JPS5978121A - Preparation of substance for hyperfunctioning phagocytic cell and product - Google Patents

Abstract

NEW MATERIAL:A substance hyperfunctioning phagocytic cells Aa-50 having the following properties; [alpha]<20>D:-57.1 deg. (c=0.5, 0.3M NaCl). Constituent amino acids: Asp, Thr, Ser, Glu, Pro, Gly, Ala, Val, Ile, Leu, Try, Phe, Lys, His and Arg; Elementary analysis: C, 45.53%; H, 6.35%; N, 5.22%. Disk polyacrylamide gel electrophoresis (9.5pH, 7% gel): five bands. Disk sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis (7.2pH, 10% gel): five bands. USE:Substances hyperfunctioning phagocytic cells. PROCESS:A leaf of a wild aloe in the growth or flowering period is stored for a given number of days in a cold dark place, and then ground and centrifuged to give a supernatant liquid, which is freeze-dried, dialyzed and fully desalted to give an internal liquid (undialyzed part). The resultant internal liquid is then freeze-dried to afford the aimed yellowish white powder Aa-50.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a new substance Aa-so obtained from aloe leaves that has the effect of enhancing bacterial cell function and a method for producing the same.

Aloe is a typical folk medicine that has been prized as a panacea since ancient times. This aloe is a plant of the genus Aloe of the family Liliaceae, such as Aioe arborescen
sMIL var, natalensis Ber
ger), Noko] U70 (A perryi)
Baker), Curacao YO Engineering IA Barbad
ensis Miller), Cape Aloe (A.
,, f e r o, xMiller or this and A a
fricans Miller and Aspicata
The existence of the Socotra aloe was already known by the Greeks in 400 B.C.E., and Alexander the Great led the Girinyan people to the Sokotra Aloe off the coast of East Africa.
ra) It is recorded that he was dispatched to the island and made efforts to cultivate and preserve it.

Therefore, the components of aloe have been attracting the attention of many pharmacologists for a long time. Various components such as an agglutinating active substance (alloctin A) and a hemagglutinating active substance (allottin B) have been isolated.

The present inventors explored the infection-preventing substance contained in aloe using the phagocytosis of peripheral blood leukocytes in humans (adult bronchial asthma (chronic and chronic) patients) as an indicator from a perspective different from past research. As a result, we found that the internal fluid (non-dialysed portion), which was thoroughly desalinated by dialyzing the supernatant obtained by centrifuging the crushed aloe vera powder stored in a dark place (sheath), enhanced the palatable activity. Aa
It was named -50. Based on the results of electrophoresis tests, this substance is estimated to be a mixture of at least five types of polysaccharides and proteins or glycoproteins, and exhibits the following physical and chemical properties.

I R (vKBr, cm'-'): '3300.
1730.1650. '1540.1050ax UV (λ03MNaC', C=0.65) 280
nmax Optical rotation [α] %0=-57.1° (C=0.3MNa
(!']) Constituent amino acids: aspartic acid, threonine, serine, glutamic acid, proline, glycino, alanine, valine, inoloinno, leucine, tyrosine, fluorinylalanino, lysine, histidine, and hyarginino disk electrophoresis polyacrylamide gel (p149. 5, 7% gel)
5 bands (coloring agent ``Coomanosie Brilliant 1'')
~ Blue and Periodate Dit (color former: Coomassie Brigliano 1 ~ Blue and Periodate Schimpf reagent).

Incidentally, alloctin A and alloctin B, which were published before the filing of the present invention, both have molecular weights of approximately 18,000 and 2,400.
Although it appears to be similar to each component of Aa-50 in that it is a glycoprotein of Aa-50, the latter does not contain restino, which is a constituent amino acid of the former two, so it is macroscopically different from the former. belongs to.

The experimental facts that formed the basis of the present invention will be described below.

(3) Preparation of samples (1) Wild aloe leaves in the growing or flowering stage were stored in the dark for a certain number of days at a temperature of 4°C to 30°C for 3 to 10 days, then ground and crushed at 110,000 rpm. Centrifuge for 10 minutes and lyophilize the supernatant. Next, this was placed in a cellophane tube and dialyzed against distilled water for 2 days, and the dialysis part (external solution) was
After concentration at 5° C. or lower, the non-dialyzed portion (internal solution) is freeze-dried as it is. Each sample (100 mg) had a pH of 7,
2 in 5 ml of 0.15 MIJ phosphate buffer,
Dilute multiple times when measuring.

