CN101224233A - Cassia plant extract and extracting method thereof - Google Patents

Abstract

The invention relates to an extract of cassia plant and an extraction method thereof. The extract can be used for the treatment of obesity, consists of one or a plurality of anthraquinones and contains by weight percentage: i) at least 1 percent of Orange obtusin, ii) at least 0.05 percent of obtusifolin, iii) at least 10 percent of total anthraquinone. In addition, the invention also discloses a method for extracting active component from cassia plant. The extract of the invention is a natural weight reduction combination and has good weight reduction effect, and can be produced into drugs, food replenishers, functional foods or drinks, cosmetics, etc. in various formulations.

Description

Cassia plant extract and extracting method thereof

Technical field

The present invention relates to a kind of pharmaceutical composition that is used to lose weight and preparation method thereof, said composition contains more than one anthraquinone analog compounds, specifically, the present invention relates to contain the plant extract of anthraquinone glycoside and anthraquinone aglycon, i.e. leguminous plant Semen Cassiae effective part extract and extracting method thereof.Described extract can be made medicine, dietary supplement ingredient, functional food or beverage and the cosmetics etc. that are used for the treatment of fat various dosage forms.

Background technology

Obesity is one of significant problem of health risk in the world today, and western countries have 20% population to suffer from obesity.65% is overweight among the U.S. adult, and the spending of fat-reducing and relevant health care with obesity and medicine has reached nearly hundred billion dollars.European countries' problem of obesity also is on the rise, and in Britain, 43% man and 34% women are overweight, and wherein 22% man and 23% women need treatment.Adiposis patient also shows a rising trend in minor and the developing country's population.In the prior art, the fat medicine of relevant treatment is classified as follows by its mechanism of action:

(1) suppresses energy (food) picked-up (or reducing appetite) by its effect and reduce hunger sensation brain;

(2) suppress fat absorption by fatty tip, gastrointestinal effect mechanism, lower energy and take in;

(3) consume through tip mechanism energization;

(4) stimulate fatty tip metabolism, reduce fat mass or reduce triglyceride synthetic.

Sibutramine (Sibutramine) and orlistat (Orlistat) are two kinds of fat medicines of U.S. Food and Drug Administration (FDA) (FDA) approval treatment, account for world's slimming medicine market share maximum at present.As for the natural product of treatment of obesity, although Young etc. observe extract of cassia seed can suppress rat body weight increase (Chinese clinical rehabilitation magazine, 2005,19:31), its active component or component are without discriminating.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of cassia plant extract and extracting method thereof of treatment of obesity.

Plant extract of the present invention derives from a kind of leguminous plant, contains by weight:

I) at least 1% aurantio-obtusin (aurantio-obtusin) (high performance liquid chromatogram mensuration);

Ii) at least 0.1% obtusifolin (obtusifolin) (high performance liquid chromatogram mensuration); And

Iii) at least 25% general anthraquinone (colorimetric method for determining)

Described plant optimization Cassia: 1, blunt leaf Semen Cassiae (Cassia obtusifolia); Or 2, Semen Cassiae (Cassiatora), be raw material with its seed.

Described extract is that reference substance is demarcated with aurantio-obtusin and obtusifolin.

Free aurantio-obtusin content should be no less than 1% (high performance liquid chromatogram mensuration) in the extract, if carried out hydrolysis process, can record free type and conjunction type aurantio-obtusin total amount, this total amount should be no less than 2%, by 1% words that increase progressively, the content of aurantio-obtusin can be 3%, 4%, preferably is no less than 5% or higher, as 6%, 7%, 8%, 9% also can be up to 10% or higher.

Free obtusifolin content should be no less than 0.1% (high performance liquid chromatogram mensuration) in the extract, if carried out hydrolysis process, can record free type and conjunction type obtusifolin total amount, this total amount should be no less than 0.2%, by 0.1% words that increase progressively, the content of aurantio-obtusin can be 0.3%, 0.4%, preferably be no less than 0.5% or higher, as 0.6%, 0.7%, and can be up to 0.8% or higher.

Assay adopts high-efficient liquid phase technique, and relevant assay method will describe in detail.

The content of aurantio-obtusin and two reference substances of obtusifolin should have certain ratio in the extract.By weight, the ratio of the two can be 5: 1 to 30: 1 usually, and comparatively preferred ratio is 5: 1 to 16: 1, and more preferred ratio is 8: 1 and 16: 1, and most preferred ratio is 8: 1.

These ratios might with example 1 in when from ethanol extract, separating reference substance resulting chemical compound ratio variant.When separating reference substance extractum has been divided into two parts of A, B, has partly obtained aurantio-obtusin from A respectively, partly obtained obtusifolin from B.And the present invention only adopts an extracting section thing that contains certain proportion aurantio-obtusin and obtusifolin.

The content of general anthraquinone should be no less than 25% (weight ratio) in the extract of the present invention, and comparatively preferred scheme is to be no less than 30%, as 35%, 40%, and 45%, 50% or higher (colorimetric method for determining).

