CN1915993A - Indole group alkaloid of Hainan Gouyahua, and application for preparing drug-breaking medicine - Google Patents

Abstract

This invention discloses chemical structures and extraction method of indole alkaloids including 3-(2-oxopropyl) ibogamine (EHA05) and 3-(2-oxopropyl) ibogamine hydroxyindolenine (EHA06) from Ervatamia hainanensis Tsiang. This invention also discloses the application of the indole alkaloids and their salts for manufacturing analgsics, drugs or food for treating opioid dependence.

Description

Hainan GOUYAHUA indoles alkaloid and the purposes in the preparation anti-additive medicament thereof

Technical field

The present invention relates to Natural Medicine Chemistry and medical technical field, relate to the new Hainan GOUYAHUA indoles alkaloid of extraction separation from Hainan GOUYAHUA (Ervatamiahainanensis Tsiang), and comprise that Hainan GOUYAHUA indoles alkaloid of this new alkaloids is preparing control opioid dependent drug or anodyne or Application in Food.

Background technology

Hainan GOUYAHUA (Ervatamia hainanensis Tsiang) is an Apocynaceae Ervatamia plant, claims Root of Hainan Ervatamia, only son and heir's wood, taberpsychine tree, Ai Jiaoqing, shank flower etc. again, is distributed in ground such as Hainan, Guangdong, Guangxi, Yunnan.The effect of clearing heat and detoxicating, step-down, swelling and pain relieving is arranged, be used for treatment of diseases such as essential hypertension, swelling and pain in the throat, rheumatic arthralgia.The chemical constitution study of Hainan GOUYAHUA is less, [Xiao-Zhang Feng such as Feng Xiaozhang, Christiane Kan, et al.Monomeric indole alkaloids fromErvatamia hainanensis.Planta Med.1982,44:212-214.Xiao-Zhang Feng, Gui Liu, Christiane, et al.New dimeric indole alkaloids from Ervatamiahainanensis.Journal of Natural Products 1989,52 (5): 928-933] last century, the eighties was therefrom got 13 indoles alkaloids: hat coronaridine alkali (coronaridine), the pseudo-indoles (coronaridine hydroxyindolenine) of hat coronaridine alkali hydroxyl, 3-oxo-hat coronaridine alkali (3-oxo-coronaridine), 3-(β hydroxyethyl)-hat coronaridine alkali (3-(β-hydroxyethyl) coronaridine), Hainan Ervatamine A, B (ervahaimine A, B), Hainan GOUYAHUA rice is decided alkali (ervahainamidine), Hainan coronarine A, B, C (ervahanine A, B, C), sea Nishan capsicine (heyneanine), 10-hydroxyl sea Nishan capsicine (10-hydroxyheyneanine), Pai Liwen alkali (perivine).So far do not see the report of above-claimed cpd being prevented and treated opioid psychic dependence and physical dependence and analgesic activity research.

Summary of the invention

The objective of the invention is to from natural product, seek the natural product that is used to prepare control opioid dependent drug or food safely and effectively, through continuous effort, finally from the GOUYAHUA of Hainan extraction separation to two kinds of new Hainan GOUYAHUA indoles alkaloids, the pseudo-indoles (3-(2-oxopropyl) ibogamine hydroxyindolenine) of EHA05:3-(2-carbonyl propyl group)-Yi Bojiaming (3-(2-oxopropyl) ibogamine) and EHA06:3-(2-carbonyl propyl group)-Yi Bojiaming hydroxyl; Simultaneously, inventor's extraction separation has arrived other known Hainan GOUYAHUA indoles alkaloid of q.s, find that Hainan GOUYAHUA indoles alkaloid and its esters can be used for preparation control opioid dependent drug or food or preparation analgesic, thereby finished the present invention.

The present invention finishes by following technical solution, from the root of Hainan GOUYAHUA and stem, extracted the indoles alkaloid component, and from component, be separated to 9 alkaloid monomer: EHA01: vobasine (vobasine) [C.J.Pace, S.D.Glick, I.M.Maisonneuve, et al.Novel ibogaalkaloid congeners block nicotinic receptors and reduce drugself-administration.European Journal of Pharmacology2004,492,159-167]; EHA02: coronaridine (coronaridine) [analogy sun, Liu Jikai.The chemical ingredients of two wooden fork GOUYAHUA.Yunnan plant research, 1999,21 (2): 260-264]; EHA03:3-hydroxyl coronaridine (3-hydroxyl coronaridine) [C.Thal, M.Dufour, P.Potier.Rearrangementof vobasine to ervatamine-type alkaloids catalyzed by microsomes.J.Am.Chem.Soc, 1981,103,4956-4957]; EHA04:3-(2-carbonyl propyl group)-coronaridine (3-(2-oxopropyl) coronaridine) [C.Thal, M.Dufour, P.Potier.Rearrangement of vobasine to ervatamine-type alkaloids catalyzed bymicrosomes.J.Am.Chem.Soc, 1981,103,4956-4957]; EHA07:10-methoxyl group coronaridine (voacangine) [Huang Liying, Zhou Yunli, Li Chaoming etc., alkaloidal research in the two wooden fork GOUYAHUA.Herbal medicine, 1997,28 (8): 451-454]; EHA08:19-sea Nishan capsicine (19-heyneanine) [Toh-Seok Kam, G.Subramaniam, Wei Chen.Kopsiadasyrachis.Phytochemistry, 1999 (51), 159-169]; EHA09:19-Biao-Hai Nishan capsicine (19-epi-heyneanine) [Toh-Seok Kam, G.Subramaniam, Wei Chen.Kopsia dasyrachis.Phytochemistry, 1999 (51), 159-169].

