacetonitrile glycoside compound 2;
fig. 13 shows ROS imaging results in NSC34 cells.
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.
In the following examples, unless otherwise specified, methanol and ethanol are referred to as volume percentage concentrations.
The extraction raw material of the oleanolic acid type triterpenoid saponin compound and the phenylacetonitrile glycoside compound is feather cockscomb seeds (Celosiae semen), which are purchased in Yuzhou medicinal material market in Henan 10 months in 2015, identified by Yuan Wang Jun professor in Henan university, and the specimens are stored in a specimen museum of the medical institute in Henan university.
Example 1
The effective component of the seeds of feather cockscomb comprises an oleanolic acid type triterpene saponin compound and/or a phenylacetonitrile glycoside compound, wherein the structural formula of the oleanolic acid type triterpene saponin compound 1 is shown as follows:
the structural formula of the phenylacetonitrile glycoside compound 2 is shown as follows:
the preparation method of the effective components of the feather cockscomb seeds comprises the following steps:
1) performing flash extraction on 9.5kg of pulverized or fried feather cockscomb seeds with 50L 50% ethanol for 3 times (first flash extraction for 3 min, second flash extraction for 5min, and third flash extraction for 5 min), filtering after flash extraction, and concentrating the extractive solution to obtain extract; dispersing the extract in water, adsorbing with HPD100 macroporous adsorbent resin, sequentially gradient-eluting with water, 20% ethanol, and 60% ethanol, collecting 20% ethanol eluate, and recovering solvent under reduced pressure to obtain 20% ethanol extract A; collecting 60% ethanol eluate, and recovering solvent under reduced pressure to obtain 60% ethanol extract B;
2) dissolving 20% ethanol extract A with 20mL of methanol, mixing with silica gel, and performing thin-layer chromatography on the mixture by silica gel column chromatography, wherein the volume ratio is 8: eluting with dichloromethane-methanol solvent of 1, and recovering solvent to obtain fraction; eluting with 20% methanol-water by semi-preparative HPLC chromatography at flow rate of 8 ml/min for 24min to obtain phenylacetonitrile glycosides compound (35 mg) as compound 2;
3) suspending the 60% ethanol extract B in 150mL of water, passing through an MCIGELCHP20 open column, selecting a methanol-water solvent system, sequentially and respectively carrying out gradient elution by using 15% methanol, 40% methanol, 50% methanol, 60% methanol and 100% methanol, and obtaining 5 components according to the elution sequence, wherein the components are sequentially recorded as: b-1, B-2, B-3, B-4 and B-5. Dissolving the component B-5 in 15mL of methanol, mixing the mixture with silica gel, and performing thin-layer chromatography on the mixture by silica gel column chromatography, wherein the volume ratio is 5: 1, and eluting with a methanol solvent to obtain a single component, namely the oleanolic acid type triterpenoid saponin compound (56 mg), which is marked as a compound 1.
The relevant test data for compound 1 and compound 2 are given below (see in particular figures 1 to 12).
Compound 1
White powder, readily soluble in methanol, TOF-MS: M/z 826.4575[ M + NH ]4]+，831.4112[M+Na]+，847.3884 [M+K]+. UV (MeOH) showed a clear UV absorption peak at 203 nm.1H NMR and13c NMR data are shown in table 1.
TABLE 1 assignment of 100MHz carbon and 400MHz hydrogen spectra data for Compound 1
Compound 2
White powder, readily soluble in methanol. HR-TOF-MS:m/z 440.1546[M-H]-，476.1310[M+Cl]-，486.1600 [M+HCOOH-H]-. IR (KBr) v max 3425, 2923, 2254, 1591, 1512, 1236; UV (MeOH) showed a clear UV absorption peak at 220 nm.1H NMR and13see table 2 for C NMR data.
TABLE 2 100MHz carbon and 400MHz hydrogen spectra data for Compound 2
The structure of compound 1 was analyzed.
