CN105878316A - Extraction method and content determination method for total cardiac glycoside in yellow oleander leaves - Google Patents

Abstract

The invention provides an extraction method and a content determination method for total cardiac glycoside in yellow oleander leaves. The extraction method comprises steps as follows: the yellow oleander leaves are subjected to high-temperature enzyme killing, drying and grinding and then are subjected to warm immersion with a 95% ethanol solution, an extracting solution is obtained, Na2CO3 is added to the extracting solution to regulate pH to neutral, decompressing ethanol recovery is performed at the temperature of 40-50 DEG C until the alcohol content is 10%-20%, the extracting solution is left to stand and subjected to colloid separation at the temperature of 5-15 DEG C and then is left to stay overnight, a supernatant is taken, subjected to decompressing concentration and extracted with dichloromethane, an aqueous layer is subjected to freeze drying to constant weight, a product is obtained, and the product extraction efficiency is high. The content determination method for the total cardiac glycoside is applied to nuclear magnetic resonance and used for determining the content of the total cardiac glycoside in plants, the specificity and the operability are high, complex pretreatment and preseparation are not needed, and the content determination method for the total cardiac glycoside has broad application prospect.

Description

The extracting method of total cardiac glycoside and content assaying method in Luckynut Thevetia Seed leaf

Technical field

The present invention relates to a kind of Traditional Chinese medicine extraction method and content assaying method, especially chrysanthemum folder The extracting method of total cardiac glycoside and content assaying method in bamboo peach leaf.

Background technology

Cardiac glycoside refers to that the class that nature exists has the steroidal glycoside of notable physiologically active to heart Class.Cardiac glycoside has pharmacological action widely, has a cardiac stimulant, and toxicity is antitumor, effect Nervous centralis, diuresis, go out spiral shell isoreactivity.Discovered in recent years cardiac glycoside has the most anticancer Activity so that increasing scholar furthers investigate cardiac glycosides.Cardiac glycoside is main in plant The Alstonia of Apocynaceae to be distributed in, Thevetia, sea fruit genus, bluish dogbane In the platymisciums such as genus, Rope, Funing Calamus, Tian Bao Pittosporum.Extract strong from medicinal material Heart glycosides is relatively difficult, is primarily due to cardiac glycoside comparision contents low, cardiac glycoside usually with The impurity such as many carbohydrate, saponin(e, tannins coexist, thus affect its solubility, and extract During cardiac glycoside easily affected by soda acid or enzyme, make biologically active reduce, therefore, extract Time acid-base value to be controlled and the activity of inhibitory enzyme.

Luckynut Thevetia Seed has cardiac stimulant diuresis, eliminating phlegm and relieving asthma, effect of the stasis of blood of dispelling analgesia.Main use In treatment heart failure, cough pant, traumatic injury, through closing.Its root, branch, leaf, Kernel, seed all contain multiple cardiac glycosides, separates the cardiac glycoside identified at present and be A type cardiac glycoside, mainly has thevetoside first, second, and hydrolysate encordin, Huang Folder time glycosides second, Ruvoside, Cerberin, perusitin.

Proton magnetic resonance (PMR) (Nuclear magnetic resonance,¹H-NMR) because of it Unique advantage and obtain increasing concern, be widely used in medicine, food, agricultural In field, and 2010 editions pharmacopeia of China will¹H-NMR records in annex.¹H-NMR is used for Qualitative information that the sharpest edges of Pharmaceutical Analysis are compound structure can be provided to confirm simultaneously and The quantitative information of assay, and method is simple, specificity is strong, it is not necessary to place before complicated Reason and pre-separation.

Summary of the invention

The technical problem to be solved is to provide total strong in a kind of Luckynut Thevetia Seed leaf The extracting method of heart glycosides.

Another technical problem to be solved by this invention is to provide in above-mentioned Luckynut Thevetia Seed leaf The content assaying method of total cardiac glycoside.