We have also obtained two new alkaloids EHA05:3-(2-carbonyl propyl group)-Yi Bojiaming (3-(2-oxopropyl) ibogamine), and [electrospray ionization mass spectrum (ESI-MS) provides molecular weight 336 (M+1 337), molecular formula C to the wave spectrum appraising datum ₂₂H ₂₈N ₂O.Proton nmr spectra ( ¹HNMR) (CD ₃OD, δ): 3.77 (2H, br, 3-H), 3.11 (1H, m, 5-H), 3.56 (1H, m, 5-H), 2.79 (1H, m, 6-H), 3.48 (1H, m, 6-H), 7.53 (1H, d, J=7.7Hz, 9-H), 7.1 2 (1H, t, J=7.5Hz, 10-H), 7.18 (1H, t, J=7.2Hz, 11-H), 7.39 (1H, d, J=8.0Hz, 12-H), 1.80 (1H, m, 14-H), 1.49 (1H, m, 15-H), 1.78 (1H, m, 15-H), 1.63 (1H, m, 16-H), 1.81 (1H, m, 1 7-H), 2.32 (1H, m, 17-H), 1.09 (3H, t, J=7.5Hz, 18-H), 1.65 (1H, m, 19-H), 1.77 (1H, m, 19-H), 3.16 (1H, m, 20-H), 2.98 (1H, br s, 21-H), 2.74 (1H, dd, J=16.0,8.0 Hz, 24-Ha), 2.94 (1H, dd, J=16.0,4.5Hz, 24-Hb), 2.33 (3H, s, 26-H); Carbon-13 nmr spectra ( ¹³CNMR) (CDCl ₃, δ): 143.48 (C-2), 55.20 (C-3), 53.87 (C-5), 22.08 (C-6), 108.97 (C-7), 131.26 (C-8), 118.77 (C-9), 119.58 (C-10), 121.67 (C-11), 111.42 (C-12), 136.95 (C-13), 31.71 (C-14), 28.19 (C-15), 42.34 (C-16), 37.62 (C-17), 12.60 (C-18), 28.81 (C-19), 43.12 (C-20), 60.69 (C-21), 48.60 (C-24), 212.52 (C-25), 31.77 (C-26) ,].

The pseudo-indoles (3-(2-oxopropyl) ibogamine hydroxyindolenine) of another new alkaloids EHA06:3-(2-carbonyl propyl group)-Yi Bojiaming hydroxyl, [electrospray ionization mass spectrum (ESI-MS) provides molecular weight 352 (M+1 353), molecular formula C to the wave spectrum appraising datum ₂₂H ₂₈N ₂O ₂Proton nmr spectra ( ¹HNMR) (CD ₃OD, δ): 3.26 (2H, br, d, J=11.5Hz, 3-H), 2.47 (1H, dd, J=16.8,4.2Hz, 5-H), 2.75 (1H, ddd, J=16.8,12.4,6.4 5-H), 2.06 (1H, m, 6-H), 7.49 (1H, m, 9-H), 7.54 (1H, m, 10-H), 7.51 (1H, m, 11-H), 7.40 (1H, m, 12-H), 1.59 (1H, m, 14-H), 1.49 (1H, m, 15-H), 1.78 (1H, m, 15-H), 3.81 (1H, br d, J=14.7Hz, 16-H), 2.31 (1H, m, 17-H), 2.36 (1H, m, 17-H), 1.12 (3H, t, J=7.3Hz, 18-H), 1.65 (1H, m, 19-H), 1.77 (1H, m, 19-H), 1.67 (1H, m, 20-H), 3.89 (1H, br s, 21-H), 2.88 (1H, dd, J=16.8,4.2Hz, 24-Ha), 3.46 (1H, m, 24-Hb), 2.29 (3H, s, 26-H); Carbon-13 nmr spectra ( ¹³CNMR) (CD ₃OD, δ): 196.23 (C-2), 54.04 (C-3), 48.09 (C-5), 33.71 (C-6), 88.53 (C-7), 144.07 (C-8), 122.97 (C-9), 120.57 (C-10), 130.51 (C-11), 127.74 (C-12), 153.02 (C-13), 32.75 (C-14), 28.58 (C-15), 44.70 (C-16), 36.30 (C-17), 12.44 (C-18), 28.48 (C-19), 41.59 (C-20), 55.54 (C-21), 48.05 (C-24), 212.00 (C-25), 31.06 (C-26)].