White powder, readily soluble in methanol, HR-TOF-MS: M/z 826.4575[ M + NH ]4]+，831.4112[M+Na]+，847.3884[M+K]+As shown in fig. 6. UV (MeOH) showed significant UV absorption at 203 nm.1H-NMR(CD3OD) spectrum, one aldehyde group hydrogen signal delta 9.44 (1H, s, H-23); the terminal hydrogen signals for both sugars δ 4.50 (1H, d, J =7.5Hz, H-Xyl-1), 4.38 (1H, d, J =8.0Hz, H-GlcA-1); an olefin hydrogen signal δ 5.24 (1H, s, H-12); six methyl proton signals, delta 0.81 (3H, s, H-29), 0.89 (3H, s, H-30), 0.93 (3H, s, H-26), 1.16 (3H, s, H-27), 1.28 (3H, s, H-25), 1.32 (3H, s, H-24).13C-NMR(CD3OD) spectrum, the low field region gives the three carbonyl carbon signals δ 208.8 (C-23), 181.8 (C-28), 171.1 (C-GlcA-6); olefin carbon signals delta 145.3 (C-13), 123.4 (C-12) and two sugar-terminal carbon signals delta 105.7 (C-Xyl-1), 104.6 (C-GlcA-1). Carbon and hydrogen signals were assigned by HSQC and HMBC spectra, where δ 2.83 (H-18) was assigned remotely to 181.8 (C-28), 145.3 (C-13), 123.4 (C-12), δ 1.16 (H-27) was assigned remotely to 145.3 (C-13), δ 5.24 (H-12) was assigned remotely to δ 24 (C-11), C-28 was presumed to be a carbonyl carbon, C-12, 13 to be an olefinic double bond, δ 4.24 (H-2) was presumed remotely to 37 (C-10), 55 (C-4), C-2 was presumed to be hydroxyl substituted, δ 9.44 (H-23) was presumed to be assigned remotely to 55 (C-4), 11.7 (C-24), δ 3.85(H-3) was assigned remotely to 208.8 (C-23) in the mother nucleusIn relation to the equation, it is assumed that C-23 is replaced by an aldehyde group, and that Δ 3.85(H-3) is remotely related to 104.6 (C-GlcA-6) so that glucose forms a glycosidic bond with C-3, and Δ 3.50 (H-GlcA-3) is remotely related to 105.7 (C-Xyl-1) so that xylose is linked to C-3 of glucose. The structural similarity of the compound with the celosin L is presumed by comparing the carbon spectrum and hydrogen spectrum data with the known compound, and the compound has one CH more than the celosin L by analyzing from the mass spectrum data2In the HMBC spectra, δ 171.1 (C-GlcA-6) is remotely correlated with the hydrogen signal 3.75(s) of-OCH 3, so the carboxylic acid structure methyl esterification on glucose is presumed, the main HMBC correlation signal, as shown in FIGS. 1 to 4. From NOESY, it can be seen that H-C (2) has an NOE effect with H-C (3), Me (24) and Me (25), H-C (3) has an NOE effect with Me (24) and Me (25), H-C (5) has an NOE effect with CHO (23), Me (25) has an NOE effect with Me (26), CH2 (19) has an NOE effect with Me (27) and Me (29), from which it can be concluded that H-C (2), H-C (3), Me (24), Me (25) and Me (26) are at the beta bond, CH2 (19) is at the beta bond, and Me (27) is at the beta bond2(19) Me (27) and Me (29) are in the alpha bond. The main NOESY related signals are shown in figure 5. The compound 1 is presumed to be a new compound by the query of a scifinider database, and the structure is 2 alpha-hydroxy-23-aldehyde-3 alpha-O- [ beta-D-xylopyranosyl- (1 → 3) -beta-D-glucopyranosyl methyl ester]-oleanolic acid. The nuclear magnetic data are shown in table 1.
The structure of compound 2 was analyzed.