Described extract must be through refining, and extract is no more than 10% of raw material weight, and the yield of comparative optimization is to be no more than 4% to 2%, and most preferred yield is to be no more than 1%.

This extract can be made medicine, health food or the cosmetics of various dosage forms.

The inventor herein obtains unique extract of the present invention after addressing the problem.

1 sets up the raw-material method of quality control of system;

2 explore the extraction and purification process that makes new advances;

3 determine the quality standard of extract;

4 set up the assay method of reference substance in raw material and the extract;

5 set up finger printing;

6 preparation reference substances;

7 determine active site and characteristic thereof;

8 activity and preparation research

9 toxicologic studies

Described dosage form can be powder, electuary, granule, solution, suspension, tablet, capsule, nasal spray, eye drop, injection, and wherein capsule is the most preferred.

The day dose of this product extract dried powder can be 50-5000mg, and optimal dose is 500mg every day.

Raw materials used is blunt leaf Semen Cassiae of cassia plant and Semen Cassiae.The inventor herein finds that it is optimum raw material that blunt leaf Semen Cassiae is produced in Hubei, and wherein the content of aurantio-obtusin is higher than other regional products, reaches 0.08%.The blunt leaf Semen Cassiae of surrounding area, Hubei such as real estates such as Shaanxi, Anhui also can be used as raw material.

Best medicinal part is a seed.

Optimum extracting method is an alcohol reflux, dispels oils and fats and resin or high concentration ethanol with centrifugal or flux such as petroleum ether, chloroform etc. then and dispels the water solublity colloid.

The preferred resin adsorption method of separation is made with extra care extract.The D101 type resin that adopts Tianjin insecticide factory to produce.

Technical data describes a clear and definite best preparation process condition in detail, comprises following operation:

1.50-80% ethanol is solvent extraction;

2. reflux, extract, method screening;

3. the pretreatment of purification resin bed;

4. resin bed parameter screening is 1 as the resin bed blade diameter length ratio: 5-1: 50;

5. eluting remove impurity conditional filtering is as volume/concentration/speed.Wash earlier, continue and wash with 20% ethanol of 1-8 times of resin bed volume with the distillation of 2 times of resin bed volumes, speed be 1-5 times of resin bed volume/hour.

6. anthraquinone component separation condition control, promptly with 70% ethanol elution of 1-10 times of resin bed volume, speed be 1-5 times of resin bed volume/hour.

Extract of the present invention can further be applied to medicine, foods and cosmetics production, and wherein preferred preparation is that extract powder directly incapsulates.

The present invention further specifies the application of this leguminous plant extract aspect products such as preparation slimming agents, cosmetics and health product.

The inventor herein has carried out following systematic study to Semen Cassiae: 1, resource investigation and pharmacognostical study; 2, effective ingredient isolation identification and active testing; 3, pharmacology and toxicological study; 4, study of pharmacy; 5, quality standard research; 6, preparation research, the effectiveness of homemade Semen Cassiae aspect treatment of obesity in having proved conclusively.

Description of drawings

Fig. 1 is the flow chart that separates reference substance aurantio-obtusin (aurantio-obtusin) and obtusifolin (obtusifolin) from Semen Cassiae;

Fig. 2 is reference substance high performance liquid chromatogram figure;

Fig. 3 is a kind of extraction process flow chart of the present invention;

Fig. 4 is extract high performance liquid chromatogram figure;

Fig. 5 is extract thin-layer chromatogram (365nm), wherein: 1 aurantio-obtusin, 2 obtusifolin, 3 samples, 1,4 sample, 2,5 samples, 3,6 samples, 4,7 aurantio-obtusins, 8 obtusifolin;

Fig. 6 is extract thin-layer chromatogram (254nm), wherein: 1 aurantio-obtusin, 2 obtusifolin, 3 samples, 1,4 sample, 2,5 samples, 3,6 samples, 4,7 aurantio-obtusins, 8 obtusifolin.

The specific embodiment

Below in conjunction with accompanying drawing relevant embodiment is described in further detail.

1.0 medicinal raw material:

1.1 background information

Semen Cassiae (comprising blunt leaf Semen Cassiae and Semen Cassiae) contains anthraquinone, lactone, aminoacid and trace element.Main effective ingredient is the anthraquinone class, contains the aglycon of 0.8-1.6% anthraquinone glycoside and 0.06-0.2%.

Semen Cassiae is often referred to the mature seed of blunt leaf Semen Cassiae (Cassia obtusifolia) and Semen Cassiae (Cassia tora), aboundresources.The seed of Herba cassiae occidentalis (Cassia occidentalis) is indivedual regional as cassia seed medicated usefulness in south.

Usually Semen Cassiae is distributed in south, and wild is main.Semen Cassiae does not distribute to the north of the Changjiang river, can not yield positive results even have yet.All there is distribution in the blunt leaf Semen Cassiae whole nation, and all there is plantation in south, the north.Document is put down in writing above-mentioned 3 kinds and is all made Semen Cassiae ancient times and use.