9 alkaloidal chemical structures are as follows:

EHA02：R ₁＝H；R ₂＝COOCH ₃

EHA03：R ₁＝OH；R ₂＝COOCH ₃

EHA04：R ₁＝CH ₂COCH ₃；R ₂＝COOCH ₃

EHA05：R ₁＝CH ₂COCH ₃；R ₂＝H

EHA08 R ₃＝OH， R ₄＝H

EHA09 R ₃＝H， R ₄＝OH

We have carried out the research of relevant medicinal use, adopt mouse Srm-Rhotaard conditioned place preference drug dependence model; Srm-Rhotaard and naloxone hydrochloride dosage escalation physical dependence model trial, found that indoles alkaloid (total Ervatamiahainanensis alkaloids is called for short tEHA) has control opioid psychic dependence and physical dependence and analgesic activity in Hainan GOUYAHUA (Ervatamia hainanensis Tsiang), therefore, indoles alkaloid (tEHA) in the GOUYAHUA of Hainan can be used to prepare the purposes of control opioid dependent drug or food.

Embodiment:

With the pharmacological activity test and the result of Hainan GOUYAHUA indole alkaloid (Ervatamia hainanensisalkaloids is called for short tEHA) the present invention is described below by embodiment.

Embodiment 1 ethanol-extracted legal system is equipped with Hainan GOUYAHUA total alkaloids

Hainan GOUYAHUA dry rhizome 12kg pulverizes the back and extracts three times with 8 times of amounts (W/W), 95% alcohol heating reflux, and the concentrating under reduced pressure extracting solution gets alcohol extract to there not being ethanol.After extract dissolves in order to 2% hydrochloric acid 2000ml, elimination sour water insolubles.Acid liquid transfers to pH 8～10 with strong aqua, promptly produces precipitation, adds chloroform 500ml extraction three times, and chloroform is removed in decompression behind the combining extraction liquid, promptly gets Hainan GOUYAHUA total alkali (Ervatamia hainanensis alkaloids is called for short tEHA) of purifying.

Embodiment 2 silica gel column chromatographies prepare Hainan GOUYAHUA alkaloid monomer

Hainan GOUYAHUA total alkali 15g, and the silica gel column chromatography separation (φ 6*80cm, 450g), chloroform-methanol (100: 1,50: 1,25: 1,10: 1,8: 1,5: 1) gradient elution, every 1000ml collects, and is divided into part into 1-24.Behind the decompression and solvent recovery, from the 4th, can obtain compd E HA-02,04 in 6,9 and 12 parts, 05 with 07 crystallization. the 7th part (2.5g) silica gel column chromatography once more separates (φ 4*40cm, 75g), and petroleum ether-ethyl acetate (20: 1,10: 1,5: 1) can obtain compd E HA-01,03 and 06 behind the gradient elution.The 14th part (1.5g) silica gel column chromatography once more separates that (φ 2*40cm 35g), can obtain compd E HA-08 and 09 behind petroleum ether-ethyl acetate (5: 1,3: 1,1: the 1) gradient elution.

The influence of 3 pairs of Srm-Rhotaard psychic dependences of embodiment and the psychic dependence of itself experiment

3.1 materials and methods

3.1.1 animal: male Sprague-Dawley rat: 160～200g is available from west, Shanghai pul-Bi Kai laboratory animal company limited.Male Kunming strain mouse (cleaning level): 18～22g, the department of the Chinese Academy of Sciences of laboratory animal section of Fudan University provides.Animal rearing is in the Animal House of natural lighting, ad lib and drinking-water.

3.1.2 medicine and reagent

Srm-Rhotaard (Morphine Hydrochloride is called for short morphine, code name Mor in the table): the physiological saline with pH 3.5～4 is made into application liquid.

Hainan GOUYAHUA alkaloid (Ervatamia hainanensis alkaloids is called for short tEHA): the physiological saline with pH3.5～4.0 is made into application liquid.

Hydrocortisone: be made into application liquid with physiological saline.

3.2. medication

3.2.1 the medication of Srm-Rhotaard

Subcutaneous injection in morning Srm-Rhotaard 4.0mgkg ^-1After put into white box (companion's medicine-chest); Subcutaneous injection in afternoon physiological saline 10mlkg ^-1After put into black box (non-companion's medicine-chest).In case, stopped 50 minutes continuous 5 days at every turn.

3.2.2 medication

Mouse random packet, i.e. physiological saline group, independent Srm-Rhotaard 4.0mgkg ^-1Group, Srm-Rhotaard+tEHA10mgkg ^-1Group, behind each subcutaneous injection Srm-Rhotaard, intraperitoneal injection tEHA immediately, continuous 5 days.