(2) Fresh aloe leaves collected during the growth period were stored in the dark at 4°C for 5 days or in the light at 15°C for 5 days, and then carefully separated into the cortex and jelly-like pulp, and each was separated into ( 1
) Prepare the measurement sample in the same way.

FB) Preparation of measurement sample: Add 4 ml to 20 ml of venous blood sample from an adult asthmatic patient.
% Dextran B (molecular weight 1.7X105) and 5
Add a saline solution containing 00 units of heparino,
Leave at room temperature for a minute to precipitate red blood cells. The leukocyte-rich supernatant is collected by squeezing, washed twice with Harmonic Hanox's solution, and used for neutrophil measurement. That is, after Giemsa staining, neutrophils are counted, and a sample solution is prepared such that 2 x 10 7 neutrophils are malignant in 1 ml of a certain volume of harmonic Hannoch's solution.

(C) Measurement of glucocorticoid activity Alexander et al.
orst & Good, , Lab, CI in,
Med, 72.136-148 (1968))
According to the method of 2 x 107 neutrophils and 5
% calf serum and 0.05mA! Prepare a sample solution containing an aloe sample, and ink this sample solution at 37° C. for 7 minutes. - 10,000, dilute yeast to contain 25 x 107 cells per ml of physiological saline, and incubate at 100°
Heat at ℃ for 30 minutes. Old ml of this sterilized yeast cell suspension
Add 0.1 rnl of the above incubate solution to 37°
Incubate at C for 60 minutes, and determine the bactericidal coefficient of the test neutrophils according to the following formula.

In the control, 0.05. 0 of nl
．． Add 15M phosphate buffer (pH 7.2).

FD+ analysis method (1) Osmotic pressure: The osmotic pressure of the aloe sample was measured using a Friske O5 osmometer (Friske As5ociates).
Inc.

Measured using U, S, A).

100 my of the crude sample dissolved in 5 ml of O, 15M phosphate buffer (pH 7.2) shows 325 m05 m/l (saline: 308 m05 m/1).

(2) Physical and spectroscopic analysis: Infrared spectrum is K
Ultraviolet spectra were measured using an OKEN DS-301 spectrometer and a Hitachi 200-10 spectrometer 4.

Optical rotation was measured using a JASCODIP-4 polarimeter. For amino acid analysis, samples were incubated with 6N hydrochloric acid in a vacuum sealed tube for 20 min.
Heated to 110°C for an hour and measured using a Hitachi 835 amino acid analyzer. Disk electrophoresis was performed on (i) I) 7% polyacrylamide gel in H9,5 and (il) PH7.
, 2 using a 10% 5DS-polyacrylamide gel.

Coloring agent: Coomaf No Brilliant Pur.

- and periodate Schimpf reagent (Zanocarius, Zell, Morinon, and Unodoronk (1969)).

(3) Chromatography didialysate ly 0.0
Dissolve 2MNH4HC0.30mZ, DEAE-Se#.

4×25 filled with Fine (Chinono Co., Ltd.) 500-
Load the column with cIn and add 0.02M NH,H to the column.
The CO3 liquid is flowed at a flow rate of 20 d/h. Eluate 324rn
After dialysis against distilled water and freeze-drying, 30 ml of a colorless, soft, thick substance is obtained.

Next, the column was flushed with 0.3M NaCl, and the eluate 234d was dialyzed against distilled water and lyophilized to obtain 1100In as a pale brown powder. The eluate from the column was directly measured at 280 nm using a Hitachi spectrophotometer, and at 490 nm using the fluorinated norsulfuric acid method (Dubois et al. (1956)).
Monitor with nm.

+Ej Bactericidal action of the substance of the present invention (1) Effects of aloe extract harvested during the growing season As shown in Table 1 below, the extract obtained from the material stored in the dark at 20°C for 5 to 10 days have the greatest effect.

Samples stored at 25°C for 3 days appear to have the greatest potency, but this is likely due to experimental error.

Table 1 (2) Effects of aloe extract harvested during flowering period are shown in Table 2 below. The influence of each sample on the bactericidal action shows the same tendency as shown in Table 1 above, but the bactericidal coefficient is 1.00.
It is lower than 0, and rather tends to inhibit the normal bactericidal action.