The early stage Japanese scientist of Semen Cassiae chemical constitution study does, and adopting Japan to produce Semen Cassiae is raw material.Some main components that from Japanese Semen Cassiae, are separated to, as chrysophanic acid (rhein), aloe-emodin (aloe-emodin), in fail to detect in the homemade Semen Cassiae.The research of rhubarb anthraquinone component content, the research of homemade Semen Cassiae chemical constituent separation of centering so far and discriminating also seldom in minority mensuration Semen Cassiae.Comprising that the quality control index of relevant Semen Cassiae medical material and preparation all rests in Chinese Pharmacopoeia and the current new drug research measures chrysophanol and this aspect of general anthraquinone, does not have special footpath property and specificity.

The inventor herein has carried out systematic study to the Semen Cassiae chemical constituent, finds that Semen Cassiae mainly contains chrysophanol (chrysophanol), physcione (physcion), obtusifolin (obtusifolin), emodin (emodin), aurantio-obtusin compositions such as (aruantio-obtusin).Wherein these two kinds of compositions of obtusifolin and aurantio-obtusin be in the special footpath composition of homemade Semen Cassiae, and aurantio-obtusin is a main component, and has good antiobesity action.

1.2 medical material is selected

The inventor herein discovers that effective ingredient is than the active constituent content height in the Semen Cassiae, so select blunt leaf Semen Cassiae as the research medical material in the blunt leaf Semen Cassiae.

Different regions product Semen Cassiae effective ingredient aurantio-obtusin and total anthraquinones content are widely different.Select Guangxi, Anhui and Hubei to produce Semen Cassiae and compare research, screening medical material producing region.The result is as shown in table 3.

The different place of production of table 3 Semen Cassiae active constituent content measuring result

The place of production	General anthraquinone (%)	Aurantio-obtusin (%)
			Guangxi	0.8	0.051
Anhui	1.1	0.071
			Hubei	1.4	0.080

As shown in table 3, effective ingredient is variant in the raw material of the different places of production, and it is higher that the Semen Cassiae active constituent content is produced in Hubei.

2.0 chemical compound

Used reference substance:

I) aurantio-obtusin

Chemical abstracts registry no sign indicating number (Registry Number): 67979-25-3

Ii) obtusifolin

Chemical abstracts registry no sign indicating number (Registry Number): 477-85-0

3.0 extract

3.1 the extraction separation of aurantio-obtusin and obtusifolin

With reference to Fig. 1, get 1kg Semen Cassiae ethanol extraction, filter, reclaim ethanol, get extractum (190g).Continue with chloroform extraction, get chloroform extract A and mother solution B.

A (20g) is with silica gel column chromatography, the chloroform methanol gradient elution, methanol crystallization, the 0.1g obtusifolin.

B is through macroporous resin adsorption, ethanol elution, the further silica gel column chromatography of washings, the chloroform methanol gradient elution, methanol crystallization, the 0.8g aurantio-obtusin.

3.1.1 quantitative analysis

3.1.2 instrument reagent

Agilent 1100 type high performance liquid chromatogram instrument (HPLC Agilent 1100), diode array detector (DAD).

Methanol, second cyanogen (chromatographically pure), deionized water, phosphoric acid (analytical pure).

3.1.3 method and result

Gradient elution sees Table 4.

Table 4 eluent gradient

Time (branch)	Second cyanogen (%)	0.1％H 3PO 4	Flow velocity (ml/min)
				0	40	60	0.8
5	50	50	0.8
				20	100	0	0.8

278nm DAD detects, reference wavelength 360nm.

Chromatographic column: carbon 18 bonded phase chromatography posts (lunar C18)

Column temperature: 25 ℃

Running time: 25mins.

3.1.4 reference substance solution preparation

Precision takes by weighing aurantio-obtusin and obtusifolin, places the 10ml volumetric flask respectively, adds methanol to scale and dissolving, promptly.

The obtusifolin retention time is 14.51 minutes, and the aurantio-obtusin retention time is 11.48 minutes (Fig. 2).

The table 5 reference substance range of linearity is investigated

Chemical compound	Linear relationship	Correlation coefficient	Scope (μ g)
				Aurantio-obtusin	Y＝93.3885334x-15.328363	0.99999	0.092～1.580
Obtusifolin	Y＝123.192564x-3.4891702	0.99995	0.008～0.580

3.2 extract preparation (optimised process)

With reference to Fig. 3 preparation method of extract of the present invention, comprise the steps:

3.2.1 Semen Cassiae (25Kg) is ground into coarse powder;

3.2.2 with 4-20 times of 50-80% alcohol reflux 0.5-3 hour;

3.2.3 repeat 2-4 time;

3.2.4 filter, decompression recycling ethanol gets crude extract;

3.2.5 centrifugal, remove impurity and upper strata oils and fats;