3.3. experimental technique

3.3.1 morphine drug dependence model, and usefulness mouse conditioned place preference [Li Wanhai .N-nitro-L-arginine is to the restraining effect of mouse morphine and Dihydroetorphine psychic dependence for ten thousand prosperities, Huang Mao. The 2nd Army Medical College journal 1999; 20 (2): 105-107]

The condition bit offset that this experiment of conditioned place preference case is adopted likes that shuttle box adopts two casees designs of " straight line " type, and material is the black synthetic glass, wall thickness 1cm.About two the casees one black one white, the internal diameter of two chests of black and white is 15cm * 15cm * 35cm, a size of shuttling back and forth is 5cm * 5cm,, 5cm place, top is mounted with respectively one on smooth, coarse wire netting respectively at the bottom of the case; Each one at the door of shuttling back and forth is arranged in the middle of the grey black, lime two casees, be the baffle plate that to twitch.Inspection box has every light, sound damping effect, and sound intensity decay is 20-25dB.At the top of each chest, white-light illuminating lamp and infrared LED are installed, and vent passages and small fan are arranged; Black, white box all is equipped with 1 little camera, and the joint of DC 12V supply lead and A/V video line is arranged; The case bottom is lined with a backing plate, can effectively prevent the reflection of light, makes imaging clear.

Select laboratory animal to select male mice for use.Open two Room gates, under the gate, carry out video acquisition simultaneously, sample rate: in the middle of mouse is put in 15 frame/seconds.And allow to explore black and white case 2min altogether, write down the residence time in inherent black box of mouse 15min and the white box then.Long run test 3d, selecting obviously, the mouse of preference black box is a laboratory mice.

Animal training with laboratory mice by residence time of black box (non-companion's medicine-chest) with the table of random number random packet.Subcutaneous injection in morning Srm-Rhotaard 4mgkg ^-1After put into white box (companion's medicine-chest), put into black box (non-companion's medicine-chest) behind subcutaneous injection in the afternoon physiological saline.In case, stop 50min, continuously 7d at every turn.Mouse will change its natural preference through the Srm-Rhotaard training, transfers the preference white box to by the preference black box.

The observation animal is finished the mouse of training and did not give any medicine in the 8th day, with its single only being put in the shuttle box, opens two Room doors, allows to explore black and white case 2min, observes the residence time of mouse 15min inherence preference side (black box) afterwards.

The model 3.3.2 the mouse morphine is reverted to take drugs

Utilize above-mentioned conditioned place preference case and method, subcutaneous injection every day Srm-Rhotaard 4mgkg ^-1, training mouse 7 days.Cutoff Srm-Rhotaard on the 8th day, observe the residence time of mouse in preference side (black box) with aforesaid method, visible mouse is obviously had a preference for white box (mixing medicine-chest).Observed once every 1 day afterwards, observe the position CPP effect and decorporate up to cutofffing the 7th day of Srm-Rhotaard.And then, the 11st day of morphine mouse carried out vola electric shock (foot shock) or subcutaneous injection hydrocortisone 20mgkg in cutofffing every 4 days ^-1After, put into the conditioned place preference case and observe at once.

The method of electric shock: with there being lid to be of a size of the chest of L 35cm * H 28cm * W 23cm, mouse is placed stainless steel grid (0.5cm at interval) at the bottom of the case, by transmitting the galvanic current foot-shock, the mouse of being shocked by electricity sends cry or jumps.Galvanic current is by the output of CH-2 type high voltage and constant current electrical stimulator, and constant voltage is 36V, and initial galvanic current is 0.1mA, increases 0.1mA in per 10 minutes, and common-battery hit 30 minutes.

The blocking effect that model and tEHA reappear morphine mouse position preference 3.3.3 mouse is reverted to take drugs

3.4 the statistical procedures of data

Relatively adopt Student t-test between administration group and physiological saline control group or between the different dosing group.All data are all with mean+SD (x ± s) expression.

3.5 experimental result

3.5.1 tEHA and EHA monomer are to the influence of Srm-Rhotaard conditioned place preference and the psychic dependence of tEHA itself

3.5.1.1 give tEHA to the influence of mouse Srm-Rhotaard conditioned place preference and the psychic dependence of tEHA itself

Simple tEHA 10mgkg ^-1Residence time indifference before and after the administration of group mouse in white box.TEHA5mgkg ^-1, tEHA 10mgkg ^-1And TEHA 20mgkg ^-1Residence time indifference before and after the administration of group mouse in white box.The results are shown in Table 1.

Table 1.tEHA is to the influence of mouse Srm-Rhotaard conditioned place preference and the psychic dependence of tEHA itself.X ± s.**P＜0.01 is with comparison before the administration; Compare with the physiological saline group ##P＜0.01.

Drugs	Dose(mg/kg)		Time in white box
					Mor(sc)	tEHA(ip)	Pre-drug	Post-drug
	Saline	--	--	235±75					254±183
Mor					4	--	236±96	542±198**##
	tEHA	--	10	162±61					229±168
Mor+tEHA					4	5	179±93	367±187
	Mor+tEHA	4	10	191±96					270±250
Mor+tEHA					4	20	189±72	292±202
	Mor+EHA01	0.4	10	275±187					433±383
Mor+EHA02					0.4	10	243±74	360±312

Mor is a Srm-Rhotaard; Code name is a route of administration in the bracket, and sc is subcutaneous injection, and ip is intraperitoneal injection.