For solving above-mentioned technical problem, the technical scheme is that

In a kind of Luckynut Thevetia Seed leaf, the extracting method of total cardiac glycoside, specifically comprises the following steps that

(1) by 60-80 DEG C of high temperature enzyme killing and drying of Luckynut Thevetia Seed leaf to dry, pulverize；

(2) accurately weighed, by 40-60 DEG C of temperature of 95% ethanol solution of 15-20 volume times amount Extraction takes 2 times, merges extract；

(3) extract will add Na₂CO₃Adjust pH the most neutral, in 40-50 DEG C of recovered under reduced pressure Ethanol to alcohol content is 10%-20%, stands analysis glue in 5-15 DEG C, overnight；

(4) Aspirate supernatant next day, reduced pressure concentration ethanol is to without alcohol taste；

(5) extract with dichloromethane, by water layer freeze-drying to constant weight, obtain powder solid, Obtain.

Preferably, the extracting method of total cardiac glycoside, concrete steps in above-mentioned Luckynut Thevetia Seed leaf As follows:

(1) by 70 DEG C of high temperature enzyme killing and dryings of Luckynut Thevetia Seed leaf to dry, pulverize；

(2) accurately weighed, with the 95% ethanol solution 40 DEG C temperature extraction of 15 volume times amount Take 2 times, merge extract；

(3) extract will add Na₂CO₃Adjust pH the most neutral, in 40 DEG C of decompression recycling ethanols It is 15% to alcohol content, stands analysis glue in 10-15 DEG C, overnight；

(4) Aspirate supernatant next day, reduced pressure concentration ethanol is to without alcohol taste；

(5) extract with dichloromethane, by water layer freeze-drying to constant weight, obtain powder solid, Obtain.

The content assaying method of total cardiac glycoside in above-mentioned Luckynut Thevetia Seed leaf, concretely comprises the following steps:

(1) preparation of inner mark solution

Precision weighs K₂HPO₄、NaH₂PO₄With internal standard compound TSP in volumetric flask, use D₂O dissolves And it is settled to graduation mark, and wherein, K in every 10ml volumetric flask₂HPO₄ 0.8709g、NaH₂PO₄ 0.6000g、TSP 5.03mg；

(2) preparation of need testing solution

Accurately weighed total cardiac glycoside extract, adds 400 in every 20mg total cardiac glycoside extract μ l pyridine and the 100 μ l heavy water containing 2.92mmol/L TSP, vortex, obtain test sample Solution, transfers them to 5mm nuclear magnetic tube, is used for¹H-NMR tests；

(3)¹The condition determination of H-NMR collection of illustrative plates

Nuclear magnetic tube is placed in NMR, uses Water suppression pulse train to measure sample Solution¹H-NMR collection of illustrative plates, wherein test parameter and condition are: press down water peak sequence zgcppr, Observing frequency 600.23MHz, mensuration temperature 300.6K, spectrum width 12335.5Hz, 90 ° Pulse width 13.5400 μ s, sampled data points 65536, scanning times 16 times, postpone Time 10s, gain acceptance in 32；

(4)¹H-NMR collection of illustrative plates processes

Use MetresNova software to obtaining¹H-NMR collection of illustrative plates carries out phase place tune successively Whole, baseline correction, calibration；

(5) total cardiac glycoside assay

Gather and process collection of illustrative plates, selecting the A type cardiac glycoside C-22 position disturbed without other proton peak The width of alkene hydrogen is unimodal for quantitative peak, to internal standard TSP methyl and the letter of cardiac glycoside C-22 position alkene hydrogen Number peak is integrated respectively, and the amount of the material calculating total cardiac glycoside material according to below equation is dense Degree:

$M_X=\frac{A_X{n}_{TSP}{M}_{TSP}}{A_{TSP}{n}_X}$

Wherein, A_TSPAnd A_xIt is TSP methyl peak and the integral area at sample amounts peak respectively, n_TSPAnd n_xRepresent the proton number at TSP methyl peak and sample amounts peak, M respectively_TSPAnd M_x It is the substance withdrawl syndrome of TSP and sample respectively.

In above-mentioned Luckynut Thevetia Seed leaf in the content assaying method of total cardiac glycoside, Luckynut Thevetia Seed In mainly contain A type cardiac glycoside, there are five yuan of unsaturated lactone ring (Δs^αβ-gamma lactone Ring) high conserved structure feature, in this fragment, C-22 position alkene hydrogen exists¹δ in H-NMR collection of illustrative plates Diagnostic protons signal peak is shown, by C-17 position and C-21 position proton hydrogen between 5.6～6.0 Impact that coupling is split point and in wider unimodal, this signal peak disturbs without other proton peak and is applicable to Calculate the content of total cardiac glycoside.