3.2.6 adding distil water is regulated concentration to 1: 1-1: 10 (medical materials: solution);

3.2.7 last D101 macroporous resin column, resin column blade diameter length ratio are 1: 2-20;

3.2.8 applied sample amount is no more than 1-4 times of resin bed volume, flow velocity be 1-2 times of resin bed volume/hour;

3.2.9 with 1-8 times of resin bed volume distilled water eluting, then with 20% ethanol elution, flow velocity is 1-5 times of resin bed volume earlier;

3.2.10 with 70% ethanol elution of 1-10 times of resin bed volume, flow velocity is a 1-5 times of resin bed volume at last;

3.2.11 collect 70% ethanol elution, decompression recycling ethanol, drying;

3.2.12 with gained dry extract 95% dissolve with ethanol, filter, reclaim ethanol to the thick paste shape;

3.2.13 vacuum drying is pulverized, and promptly gets exquisite extract.

Extract obtained is dark brown brown powder, bitter in the mouth.Water-soluble, ethanol, methanol.

This optimised process draws through following screening experiment research.

3.3. concentration of alcohol is selected

Test is screened optimum condition by 10% gradient 0%-80% ethanol extraction.

Method: take by weighing the ethanol that 50g Semen Cassiae coarse powder adds the 300ml experimental concentration respectively, reflux, extract, 1.5 hours repeats 3 times, filters, and adjusted volume is 1000ml.

Measure: content and the extractum weight of measuring aurantio-obtusin, general anthraquinone.The results are shown in Table 6.

Table 6. concentration of alcohol is to the influence of Semen Cassiae extraction efficiency

Concentration of alcohol	Aurantio-obtusin content (%)	Total anthraquinones content (%)	Extractum weight (g)
				0	0.16	5.62	8.61
10	0.18	5.56	8.71
				20	0.17	5.58	7.91
30	0.19	6.95	8.58
				50	0.20	8.94	8.54
60	0.21	9.48	8.81
				70	0.21	9.20	8.25
80	0.22	9.95	8.49

Table 6 result shows that along with concentration of alcohol increases, aurantio-obtusin and total anthraquinones content increase, and extractum weight reduces.Comprehensive optimum condition is 60% ethanol.

3.4 ethanol extraction method screening

To 3 kinds of ethanol extraction methods commonly used, promptly percolation, cold-maceration and circumfluence method compare research.

Percolation: take by weighing 50g Semen Cassiae coarse powder, add 100ml 60% ethanol, placed 12 hours,, collect ethanol percolation liquid, be adjusted to 1000ml with ethanol with the 500ml ethanol percolation.

Cold-maceration: take by weighing 50g Semen Cassiae coarse powder, add 100ml 60% ethanol, placed 12 hours, filter; Soak at twice with 500ml ethanol again, each 12 hours, filter, merge all filtrates, be adjusted to 1000ml with ethanol.

Circumfluence method: take by weighing 50g Semen Cassiae coarse powder, add 300ml 60% ethanol, reflux, extract, 1.5 hours is filtered, residue reuse 300ml 60% ethanol, and reflux, extract, 1 hour is filtered, and merges filtrate twice, is adjusted to 1000ml with ethanol.

The results are shown in Table 7.：

Table 7 Different Extraction Method is to the influence of Semen Cassiae extraction efficiency

Extracting method	Aurantio-obtusin	General anthraquinone	Extractum weight
				Percolation	0.38	2.84	8.37
Merceration	0.23	1.48	5.12
				Reflux	0.86	5.92	23.47

As seen, reflux extraction gained extractum and active constituent content all are higher than other extraction methods.

3.5 solvent load, return time and extraction time analysis

Through orthogonal test research, have only extraction time that extraction efficiency is had appreciable impact.Optimum extraction process is with 8 times of amount ethanol, refluxes 1 hour, extracts 3 times.

3.6 purification

After removing degrease, crude extract is with following method purification:

3.6.1 crude extract is carried out separation and purification with D101 type macroporous resin column;

3.6.2 the resin column blade diameter length ratio is 1: 20;

3.6.3 with 1 times of resin bed volume/hour speed add the extracting solution of 1.4 times of resin bed volumes, dynamic adsorption;

3.6.4 earlier with the distilled water eluting of 2 times of resin bed volumes (2BV), continue with 4 times of resin bed volume 20% ethanol with 2 times of resin bed volumes/hour the speed eluting.(removing the water solublity colloid);

3.6.5 with 4 times of resin bed volumes (4BV), 70% ethanol with 2 times of resin bed volumes/hour the speed eluting, collect 70% ethanol elution;

3.6.6 reclaim ethanol and with the residue vacuum drying;

3.6.7 the gained dry extract with 95% dissolve with ethanol, filters decompression recycling ethanol;

3.6.860 vacuum drying under ℃ condition is ground into the granule of suitable size.