3.5.1.2 the single intraperitoneal injection gives the influence of 5 EHA monomers to mouse Srm-Rhotaard conditioned place preference

The single intraperitoneal injection gives 5 EHA monomer EHA-03, EHA-04, EHA-05, EHA-06 or EHA-07 with 20mgkg respectively ^-1The group mouse significantly increases the residence time of mouse in white box after the administration, promptly the conditioned place preference of morphine induction is reversed by Hainan GOUYAHUA alkaloid.The results are shown in Table 2.

Table 2. single gives the influence of 5 EHA monomers to mouse Srm-Rhotaard conditioned place preference.

Every group of number of mice=8, x ± s.With comparison before the administration, ^aP＞0.05, ^bP＜0.05, ^cP＜0.01.Compare with the Srm-Rhotaard group, ^dP＞0.05, ^eP＜0.05, ^fP＜0.01.

Drugs	Dose(mg·kg -1) Mor(sc) Alkaloids(ip)		Time in the white box( x±s，s)
					Pre-drug	Post-drug
			Saline Mor4 Mor+EHA-03 20 Mor+EHA-04 20 Mor+EHA-05 20 Mor+EHA-06 20 Mor+EHA-07 20	-- 4 4 4 4 4 4			-- -- 20 20 20 20 20	363±43 363±57 378±77 d 354±73 d 374±57 d 360±44 d 361±71 d	393±58 ad 533±65 c 651±208 cf 722±232 cf 679±75 cf 765±57 cf 763±107 cf

3.5.1.3 intraperitoneal injection gives 5 monomer EHA-03, EHA-04, EHA-05, EHA-06 and EHA-07 the influence to the formation of morphine inductive mouse position preference continuously

Each 5mgkg of EHA-03, EHA-04, EHA-05, EHA-06 and EHA-07 of the continuous intraperitoneal injection smaller dose of difference ^-1, can significantly suppress the formation of the mouse position preference of morphine induction.The results are shown in Table 3.

Table 3. intraperitoneal injection continuously gives monomer EHA-03, EHA-04, EHA-05, EHA-0 and EHA-07 the influence to the formation of morphine inductive mouse position preference.Every group of number of mice=8, x ± s.With comparison before the administration, ^aP＞0.05, ^bP＜0.05, ^cP＜0.01.Compare with the Srm-Rhotaard group ^dP＞0.05, ^eP＜0.05, ^fP＜0.01.

	Dose(mg·kg -1) Mor(sc) Alkaloids(ip)		Time in the white box( x±s，s)
					Pre-drug	Post-drug
			Saline Mor4 Mor+EHA-03 5 Mor+EHA-04 5 Mor+EHA05 5 Mor+EHA-06 5 Mor+EHA-07 5	-- 4 4 4 4 4 4			-- -- 5 5 5 5 5	410±51 408±57 418±74 d 420±75 d 412±61 d 418±56 d 412±53 d	421±53 ad 538±65 c 583±47 cf 555±61 cf 534±77 cf 561±47 cf 578±81 cf

3.5.2 the blocking effect that tEHA " reverts to take drugs " to morphine

3.5.2.1 disappearing of morphine-dependent mice conditioned place preference effect

After 7 days morphine training periods finished 1～3 day, mouse keeps the conditioned place preference effect of same intensity; Conditionality position CPP effect began to weaken in the 5th day, disappeared substantially, and the results are shown in Table 4 in the 7th day.Thereby select training to finish the 11st day the mouse in back and bring out the reproduction of conditioned place preference effect.

Table 4. mouse morphine training period end back on the same group reacts in the position of different time preference.

Every group of number of mice=9, x ± s. with give before the morphine relatively, ^aP＞0.05, ^bP＜0.05, ^cP＜0.01; Compare with the physiological saline group, ^dP＞0.05, ^eP＜0.05, ^fP＜0.01.

Medicine (subcutaneous injection)	Dosage (mgkg -1)	Before giving morphine	In the preference side residence time (x ± s, second)
								Cutoff different time after the morphine
			The 1st day	The 3rd day	The 5th day	The 7th day	The 9th day
								The physiological saline Srm-Rhotaard	-- 4	602±47 560±44 d	564±46 a 352± 137 c，f	545±63 a 362± 123 c，f	560±66 363± 87 c，f	548±64 a 435± 144 b，d	550±65 a 497± 99 a，d

3.5.2.2 tEHA brought out the 11st day that blocking effect that position preference that mouse disappears reappears finishes in the morphine training vola electric shock, the mouse of having disappeared is loved in the contraposition offset, and the vola electric shock can make the 4mgkg that has disappeared ^-1The position CPP effect that Srm-Rhotaard causes is reproduced, and physiological saline does not change its choice of location.Show that single gives tEHA 40mgkg ^-1This position capable of blocking preference is reappeared the psychic dependence that Srm-Rhotaard promptly capable of blocking causes.The results are shown in Table 5.