The invention has the beneficial effects as follows:

In above-mentioned Luckynut Thevetia Seed leaf, the extracting method of total cardiac glycoside is simple, low cost Honest and clean, to producing, equipment requirement is low, and extraction efficiency is high, it is easy to large-scale industrial production； The content assaying method of total cardiac glycoside in Luckynut Thevetia Seed leaf, application nuclear magnetic resonance technique measures The content of total cardiac glycoside in plant, specificity and workable, it is not necessary to complicated pre-treatment and Pre-separation, has broad application prospects.

Accompanying drawing explanation

Fig. 1 is the process chart of extracting method of the present invention.

Fig. 2 is¹H-NMR measures the figure of total cardiac glycoside content.(A is A type cardiac glycoside Δ^αβ- The peak of C-22 position alkene hydrogen in gamma lactone ring)

Fig. 3 is for linearly investigating figure.

Detailed description of the invention

In order to make those skilled in the art be better understood from technical scheme, below In conjunction with detailed description of the invention, technical scheme of the present invention is described in further detail.

Embodiment 1:

1, Luckynut Thevetia Seed leaf picks up from Guangzhou, Guangdong in April, 2015；

2, by 70 DEG C of high temperature enzyme killing and dryings of leaf of Luckynut Thevetia Seed to dry, pulverize, standby.

3, the extracting method of total cardiac glycoside in Luckynut Thevetia Seed leaf:

A methyl alcohol cold soaking extraction process:

(1) the Luckynut Thevetia Seed leaf that 50g high temperature enzyme killing and drying is pulverized is weighed, with 15 The methanol solution cold soaking of times amount extracts 2 times, merges extract；

(2) being evaporated to do in less than 60 DEG C by extract, add water ultrasonic dissolution；

(3) cross macroreticular resin and remove pigment；

(4) it is evaporated to do in less than 60 DEG C by the aqueous solution, washs with ether；

Dry to constant weight, obtain powder cardiac glycoside dry powder 7.1657g for (5) 70 DEG C.

B 70% ethanol heating and refluxing extraction technique:

(1) the Luckynut Thevetia Seed leaf that 50g high temperature enzyme killing and drying is pulverized is weighed, with 15 70% ethanol solution heating and refluxing extraction of times amount 2 times, merges extract；

(2) being evaporated to do in less than 60 DEG C by extract, add water ultrasonic dissolution；

(3) cross macroreticular resin and remove pigment；

(4) aqueous solution is evaporated on a small quantity, extracts with dichloromethane；

(5) by water layer freeze-drying to constant weight, powder cardiac glycoside dry powder 8.1586 is obtained g。

C 70% ethanol cold soaking extraction process:

(1) the Luckynut Thevetia Seed leaf that 50g high temperature enzyme killing and drying is pulverized is weighed, with 15 70% ethanol solution cold soaking of times amount extracts 2 times, merges extract；

(2) extract will add Na₂CO₃Adjust pH the most neutral, in less than 60 DEG C decompressions Reclaiming ethanol to alcohol content is 10%-20%, stands analysis glue in less than 15 DEG C, overnight；

(3) Aspirate supernatant next day, reduced pressure concentration ethanol is to without alcohol taste；

(4) extract with dichloromethane, by water layer freeze-drying to constant weight, obtain powder strong Heart glycosides dry powder 8.8590g.

D 95% ethanol temperature extraction taking technique (as shown in Figure 1):

(1) homogeneous extraction: weigh the Luckynut Thevetia Seed leaf that 50g high temperature enzyme killing and drying is pulverized Son, takes 2 times with the 95% ethanol solution 40 DEG C temperature extraction of 15 times amount, merges extract；

(2) crystallization, depigmentation are concentrated: extract will add Na₂CO₃In adjusting pH extremely Property, it is 15% in less than 60 DEG C decompression recycling ethanols to alcohol content, in 12-15 DEG C of standing Analysis glue, overnight；

(3) Aspirate supernatant next day, reduced pressure concentration ethanol is to without alcohol taste；

(4) wash, be dried: extract with dichloromethane, by water layer freeze-drying to permanent Weight, obtains powder cardiac glycoside dry powder 6.3298g.