Detailed technological parameter screening study data is as follows:

3.7 Choice of Resin

The absorbability of more several resins under dynamic and static conditions serves as to investigate index with absorption and desorption efficiency.

3.7.1 resin pre-treatment

Eluted resin is to dispel the residue in the resin.95% ethanol that adds 1/2 resin volume earlier adds macroporous resin then in chromatographic column, regulate amount of alcohol to exceeding 0.3 meter on resin top, soaks 24 hours.

With 2BV (BV, the i.e. abbreviation of BED VOLUME, resin bed volume, down together) 95% ethanol is with 2BV/h speed flushing resin, and then with 95% soak with ethanol resin 4-5 hour, 95% ethanol was with the flushing of 2BV/h speed, when eluate is diluted with 5 times of water gagings till the constant muddiness.Continue and be washed till eluate with distilled water and do not have alcohol flavor.

Reuse 2BV 5% HCl washes with 4-6BV/h, places after 2-4 hour, and is extremely neutral with distilled water flushing.

Reuse 2BV 2% NaOH HCl washes with 4-6BV/h, places after 2-4 hour, and is extremely neutral with distilled water flushing.

3.7.2 static adsorption test

Method: surface moisture is handled and removed to selected experiment with 5 kinds of resins.

Take by weighing a certain amount of resin in tool plug flask, add over test solution, shake well 24 hours makes anthraquinone component fully absorption on resin.Filter, get filtrate 1.Add 80ml95% ethanol, shake well made it desorption in 24 hours, filtered then, got filtrate 2.Measure the content of anthraquinone in filtrate 1 and the filtrate 2, calculate adsorption rate and conciliate adsorption rate.The results are shown in Table 8.

Table 8. resin is conciliate the adsorption test result to the static adsorption of anthraquinone

Resin	Sample size (mg)	Adsorption rate (%)	Desorption rate (%)
				D 101	1260	35.72	94.81
X-5	1260	27.78	88.81
				NKA-9	1260	19.05	65.45
NKA-12	1260	21.95	95.56
				D4020	1260	21.51	61.14

Table 8 result shows that the D101 resin has optimal adsorption ability reconciliation absorption property in the static adsorption test.

3.7.3 dynamic adsorption test

Method: the resin column eluent detects with the HPLC method.Experimental liquid is added till resin column to effluent detects aurantio-obtusin.Record adds the volume of experimental liquid.It is of light color earlier to be eluted to eluate with distilled water, collects eluate; It is of light color to eluate with 95% ethanol elution to continue, and collects ethanol elution.Measure content determination of three kinds of anthraquinones in above two kinds of eluents, calculate adsorption rate respectively and conciliate adsorption rate.The results are shown in Table 9.

Table 9 resin is conciliate the adsorption test result to the dynamic adsorption of anthraquinone

Resin	Sample size (mg)	Adsorption rate (%)	Desorption rate (%)
				D 101	3360	64.28	85.12
X-5	3360	56.38	96.87
				NKA-9	3360	27.18	54.76
NKA-12	3360	58.52	90.69
				D4020	3360	38.09	58.93

Table 9 result shows that the D101 resin has optimal adsorption ability reconciliation absorption property in the dynamic adsorption test.

Dynamic and static test result shows that all D101 type macroporous adsorbent resin has better absorption than other resins and conciliates absorption property, is suitable for separation and purification extract just.

3.8 column separating purification technical parameter screening

Upper prop sample solution concentration

Sample solution (the medical material: solution) make it carry out dynamic adsorption, measure the amount of absorption anthraquinone that in the resin column that 50g handles well is housed, adds variable concentrations.The results are shown in Table 10.

The different sample solution concentration of table 10 D101 macroporous resin is to the adsorbing influence of Semen Cassiae anthraquinone

Sample solution concentration	Adsorption rate (%)
		1∶1	47.22
1∶2	51.75
		1∶5	76.66
1∶8	76.61
		1∶10	76.61

Last table result shows that (medical material: adsorption rate no longer increases in the time of solution) when concentration ratio reaches 1: 5 along with concentration ratio increases the raising of resin absorption rate.

3.9.1 the influence of flow velocity

Respectively with 1,2, the D101 resin column that the speed of 3BV/h is handled well by 50g is housed carries out dynamic adsorption with sample solution.Follow-up with the distilled water eluting with 95% ethanol elution, collect ethanol elution and measure the content of anthraquinone, the results are shown in Table 11.

Table 11. flow velocity is to the influence of D101 macroporous resin adsorption Obtusifolin efficient

Flow velocity	1BV/h	2BV/h	3BV/h
				Anthraquinone amount (mg)	2140	1970	1773

The slow more adsorption rate of display speed is high more as a result.Select 1BV speed.

3.9.2 leakage plot is measured

With the dynamic adsorption condition, the D101 macroporous resin column that 50g handles well is equipped with in the sample solution adding, collect eluate with the 10ml/ group component, collect 10 parts altogether, with 0.2 μ m membrane filtration, the content with HPLC method mensuration aurantio-obtusin the results are shown in Table 12.