White powder, readily soluble in methanol. HR-TOF-MS:m/z 440.1546[M-H]-，476.1310[M+Cl]-，486.1600 [M+HCOOH-H]-as shown in fig. 11. IR (KBr) v max 3425, 2923, 2254, 1591, 1512, 1236; UV (MeOH) showed significant UV absorption at 220 nm.1H-NMR(CD3OD) spectrum shows that the terminal hydrogen signals of two sugarsδ4.72（1H，br. d，J=1.2Hz，H-Rha-1），4.88（1H，d，JH-Glc-1) =7.6 Hz; four aromatic proton signalsδ7.32（2H，d，J=8.4Hz，H-2、6），7.12（1H，d，JH-3, 5) of 8.4Hz, see fig. 7.13C-NMR(CD3OD) spectrum, six aromatic carbon signalsδ126.0（C-1），130.3（C-2、6），118.3(C-3, 5), 158.6 (C-4); terminal carbon signals of two sugarsδ102.2 (C-Glc-1), 102.1 (C-Rha-1); one nitrile carbon signalδppm: 119.9 (C-8); a secondary carbon signalδ22.7 (C-7), see FIG. 8. The carbon and hydrogen signals are assigned through HSQC and HMBC spectrums, and the end group hydrogen signals of glucose can be seen in the HMBC spectrumsδ4.88（1H，d，J=7.6Hz, H-Glc-1) andδ158.6 (C-4) remote association, so that glucose is attached at the C-4 position; terminal hydrogen signal of rhamnoseδ4.72（1H，br.d，J=1.2Hz, H-Rha-1) andδ67.8 (C-Glc-6) remote association, so rhamnose is attached at the glucose C-6 position; hydrogen signal at C-7δ3.85(s) withδ126.0 (C-1), 130.3 (C-2), 119.9 (C-8) are remotely correlated, so a-CH is presumed2The CN structure is connected to the C-1 position, and the main HMBC related signals are shown in figures 9 and 10. In the infrared spectrum of FIG. 12, the absorption peak 2254 is characteristic for cyano. Compound 10 was presumed to be a new compound by query of the scifrinder database: the structure is benzyl cyanide-4-O-αL-rhamnopyranose- (1 → 6) -O-β-D-glucopyranoside. The nuclear magnetic data are shown in Table 2.
Experiments on the protective effect of the effective components of feather cockscomb seeds (compound 1 and compound 2) on NSC34 cell damage caused by t-BHP.
1.1 laboratory instruments and materials.
6. 12, 24, 96-well cell culture plates (CoStar. Inc.), NSC34 cells were purchased from ATCC cell bank, Biofuge stratos high-speed cryogenic centrifuge (Thermo Inc.), CKX 41 microscope (OLYMPUS Inc.), HEPA Class 100 carbon dioxide incubator (Thermo Inc.) YXQ-LS-50 II full-automatic digital display steam sterilizer (Shanghai Boxun industries, Inc.), flow cytometer BD Fortessa X-20, Confocal microscope Invertzeiss LSM 880 Confocal, enzyme reader PHERAStator FS, Li-Cor imager Odyssey CLx., fetal bovine serum was purchased from PAA Laboratories GmbH, RPMI 1640 medium was purchased from Invitron Inc., and trypsin was purchased from Amres Inc. CKK-8, t-BPH, CellROX, Heochest33342 were purchased from SIGMA.
1.2 experiment for detecting NSC34 cytotoxicity by CCK-8 method
The experimental method comprises the following steps:
1) taking NSC34 cells in exponential growth phase, adding 0.25% trypsin digestive juice for digestion to make adherent cells shed, counting 1 × 105Preparing cell suspension per ml;
2) inoculating the cell suspension on a 96-well plate at a constant temperature of 37 ℃ and 5% CO at a concentration of 100. mu.l/well2Culturing in an incubator for 24 hours;
3) different concentrations of test drug were added, 10 μ l/well (final concentration: 2, 5, 10, 25, 50, 100 μ M), and culturing for 24 hours;
4) adding 10 mul of CCK-8 into each hole, and incubating for 1h in an incubator;
5) measuring by using a microplate reader, wherein the measuring wavelength is 450nm, and calculating the inhibition rate:
inhibition = [ a (control) -a (sample) ]/[ a (control) -a (blank) ] × 100%.
The results of the experiments are shown in the table below. The results indicate that compound 1 and compound 2 are safe for NSC34 cells.