The powder that above 4 kinds of extractions obtain is carried out the assay of total cardiac glycoside:

(1) instrument and reagent

Bruker Avance III 600 type NMR spectrometer with superconducting magnet, proton excitation frequency 600.23MHz, configuration BBFO positive observes broadband probe and BVT3200 numeral temperature control Instrument；5mm standard nuclear-magnetism sample cell, Norell company of the U.S.；Heavy water (D₂O) deuterated degree > 99.8%, Sigma-Aldrich；3-(trimethyl silyl) propionic acid-d₄Sodium salt (TSP) deuterated degree > 98%, Sigma-Aldrich；

(2) preparation of inner mark solution

Precision weighs 0.8709g K₂HPO₄、0.6000g NaH₂PO₄With 5.03mg internal standard Thing TSP, in 10ml volumetric flask, uses D₂O dissolves and is settled to graduation mark, is used for owning Test solvent；

(3) preparation of need testing solution

Precision weighs above 4 kinds of methods and extracts the powder 20mg obtained, every kind of method parallel 3 Individual sample, adds 400 μ l pyridines and the 100 μ l heavy water containing 2.92mmol/L TSP, whirlpool Rotation, obtains need testing solution.Transfer them to 5mm nuclear magnetic tube, be used for¹H-NMR surveys Examination；

(4)¹The condition determination of H-NMR collection of illustrative plates

Nuclear magnetic tube is placed in NMR, uses Water suppression pulse train to measure sample Solution¹H-NMR collection of illustrative plates, wherein test parameter and condition are: press down water peak sequence Zgcppr, observing frequency 600.23MHz, measure temperature 300.6K, spectrum width 12335.5 Hz, 90 ° of pulse width 13.5400 μ s, sampled data points 65536, scanning times 16 times, Time delay 10s, gain acceptance in 32；

(5)¹H-NMR collection of illustrative plates processes

Use MetresNova software to obtaining¹H-NMR collection of illustrative plates carries out phase place tune successively Whole, baseline correction, calibration；

(6)¹H-NMR measures content

Luckynut Thevetia Seed mainly contains A type cardiac glycoside, there are five yuan of unsaturated lactone rings (Δ^αβ-gamma lactone ring) high conserved structure feature, in this fragment, C-22 position alkene hydrogen exists¹H- NMR spectra shows diagnostic protons signal peak between δ 5.6～6.0, by C-17 position and C- Impact that the coupling of 21 proton hydrogen is split point and in wider unimodal, this signal peak is without other proton peak Interference is applicable to calculate the content of total cardiac glycoside.Gather under these experimental conditions and process figure Spectrum, as in figure 2 it is shown, divide the signal peak of internal standard TSP methyl and cardiac glycoside C-22 position alkene hydrogen It is not integrated, according to the substance withdrawl syndrome of below equation calculating total cardiac glycoside material:

$M_X=\frac{A_X{n}_{TSP}{M}_{TSP}}{A_{TSP}{n}_X}$

Wherein, A_TSPAnd A_xIt is TSP methyl peak and the integral area at sample amounts peak respectively, n_TSPAnd n_xRepresent the proton number at TSP methyl peak and sample amounts peak, M respectively_TSPWith M_xIt is the substance withdrawl syndrome of TSP and sample respectively.

Described in embodiment 1, four kinds of extraction process extraction cardiac glycosides the results are shown in Table 1.

Table 1

Conclude that 95% ethanol temperature extraction follows the example of the total cardiac glycoside amount obtained at most, be one Plant the best approach of total cardiac glycoside content in rapid extraction Luckynut Thevetia Seed leaf.

(7) linearly investigate

The total cardiac glycoside that accurately weighed 70% ethanol cold soaking extracts, makes as stated above for examination Product solution, concentration be followed successively by 0.2507mmol/L, 0.2821mmol/L, 0.3134 Mmol/L, 0.3447mmol/L, 0.3761mmol/L, by above-mentioned¹H-NMR tests ginseng Number and condition test¹H-NMR collection of illustrative plates, records quantitative peak integral area, with mass concentration (mmol/L) being abscissa (X), quantitative peak is ordinate (Y) with the ratio of internal standard compound peak area Carry out linear regression, as it is shown on figure 3, obtaining regression equation is y=2.2016x-0.2293, R²=0.9997.The linear investigation of other extracting methods also can be carried out according to the method described above.