The leakage plot of table 12. Semen Cassiae extract in D101 macroporous resin adsorption process

Eluent ml	10	20	30	40	50 (1BV)	60	70	80	90	100
											Aurantio-obtusin mg	0	0	0	0	0	0	0	45.00	96.75	97.75

From last table result as can be known, when surpassing 1.4BV, sample solution begins to occur leaking when adding.

3.10 eluent concentration screening

With the dynamic adsorption condition, sample solution added 4 D101 macroporous resin column that 50g handles well are housed, be eluted to earlier of light colorly respectively with distilled water, continue respectively with 30%, 50%, 70% and 90% is eluted to of light colorly, collects ethanol elution, measures anthraquinone content.The results are shown in Table 13.

Table 13 different ethanol concentration is to the elute effect of anthraquinone

Concentration of alcohol	30％	50％	70％	90％
					General anthraquinone mg	1587	1907	2140	1733

The result shows 75% ethanol the best.

3.11 elution speed screening

Dynamic adsorption sample solution in 3 resin columns that 50g handles well are housed, use then 70% ethanol respectively with 1,2,3BV/h speed eluting, measure the amount of anthraquinone in the eluent.The results are shown in Table 14.

The different elution speeds of table 14. are to the influence of anthraquinone elution efficiency

Elution speed	1BV/h	2BV/h	3BV/h
				Anthraquinone amount (mg)	2180	2120	1960

Last table result shows that the slow more elution efficiency of elution speed is high more, but the elution efficiency difference of 1BV/ hour and 2BV/ speed at one hour rating is little, considers that from producing actual needs 2BV/ hour is best elution speed.

3.12 desorption curve

With above-mentioned optimum condition, sample solution joined be equipped with in the D101 resin column that 50g handles well, after the washing, ethanol elution.Collect ethanol elution, every part of 10ml, after the 15th part, every part of 20ml.Content with HPLC method mensuration aurantio-obtusin the results are shown in Table 15.

Aurantio-obtusin is measured at D101 macroporous resin upper bound adsorption curve in table 15 Semen Cassiae extract

Sample number	1	2	3	4	5	6	7
								Aurantio-obtusin (mg)	0.76	27	250	281	60	40	26
Sample number	8	9	10	11	12	13	14
								Aurantio-obtusin (mg)	2.84	1.84	1.2	0.76	0.48	0.0372	0.0276
Sample number	15	16	17	18	19	20
								Aurantio-obtusin (mg)	0.0108	0.0081	0.0062	0.0052	0.0030	0.0040

Last table as can be known, the content of aurantio-obtusin is very low in the 18th component eluent, is the eluting terminal point so determine No. 18 (200ml), promptly uses the 4BV70% ethanol elution.

3.13pH influence to the anthraquinone yield

Testing liquid is regulated pH4 respectively, 5, and 6 (first extracting solution is pH5) investigates the influence of pH to anthraquinone content, the results are shown in Table 16.

Table 16pH is to the influence of anthraquinone yield

pH	4	5	6
				General anthraquinone (mg)	2240	2220	1740

PH4 and 5 tells on does not as can be known from the above table have significant difference, all than pH6 better effects if, therefore, does not need to regulate pH.

3.14 the resin bed blade diameter length ratio is to the influence of anthraquinone yield

Based on above-mentioned absorption and elution requirement, get 3 posts and load the macroporous resin that 17ml, 34ml, 50ml handle well respectively blade diameter length ratio was respectively 1: 5,1: 10,1: 20.Add the 1.4BV sample solution respectively, method eluting and mensuration according to above selection the results are shown in Table 17.

Table 17. resin bed blade diameter length ratio is to the influence of anthraquinone yield

The resin bed blade diameter length ratio	1∶5	1∶10	1∶20
				General anthraquinone (mg)	1424	1773	2081

The result shows that blade diameter length ratio reaches 1: 20 o'clock anthraquinone yield height.

3.15 repurity

Semen Cassiae contains the water solublity colloid.These colloids can water or 20% ethanol wash the resin bed that has adsorbed extracting solution in advance and remove, anthraquinone component is kept under this condition.

In addition, can remove remaining water solublity colloid with 95% ethanol redissolution extractum.

3.16 test is extracted in pilot scale

Carry out 3 batches of pilot experiments based on above-mentioned condition, investigate the adaptability of selected parameter and large-scale production.The results are shown in Table 18.

Table 18 Semen Cassiae extract pilot plant test result

Lot number	General anthraquinone %	Aurantio-obtusin %	Productive rate %
				030815	45.5	4.5	0.97
030818	51.0	5.4	1.07
				030821	53.4	5.8	0.98
On average	50.0	5.2	1.01

Above process conditions can produce contain general anthraquinone greater than 45% and aurantio-obtusin content be higher than 4.5% extract.Detailed quality and technical index is as described in following 4.0.