1.2 CCK-8 method for detecting cell viability experiment
The experimental method comprises the following steps:
1) taking NSC34 cells in exponential growth phase, adding 0.25% trypsin digestive juice for digestion to make adherent cells shed, counting 1 × 105Preparing cell suspension per ml;
2) inoculating the cell suspension on a 96-well plate at a constant temperature of 37 ℃ and 5% CO at a concentration of 100. mu.l/well2Culturing in an incubator for 24 hours;
3) the administration groups were added with different concentrations of test drug, 10 μ l/well (final concentration: 2, 5, 10 μ M), positive group was added with 2 μ l/well (final concentration: 200 μ M) vitamin E; culturing for 24 hours;
4) adding 10 mul of culture solution into each hole of the control group, respectively adding 10 mul of t-BHP with final concentration of 100 mul into each hole of the model group, the positive group and the administration group, and incubating for 6h in an incubator;
5) adding 10 mul of CCK-8 into each hole, and incubating for 1h in an incubator;
6) measuring by using a microplate reader, wherein the measuring wavelength is 450nm, and calculating the activity:
viability =100% - [ a (control) -a (sample) ]/[ a (control) -a (blank) ] × 100%;
results are expressed as mean ± s.d. of each experiment (n = 3).
The experimental results are as follows:
compared with the control group, the compound of the formula,#p <0.01 has significant differences. Compared with the model group, P is less than 0.05, P is less than 0.01, and the differences are significant.
The experimental result shows that the high, medium and low dose groups of the compound 1 and the compound 2 can improve the activity of NSC34 cells and have a protective effect on NSC34 cells.
1.3 flow assay for cellular ROS levels
The experimental method comprises the following steps:
1) taking NSC34 cells in exponential growth phase, adding 0.25% trypsin digestive juice for digestion to make adherent cells shed, counting 106Preparing cell suspension per ml;
2) inoculating the cell suspension on a 24-well plate at 500 μ l/well, maintaining the temperature at 37 deg.C and 5% CO2Culturing in an incubator for 24 hours;
3) the administration groups were added with test drugs at different concentrations, 100. mu.l/well (final concentration: 2, 5, 10 μ M) for 24 hours; the positive group was cultured for 24 hours with addition of 10. mu.l/well of vitamin E (final concentration: 200. mu.M);
4) adding 10 μ l culture solution into each well of control group, adding 10 μ l t-BHP with final concentration of 200 μ M into each well of model group, positive group and administration group, and incubating for 0.5h in incubator;
5) adding CellROX reagent into each well, and incubating for 0.5h in an incubator, wherein the final concentration is 5 mu M; then washing with PBS for 2 times;
6) adding 0.25% trypsin digestion solution, collecting cells, adding 500 μ l PBS, and making into cell suspension;
7) measurement with flow cytometer 105Cells, detection wavelength 670 nm;
the experimental results of the fluorescence intensity of ROS are shown in the following table:
compared with the control group, # P < 0.01; p <0.01 in comparison to model groups
The results show that: the compound 1 and the compound 2 can obviously reduce the fluorescence intensity of ROS in NSC34 cells, and have an antioxidant protection effect on NSC34 cells.
1.4 intracellular ROS imaging
The experimental method comprises the following steps:
steps 1) -5) are the same as steps 1) -5) of 1.3 above;
6) adding 4% paraformaldehyde, fixing for 15min, removing paraformaldehyde by suction, washing with PBS for 2 times, adding 300 μ l Heochest33342 with final concentration of 10 μ g/ml, culturing for 10min, removing paraformaldehyde by suction, and washing with PBS for 2 times;
7) and (5) preparing a film, and observing the film by using a confocal microscope.
The experimental results are as follows:
the results are shown in FIG. 13, blue for nuclei and red for intracellular ROS. Compared with a model group, the red fluorescence intensity of the ROS in the cells of the administration group is obviously reduced, which shows that the compound 1 and the compound 2 inhibit t-BHP to induce NSC34 cells to generate ROS, and the anti-oxidation effect is proved.
1.5 apoptosis Rate
The method comprises the following steps: the cell culture method is the same as '1.2', and the detection method adopts Annexin V-FITC apoptosis staining and detection kit (abcam, USA) to detect the apoptosis rate; the specific experimental operation refers to the kit instruction. The results of the experiments are shown in the following table.
Compared with the control group, the compound of the formula,# P<0.01; compared with the model groupP<0.05
The results show that: both compound 1 and compound 2 were able to significantly reduce the apoptosis rate of NSC 34.
In summary, it can be seen that: the celosia seeds effective component containing the oleanolic acid type triterpenoid saponin compound and/or the phenylacetonitrile glycoside compound has a good protection effect on a t-BHP induced nerve cell NSC34 damage model, and is specifically characterized by no cytotoxicity, improved cell activity, reduced intracellular ROS level and reduced apoptosis rate.
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