(8) mixed mapping determines error rate

Accurately weighed digoxin, foxalin, Deslanoside 0.95mg, press respectively Said method makes need testing solution, and concentration is 0.41mg/ml, by above-mentioned¹H-NMR surveys Examination parameter and condition test¹H-NMR collection of illustrative plates, these three cardiac glycoside is A type cardiac glycoside, C-22 position alkene hydrogen exists¹H-NMR collection of illustrative plates δ 6.2 shows diagnostic protons signal peak, in wider list Peak, records quantitative peak integral area, calculates cardiac glycoside content.Calculate by mistake according to below equation Rate:

Show that error rate is 0.99%.

Embodiment 2:

1, Luckynut Thevetia Seed leaf picks up from Guangzhou, Guangdong in October, 2015；

2, by 70 DEG C of high temperature enzyme killing and dryings of Luckynut Thevetia Seed leaf to dry, pulverize, standby.

3, use 95% ethanol temperature extraction taking technique extract total cardiac glycoside:

(1) the Luckynut Thevetia Seed leaf that 50g high temperature enzyme killing and drying is pulverized is weighed, with 15 times The 95% ethanol solution 40 DEG C temperature extraction of amount takes 2 times, merges extract；

(2) extract will add Na₂CO₃Adjust pH the most neutral, in 40 DEG C of recovered under reduced pressure second Alcohol to alcohol content is 17%, stands analysis glue in 13-15 DEG C, overnight；

(3) Aspirate supernatant next day, reduced pressure concentration ethanol is to without alcohol taste；

(4) extract with dichloromethane, by water layer freeze-drying to constant weight, obtain powder cardiac stimulant Glycosides dry powder 7.7893g.

(5) measuring the content of its total cardiac glycoside by above-mentioned content assaying method is 0.7271±0.0023mol/g。

Embodiment 3

1, Luckynut Thevetia Seed leaf picks up from Guangzhou, Guangdong in October, 2015；

2, by 80 DEG C of high temperature enzyme killing and dryings of Luckynut Thevetia Seed leaf to dry, pulverize, standby.

3, use 95% ethanol temperature extraction taking technique extract total cardiac glycoside:

(1) the Luckynut Thevetia Seed leaf that 50g high temperature enzyme killing and drying is pulverized is weighed, with 20 times The 95% ethanol solution 45 DEG C temperature extraction of amount takes 2 times, merges extract；

(2) extract will add Na₂CO₃Adjust pH the most neutral, in 50 DEG C of recovered under reduced pressure second Alcohol to alcohol content is 10%, stands analysis glue in 5-10 DEG C, overnight；

(3) Aspirate supernatant next day, reduced pressure concentration ethanol is to without alcohol taste；

(4) extract with dichloromethane, by water layer freeze-drying to constant weight, obtain powder cardiac stimulant Glycosides dry powder 7.3243g.

(5) measuring the content of its total cardiac glycoside by above-mentioned content assaying method is 0.7413±0.0026mol/g。

Embodiment 4

1, Luckynut Thevetia Seed leaf picks up from Guangzhou, Guangdong in October, 2015；

2, by 60 DEG C of high temperature enzyme killing and dryings of Luckynut Thevetia Seed leaf to dry, pulverize, standby.

3, use 95% ethanol temperature extraction taking technique extract total cardiac glycoside:

(1) the Luckynut Thevetia Seed leaf that 50g high temperature enzyme killing and drying is pulverized is weighed, with 17 times The 95% ethanol solution 60 DEG C temperature extraction of amount takes 2 times, merges extract；

(2) extract will add Na₂CO₃Adjust pH the most neutral, in 40 DEG C of recovered under reduced pressure second Alcohol to alcohol content is 20%, stands analysis glue in less than 10-12 DEG C, overnight；

(3) Aspirate supernatant next day, reduced pressure concentration ethanol is to without alcohol taste；

(4) extract with dichloromethane, by water layer freeze-drying to constant weight, obtain powder cardiac stimulant Glycosides dry powder 7.6352g.

(5) measuring the content of its total cardiac glycoside by above-mentioned content assaying method is 0.7833±0.0018mol/g。

Above-mentioned reference detailed description of the invention is to the extraction side of total cardiac glycoside in this Luckynut Thevetia Seed leaf The detailed description that method and content assaying method are carried out, is illustrative rather than determinate, Can according to restriction scope list several embodiments, therefore overall without departing from the present invention Changing and modifications under Gou Si, within should belonging to protection scope of the present invention.