Table 5. single tEHA brings out the blocking effect of the position preference reproduction of disappearing to mouse vola electric shock.Every group of number of mice=9, x ± s.It is relatively preceding with the vola electric shock, ^aP＞0.05, ^bP＜0.05, ^cP＜0.01; Compare with electric shock back, vola, ^dP＞0.05, ^eP＜0.05, ^fP＜0.01; Compare with the physiological saline group ^gP＞0.05, ^hP＜0.05, ⁱP＜0.01.

Medicine	Dosage (mgkg -1)	In the time (x ± s, second) that the preference side stops
					Before the electric shock of vola	After the electric shock of vola	*tEHA40
		The physiological saline Srm-Rhotaard	- 4	550±61 507±97 g				552±60 a 376±36 c，i	555±64 a 688±234 f

* tEHA 40 is single intraperitoneal injection Hainan GOUYAHUA total alkaloids hydrochloride 40mgkg ^-1

3.5.2.3 single gives tEHA hydrocortisone is brought out the blocking effect that position preference that mouse disappears is reappeared

Trained the 11st day that finishes the mouse that contraposition offset love has disappeared, subcutaneous injection hydrocortisone 20mgkg at morphine ^-1, can make the 4mgkg that has disappeared ^-1The position CPP effect that Srm-Rhotaard causes is reproduced, and physiological saline does not change its choice of location.Single gives tEHA 40mgkg ^-1This position capable of blocking preference is reappeared, and proves the psychic dependence that tEHA Srm-Rhotaard capable of blocking causes once more.The results are shown in Table 6.

Table 6. single gives tEHA to hydrocortisone (subcutaneous injection, 20mgkg ^-1) bring out the blocking effect that position preference that mouse disappears is reappeared.Every group of number of mice=8, x ± s.Excite preceding comparison with hydrocortisone, ^aP＞0.05, ^bP＜0.05, ^cP＜0.01; Excite the back relatively with hydrocortisone, ^dP＞0.05, ^eP＜0.05, ^fP＜0.01; Compare with the physiological saline group, ^gP＞0.05, ^hP＜0.05, ⁱP＜0.01.

Medicine (subcutaneous injection)	Dosage (mgkg -1)	In the time (x ± s, second) that the preference side stops
					Before hydrocortisone excites	After hydrocortisone excites	*tEHA40
		The physiological saline Srm-Rhotaard	-- 4	541±74 588±98 g				595±91 a 329±138 c，i	571±81 a 766±159 f

* tEHA40 is single intraperitoneal injection Hainan GOUYAHUA total alkaloids hydrochloride 40mgkg ^-1

The analgesic activity of embodiment 4 tEHA reaches the influence to the Srm-Rhotaard analgesic activity

4.1 materials and methods

4.1.1 laboratory animal

Female Kunming strain mouse (cleaning level): 18～22g, the department of the Chinese Academy of Sciences of Fudan University in Shanghai laboratory animal section provides, and raises in the natural lighting Animal House; Room temperature: 20～25 ℃, temperature difference per day is less than 3 ℃; Take food arbitrarily and drinking-water.

4.1.2 reagent and preparation thereof

Srm-Rhotaard (Morphine Hydrochloride is called for short morphine, code name Mor in the table): Qinghai Pharmaceutic Plant's product is made into application liquid with the physiological saline of pH 3.5～4.

Naloxone hydrochloride (Naloxone Hydrochloride is called for short naloxone, code name NLX in the table): Fourth Ring, Beijing pharmaceutical factory is made into application liquid with the physiological saline of pH 3.5～4.

The hydrochloride of Hainan GOUYAHUA total alkaloids (total Ervatamia hainanensis alkaloids, be called for short tEHA) is made into application liquid with the physiological saline of pH 3.5～4.

Hydrocortisone: the Shanghai Xinyi Pharmaceutical Factory is made into application liquid with physiological saline.

4.2 medication

4.2.1 morphine

4.2.2 single gives the medication of tEHA

Mouse random packet, i.e. physiological saline group, independent Srm-Rhotaard group, Srm-Rhotaard+tEHA 20mgkg ^-1(note is made Mor+tEHA to group ₂₀), Srm-Rhotaard+tEHA 40mgkg ^-1(note is made Mor+tEHA to group ₄₀) and Srm-Rhotaard+tEHA 80mgkg ^-1(note is made Srm-Rhotaard+tEHA to group ₈₀).

4.3 experimental technique

Analgesic test---mouse hot plate (hot-plate) method, according to Xu Shuyun, Bian Rulian, Chen Xiu chief editor. pharmacological experimental methodology. second edition. Beijing: People's Health Publisher, 1991:695

Hot-plate instrument places electric-heated thermostatic water bath to form by hot plate (sheet copper material).Bath temperature is accurate to 0.1 ℃, and hot plate temperature maintains 55 ℃ ± 0.5 ℃.To the time that metapedes occurs licking (s of unit), promptly the hot plate response latency (or being called the pain reaction times) is as threshold of pain index from mouse vola contact hot plate for stopwatch (the quartzy stopwatch of goldspink board, Shanghai stopwatch factory) record.Experiment select hot plate response latency (the basic threshold of pain)＞15s for use but＜mouse of 30s.The threshold of pain, every mouse basis is this mouse mean value in 3 pain reaction times, and each interval in pain reaction times of measuring is greater than 15min; Mouse latent period after the administration＞60s person withdraws hot plate and in 60s.Room temperature maintains 23 ℃～25 ℃ during experiment.