4.0 quality standard

This product is blunt leaf Semen Cassiae seed extract, and preparation method contains aurantio-obtusin and is no less than 4.5% as mentioned above, and obtusifolin is no less than 0.25%, and general anthraquinone is no less than 50%.Pilot product shows that wherein general anthraquinone reaches 50%, and aurantio-obtusin content reaches 5.2%, and obtusifolin content reaches 0.6%..

This product can be differentiated according to the described HPLC method of 3.1.1-3.1.4.

Take by weighing the 10mg sample, add 20ml methanol and 10ml 5%HCl, refluxed 30 minutes, with the 30ml extracted with diethyl ether, volatilize ether, residue is with dissolve with methanol, and is fixed molten to 25ml, 0.22 μ m membrane filtration.

Inject 20 μ l sample solutions in the HPLC instrument, measure peak area, calculate result following (mg/g).

Table 19 extractive content measurement result

Sample number.	Aurantio-obtusin	Obtusifolin
				1	57.599	6.785
2	45.178	5.231
			3	52.433	6.189
On average	51.737	6.068

The HPLC collection of illustrative plates is seen Fig. 4.

Also available TLC differentiates.

Method:

4 batch samples are respectively got 10mg and are added the dissolving in ultrasonic 60 minutes of 10ml methanol, filter, and get sample solution.

Each is an amount of to get aurantio-obtusin and obtusifolin, uses dissolve with methanol, gets reference substance solution.

With each 1 μ l point of sample solution and reference substance solution on same lamellae, with petroleum ether (30 ℃-60 ℃): normal hexane: Ethyl formate: formic acid (1: 3: 1.5: be that developing solvent launches 0.01), take out, dry, examine down respectively at ultraviolet light 365nm and 254nm and show.

More detailed technical specification is as follows:

1. the dark brown brown powder of outward appearance, bitter in the mouth

2. aurantio-obtusin content＞5% (HPLC)

3. obtusifolin content＞0.5% (HPLC)

Loss on drying＜9.0% (1g, 105 ℃, 2h)

5. heavy metal inspection

(1) heavy metal＜10ppm

(2) arsenic＜1ppm

6. total number of bacteria＜3 * 103cfu/g

7. mycete＜1 * 103cfu/g

8. pathogenic bacterium do not detect

9. component 100% blunt leaf Semen Cassiae extract

This product is a dry extract, can be directly used in filled capsules.Every capsules contains the 250mg extract, takes 500mg every day, is convenient to take.

Animal test results shows that this product has antiobesity action.

5.0 the test of pesticide effectiveness (fat-reducing)

Materials and methods

Sample:

Sample is dark brown brown powder by the above preparation method preparation.

Laboratory animal and feedstuff:

75 of SD rats, male, body weight 140g ± 10g.Provide by the West China Experimental Animal Center.Experimental situation is temperature 22-24 ℃, relative humidity 65-70%.

Two kinds of feedstuffs:

I) normal feedstuff: Fructus Hordei Vulgaris powder 20%, dehydrated vegetables 10%, Semen Glycines powder 20%, yeast 1%, bone meal 1%, Semen Maydis powder 15%, wheat bran 16%, fish flour 10%, Sal 2%.

The high nutrient fodder of ii) high fat: in the 100g normal feedstuff, add again; Milk powder 10g, Adeps Sus domestica 10g, 1 in egg, 10 of cleum jecoris piscis concentratums, fresh Semen Glycines Germinatus 50g.

Dosage is selected:

Recommend taking in 5 times, 15 times and 30 times of dosage 0.5-0.6g/ people/day according to human body, to establish three dosage groups be that 0.05g/kg.bw/d (i.e. gram/kg body weight/sky, down with) organizes, 0.15g/kg.bw/d organizes and 0.30g/kg.bw/d organizes.

Key instrument and reagent: electronic balance (Sai Duolisi, BL610)

Experimental technique:

Rat is divided into 5 groups at random: 1, normal control group; 2, high lipid food group; With 3,3 test group.

Test group gives 0.05g/kg.bw/d respectively, the extract of 0.15g/kg.bw/d and 0.30g/kg.bw/d.

Normal group awards normal feedstuff; Other groups award the high nutrient fodder of high fat.Equal ad lib of each treated animal and drinking-water.Test group gastric infusion every day 1 time, other groups are irritated stomach feedwater 1 time, continue 36 days, put to death animal then.

Observation index:

Body weight, body fat weight (testis and perinephric fat pad), body fat/weight ratio and side effect.

Data analysis:

Adopt " Chinese medicine encyclopedia-medicostatistics " statistical package.Carry out variance analysis.

The result

Influence to body weight sees Table 20.

The influence (X+SD) of table 20 pair rat body weight

Has significant difference, * *: P＜0.01 with normal control group ratio; *: P＜0.05.

Have significant difference with fat matched group ratio, ◆ ◆: P＜0.01; ◆: P＜0.05.