Claims (3)

1. the extracting method of total cardiac glycoside in a Luckynut Thevetia Seed leaf, it is characterised in that: tool Body step is as follows:

(1) by 60-80 DEG C of high temperature enzyme killing and drying of Luckynut Thevetia Seed leaf to dry, pulverize；

(2) accurately weighed, by 40-60 DEG C of temperature of 95% ethanol solution of 15-20 volume times amount Extraction takes 2 times, merges extract；

(3) extract will add Na₂CO₃Adjust pH the most neutral, in 40-50 DEG C of recovered under reduced pressure Ethanol to alcohol content is 10%-20%, stands analysis glue in 5-15 DEG C, overnight；

(4) Aspirate supernatant next day, reduced pressure concentration ethanol is to without alcohol taste；

(5) extract with dichloromethane, by water layer freeze-drying to constant weight, obtain powder solid, Obtain.

The extraction side of total cardiac glycoside in Luckynut Thevetia Seed leaf the most according to claim 1 Method, it is characterised in that: specifically comprise the following steps that

(1) by 70 DEG C of high temperature enzyme killing and dryings of Luckynut Thevetia Seed leaf to dry, pulverize；

(2) accurately weighed, with the 95% ethanol solution 40 DEG C temperature extraction of 15 volume times amount Take 2 times, merge extract；

(3) extract will add Na₂CO₃Adjust pH the most neutral, in 40 DEG C of decompression recycling ethanols It is 15% to alcohol content, stands analysis glue in 10-15 DEG C, overnight；

(4) Aspirate supernatant next day, reduced pressure concentration ethanol is to without alcohol taste；

(5) extract with dichloromethane, by water layer freeze-drying to constant weight, obtain powder solid, Obtain.

3. the assay side of total cardiac glycoside in Luckynut Thevetia Seed leaf described in claim 1 or 2 Method, it is characterised in that: concretely comprise the following steps:

(1) preparation of inner mark solution

Precision weighs K₂HPO₄、NaH₂PO₄With internal standard compound TSP in volumetric flask, use D₂O dissolves And it is settled to graduation mark, and wherein, K in every 10ml volumetric flask₂HPO₄ 0.8709g、 NaH₂PO₄0.6000g、TSP 5.03mg；

(2) preparation of need testing solution

Accurately weighed total cardiac glycoside extract, adds in every 20mg total cardiac glycoside extract 400 μ l pyridines and the 100 μ l heavy water containing 2.92mmol/L TSP, vortex, obtain confession Test sample solution, transfers them to 5mm nuclear magnetic tube, is used for¹H-NMR tests；

(3)¹The condition determination of H-NMR collection of illustrative plates

Nuclear magnetic tube is placed in NMR, uses Water suppression pulse train to measure sample Solution¹H-NMR collection of illustrative plates, wherein test parameter and condition are: press down water peak sequence zgcppr, Observing frequency 600.23MHz, mensuration temperature 300.6K, spectrum width 12335.5Hz, 90 ° Pulse width 13.5400 μ s, sampled data points 65536, scanning times 16 times, postpone Time 10s, gain acceptance in 32；

(4)¹H-NMR collection of illustrative plates processes

Use MetresNova software to obtaining¹H-NMR collection of illustrative plates carries out phase place tune successively Whole, baseline correction, calibration；

(5) total cardiac glycoside assay

Gather and process collection of illustrative plates, selecting the A type cardiac glycoside C-22 position disturbed without other proton peak The width of alkene hydrogen is unimodal for quantitative peak, to internal standard TSP methyl and the letter of cardiac glycoside C-22 position alkene hydrogen Number peak is integrated respectively, and the amount of the material calculating total cardiac glycoside material according to below equation is dense Degree:

$M_X=\frac{A_X{n}_{TSP}{M}_{TSP}}{A_{TSP}{n}_X}$

Wherein, A_TSPAnd A_xIt is TSP methyl peak and the integral area at sample amounts peak respectively, n_TSPAnd n_xRepresent the proton number at TSP methyl peak and sample amounts peak, M respectively_TSPAnd M_x It is the substance withdrawl syndrome of TSP and sample respectively.