4.4 the statistical procedures of data

Relatively adopt Student t-test between administration group and physiological saline control group or between the different dosing group.All data are all with mean+SD (x ± s) expression.

4.5. experimental result

4.5.1 the analgesic activity of tEHA

TEHA has analgesic activity, and strengthens with the increase of dosage, compares with Srm-Rhotaard to have slow, persistent characteristics.With Srm-Rhotaard 8mgkg ^-1Compare, as seen irritate stomach tEHA 20mgkg ^-1, tEHA 40mgkg ^-1With tEHA 80mgkg ^-1, and intraperitoneal injection tEHA 40mgkg ^-1, all having significant analgesia role, the effect behind the stomach of irritating produces slow than after the intraperitoneal injection, but suitable after action intensity and the intraperitoneal injection shows that the gastric infusion post-absorption is than more completely.The results are shown in Table 7.

The analgesic activity of table 7.tEHA on the mouse hot plate method.Every group of number of mice=9, x ± s. and physiological saline group relatively, ^aP＞0.05, ^bP＜0.05, ^cP＜0.01; Compare with the Srm-Rhotaard group, ^dP＞0.05, ^eP＜0.05, ^fP＜0.01.

* medicine	Dosage (mgkg -1) Mor(sc)tEHA		Remove hind leg latent period (threshold of pain) (x ± s, second)
								Before the administration	After the administration
			30 minutes	60 minutes	120 minutes	240 minutes
							Physiological saline Srm-Rhotaard tEHA 40tEHA 20tEHA 40tEHA 80		-- 8 -- -- -- --	-- -- 40(ip) 20(ig) 40(ig) 80(ig)	24.3±2.8 d 24.3±1.9 a 24.9±1.8 a，d 25.2±1.8 a，d 24.8±2.1 a，d 24.2±3.8 a，d	25.9±2.7 f 59.3±1.4 c 41.1±12.3 c，f 34.5±7.5 c，f 35.8±5.2 c，f 37.4±7.7 c，f	25.7±3.1 f 36.7±5.7 c 36.2±9.9 c，d 34.1±1.1 c，d 44.3±11.1 c，d 49.4±9.7 c，f	25.7±1.9 d 28.7±7.7 a 30.0±7.4 a，d 34.4±3.0 c，e 35.9±8.8 c，d 36.5±8.7 c，e	25.1±2.1 d 28.5±5.7 a 29.7±5.9 b，d 30.9±3.3 c，d 30.6±6.1 b，d 31.7±6.7 b，d

* Mor is a Srm-Rhotaard, tEHA ₂₀, tEHA ₄₀, tEHA ₈₀Be respectively Hainan GOUYAHUA total alkaloids hydrochloride 20mgkg ^-1, 40mgkg ^-1Or 80mgkg ^-1Code name is a route of administration in the bracket, and sc is subcutaneous injection, and ip is intraperitoneal injection, and ig is for irritating stomach.

4.5.2 tEHA is to the influence of Srm-Rhotaard analgesic activity

With Srm-Rhotaard 4mgkg ^-1Compare tEHA 40mgkg ^-1Analgesic activity than Srm-Rhotaard is obvious.Give opiate receptor antagonist naloxone hydrochloride 1mgkg simultaneously ^-1, the analgesic activity of Srm-Rhotaard can be cancelled.TEHA 40mgkg ^-1Analgesic activity can be by intraperitoneal injection 4mgkg ^-1Naloxone hydrochloride partly block; Give Srm-Rhotaard 4mgkg when simultaneously ^-1With tEHA 10mgkg ^-1, Srm-Rhotaard 4mgkg ^-1With TEHA 20mgkg ^-1The time analgesic activity occur collaborative.The analgesic activity of results suggest tEHA may part be realized by opiate receptor.The results are shown in Table 8.

TEHA is to the influence of morphine analgesic activity on the table 8. mouse hot plate method.Every group of number of mice=9, x ± s. and physiological saline group relatively, ^aP＞0.05, ^bP＜0.05, ^cP＜0.01; Compare with the Srm-Rhotaard group, ^dP＞0.05, ^eP＜0.05, ^fP＜0.01.