As seen from Table 20, test after mid-term, the rat of the higher fatty acid high nutrient fodder of feed, its body weight is significantly higher than normal control group (P＜0.01 or P＜0.05), and fat dynamic model success is described; What each experimental group was irritated the stomach various dose was tried thing after 36 days, and 0.05g/kg.bw/d and 0.15g/kg.bw/d two dosage treated animals body weight and weight increase amount significantly are lower than fat matched group (P＜0.05).Do not see side effect such as diarrhoea, depilation, illustrate that this is tried the body weight that thing can reduce animal.

Influence sees Table 21 to rat physics fat target.

The influence (X+SD) of table 21 pair rat physical index

Group	Animal (only)	End-body heavy (g)	Body fat weight in wet base (g)	Body fat/weight ratio
					The fat matched group 0.05g/kg.bw/d of normal control group 0.15g/kg.bw/d 0.30g/kg.bw/d	15 15 15 15 15	2887.7±25.3 326.5±37.5 ** 293.1±31.1 ◆ 290.2±39.2 ◆ 318.9±51.3	6.1±1.7 8.7±2.1 ** 6.5±1.5 ◆◆ 6.1±1.9 ◆◆ 7.2±2.6	0.022±0.005 0.027±0.006 * 0.022±0.006 ◆ 0.021±0.005 ◆◆ 0.022±0.006 ◆

Has significant difference, * *: P＜0.01 with normal control group ratio.

Have significant difference with fat matched group ratio, ◆ ◆: P＜0.01; ◆: P＜0.05.

Table 21 as seen, after the off-test, the body fat weight of fat matched group and body fat/weight ratio are significantly higher than normal control group (P＜0.01), and fat animal model success is described; After result of the test finished, the body fat weight of 0.05g/kg.bw/d and two dosage treated animals of 0.15g/kg.bw/d and the body fat/weight ratio of three dosage treated animals significantly were lower than fat matched group (P＜0.01 or P＜0.05).Illustrate that this is tried the body fat weight that thing can reduce animal.

Conclusion

The body weight of fat control animals, body fat weigh and body fat/weight ratio all is significantly higher than normal control group (P＜0.01 or P＜0.05); 0.05g/kg.bw/d and the body weight of two test group animals of 0.15g/kg.bw/d and body fat is heavy, body fat/weight ratio of three test group animals all significantly is lower than fat matched group (P＜0.01 or P＜0.05), illustrate that this product has antiobesity action in zoopery.

6.0 toxicologic study

6.1 acute toxicity test

The each maximum tolerated dose of mouse stomach extract is 16.50g/kg, and twice is irritated stomach (4 hours at interval) LD50 is 20.84g/kg, is equivalent to 2511 clinical dosages (kilogram/body weight).Therefore, to take the 0.5g extract be safe to per day for adults.

6.2 long term toxicity test

26 weeks of rat oral gavage extract, and carry out biochemistry, organ exponential sum pathological examination respectively at putting to death the part test Mus behind the 4th, 16,26 all medicines.The result shows that every histopathological examination result does not have difference between test Mus and the normal mice.

Claims (11)

1. cassia plant extract is characterized in that containing by weight:

I) at least 1% aurantio-obtusin;

Ii) at least 0.05% obtusifolin; With

Iii) at least 10% general anthraquinone.

2. plant extract according to claim 1 is characterized in that: described cassia plant is blunt leaf Semen Cassiae or Semen Cassiae.

3. plant extract according to claim 1 is characterized in that: by weight, contain and surpass 5% aurantio-obtusin, surpass 0.25% obtusifolin, surpass 25% general anthraquinone.

4. according to each described plant extract of claim 1 to 3, it is characterized in that: the weight ratio of described aurantio-obtusin and obtusifolin is 5: 1 to 30: 1.

5. according to each described plant extract of claim 1 to 3, it is characterized in that: contain chrysophanol, physcione, obtusifolin, emodin and aurantio-obtusin in the described general anthraquinone.

6. a method of extracting the medical active component from cassia plant is characterized in that this method comprises the steps:

I) select medical material kind and position;

Ii) pulverize;

Iii) alcohol extraction;

Iv) separation and purification.

7. the method for extracting the medical active component from cassia plant according to claim 6, it is characterized in that: described cassia plant is selected blunt leaf Semen Cassiae for use, and medicinal part is a seed.

8. the method for extracting the medical active component from cassia plant according to claim 6 is characterized in that: described step I adopts repeatedly circumfluence method ethanol extraction in ii), and used concentration of alcohol is 50-80%.

9. according to each described method of from cassia plant, extracting the medical active component of claim 6 to 8, it is characterized in that: also contain and adopt centrifugal layering or remove greasy step with the lipophilic solvent extraction.

10. according to each described method of from cassia plant, extracting the medical active component of claim 6 to 8, it is characterized in that: described step I v) in, adopt nonpolar absorption macroporous resin column absorption and purification technology.

11. the application of each described plant extract of claim 1 to 3 in the preparation diet products.

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