*Medicine	*Dosage (mgkg -1)			Remove hind leg latent period (threshold of pain) (x ± s, second)
									Before the administration	After the administration
				Mor (sc)	NLX (ip)	tEHA (ip)
	30 minutes	60 minutes	120 minutes				240 minutes
								Physiological saline Srm-Rhotaard Srm-Rhotaard+NLX tEHA 40TEHA 40+ NLX Srm-Rhotaard+tEHA 10Srm-Rhotaard+tEHA 20		-- 4 4 4 4	-- -- 1 -- 4 -- --	-- -- -- 40 40 10 20	22.0±3.0 d 22.1±1.7 a 22.1±2.7 a，d 21.2±2.7 a，d 22.4±3.2 a，d 21.0±2.2 a，d 22.6±2.2 a，d	22.1±3.5 f 39.4±8.8 c 22.1±3.5 a，f 55.0±8.0 c，f 54.4±9.3 c，f 40.4±10.5 c，d 41.7±12.3 c，d	21.5±3.5 f 33.4±4.6 c 22.4±3.5 a，f 56.4±7.0 c，f 34.5±7.4 c，d 31.0±7.7 c，d 40.1±8.3 c，d	21.0±2.7 f 28.8±5.7 c 22.1±5.9 a，d 37.6±10.6 c，e 32.2±10.3 b，d 28.7±8.1 a，d 35.1±11.4 c，d	20.4±2.4 d 22.7±4.4 a 21.7±4.7 a，d 21.4±3.3 a，d 23.1±3.5 a，d 22.4±8.2 a，d 30.6±7.2 c，f

* Mor is a morphine, and NLX is a naloxone hydrochloride, tEHA ₁₀, tEHA ₂₀, tEHA ₄₀Be respectively Hainan GOUYAHUA total alkaloids hydrochloride 10mgkg ^-1, 20mgkg ^-1Or 40mgkg ^-1Code name is a route of administration in the bracket, and sc is subcutaneous injection, and ip is intraperitoneal injection.

Because the habituation and the treatment thereof of opiates all have close ties with its analgesic mechanism in many aspects.Whole body muscle arthralgia is one of main protracted syndrome after the heroin addiction detoxification, also is to impel one of major reason that the drug addict reverts to take drugs.Therefore, non-addicted analgesics has considerable meaning in the treatment that minimizing is reverted to take drugs.GOUYAHUA is in China's treating rheumatic ostealgia, wound or snakebite of just being used among the people.

Above analgesic experiment result shows that tEHA has significant analgesia role, and is dose-dependence, and with Srm-Rhotaard synergy is arranged; This effect of tEHA can partly be blocked by the opiate receptor antagonist naloxone hydrochloride, and this analgesic activity that shows tEHA may part be realized by opiate system.The analgesic activity of tEHA will have positive assisting therapy effect for the protracted syndrome that alleviates after the drug abuse patient detoxification.

The test-results of above embodiment 3,4 shows: in the dosage range of being tested, tEHA can suppress the conditioned place preference of morphine induction significantly when not having physical dependence and drug dependence.TEHA can block by stress stimulation (vola electric shock) and give " reverting to take drugs " behavior to morphine of mouse that exogenous glucocorticosteroid causes.TEHA has significant analgesia role.Filling stomach and intraperitoneal injection give the test of pesticide effectiveness prompting of tEHA, and tEHA is complete and rapid in the gi tract internal absorptance, suitable oral administration.From the research data of present accumulation, tEHA tentatively possesses as the primary condition that reduces the medicine of reverting to take drugs behind effective control heroin addiction detoxification treatment.Therefore, Hainan GOUYAHUA alkaloid component of the present invention and its esters can be used for preparation control opioid dependent drug or food or preparation analgesic.

Claims (2)

1. two new Hainan GOUYAHUA indoles alkaloids, 3-(2-carbonyl propyl group)-Yi Bojiaming (3-(2-oxopropyl) ibogamine, EHA05) and 3-(2-carbonyl propyl group)-the pseudo-indoles of Yi Bojiaming hydroxyl (3-(2-oxopropyl) ibogamine hydroxyindolenine, EHA06) it is characterized in that these two alkaloids have chemical structural formula (EHA05) and (EHA06) shown in chemical structure:

EHA05: R wherein ₁=CH ₂COCH ₃R ₂=H; EHA06.

2. one kind comprises two new Hainan GOUYAHUA indoles alkaloids described in the claim 1,3-(2-carbonyl propyl group)-Yi Bojiaming (3-(2-oxopropyl) ibogamine, EHA05), pseudo-indoles (3-(2-oxopropyl) the ibogamine hydroxyindolenine of 3-(2-carbonyl propyl group)-Yi Bojiaming hydroxyl, EHA06) and also comprise vobasine (vobasine, EHA01), coronaridine (coronaridine, EHA02), 3-hydroxyl coronaridine (3-hydroxyl coronaridine, EHA03), 3-(2-carbonyl propyl group)-coronaridine (3-(2-oxopropyl) coronaridine, EHA04), 10-methoxyl group coronaridine (voacangine, EHA07), 19-sea Nishan capsicine (19-heyneanine, EHA08) and 19-Biao-Hai Nishan capsicine (19-epi-heyneanine, EHA09) at interior Hainan GOUYAHUA indoles alkaloid and its esters in preparation control opioid dependent drug or anodyne or Application in Food.