Table 2 (3) Whole British extract, jelly, or part of the whole hive leaf, has an effect on enhancing the glucocorticoid activity of the dialyzed and non-dialyzed parts Samples were prepared using the method described in (A) (1) above.

As shown in Table 3 below, the celery-like extract and the whole British dialysis section showed positive behavior with respect to bacterial action. Valorbairono, which was used as a reference, exhibits negative behavior against bacterial action.

Table 3 (4) Samples for the effect of enhancing the bacteriostatic activity of the dialyzed or non-dialyzed areas from the whole body, cortical layer, or part of the jelly were prepared according to the method described in A (2) above.

As shown in Table 4 below, the effect of enhancing the bactericidal action was observed in some non-dialyzed parts of the jelly.

On the contrary, this effect was suppressed in the non-dialysis area from the cortical layer.

Table 4 (F) Physical and chemical properties of the substance of the present invention The physical and chemical properties of the non-dialysed area (Aa-50) after one week of dialysis are as follows: Knee optical rotation: [α, :] Old 0- s 7.1° (c=0.6
5,0.3M NaCl) infrared absorption spectrum (λ
KBrcm1): max' 3300 (strong, -0H), 1700 (strong, -COOH)
, 1650 (strong.

-NH2) tOH Ultraviolet absorption spectrum (ν, nm); 280
Color reaction: Positive with Lowry's reagent and phenol sulfuric acid.

Elemental analysis: C; 45.53%, N; 5.22%, H
;6.35% Constituent amino acids: ASp, Thr, S
er, Glu, Pro, Gly.

Ala, Val, T Ie, Leu+ TYr,
Phe, Lys, His, Arg.

Polyacrylamide gel and 5DS-polyacrylamide, gel disk electrophoresis: 5 bands (same when using either Kuumatsunow Brilliant Blue or Periodate Schiff reagent as coloring agents). From the above results, this substance It is estimated that it is composed of at least five types of unit components that are a mixture of saccharides and proteins or glycobrothiino. The bactericidal coefficient of this non-dialysate (Aa-50) is 1.312±0.10.
It was 6.

(G) Pharmacological effect of the substance of the present invention A purified aloe extract (Aa-50) stored in the dark at 4°C for 7 days was administered to patients of various ages. The results are shown in Table 5 below. As shown in <6285 for patients over 51 years of age.
%, but this dropped to 333% on average when younger patients were included. In addition, this extract was effective in 429% of endogenous patients, but 16% in exogenous patients.
It was only effective in 7%. Furthermore, in comparing the steroid-administered group and the non-steroid-administered group, aloe extract
Although it is more than 0% effective, the fact that it is ineffective against the former is an important finding and at the same time presents an interesting problem regarding the action of aloe extract as an infection prevention agent.

Table 5 Note) Dosage and administration method: Administer 5 ml of aloe extract diluted to 20% with physiological saline twice a day for 6 months.Patent applicant: Maruho Co., Ltd. Procedural amendment filed by the Commissioner of the Japan Patent Office [Mr. Wainu] Indication of the case 1989 Patent Application No. 189624 2, Title of the invention ``Production method and product of a substance that enhances the function of glucocorticoid cells 3,
Relationship with the case of the person making the amendment: Patent applicant 4, agent 5, date of amendment order: February 22, 1982 (shipment date) 6, number of inventions increased by amendment: 0 (attached) I can see it, but it's probably due to experimental error.

Table 1 (2) Effects of aloe extract harvested during flowering period are shown in Table 2 below. The influence of each sample on the bactericidal action shows the same tendency as shown in Table 1 above, but the bactericidal coefficient is 1.00.
It is lower than 0, and rather tends to inhibit the normal bactericidal action.

Table 2 (3) Samples of the complete extract, part of the jelly, and the bacteriostatic effect of the dialyzed and non-dialyzed parts of the complete collection were prepared using the method described in (Al (1)) above.

As shown in Table 3 below, the jelly-like extract and the dialyzed portion of the complete collection showed positive behavior against the bacillus effect. Valorviron, which was used as a reference, exhibits negative behavior against the bacillus effect.

Table 3 (4) Samples for enhancing the bacteriostatic activity of the dialyzed or non-dialyzed portions from the complete collection, cortical layer, or part of the jelly were prepared according to the method described in A (2) above.

As shown in Table 4 below, the effect of enhancing the bactericidal action was observed in some non-dialyzed parts of the jelly.

On the contrary, this effect was suppressed in the non-dialysis area from the cortical layer.

Table 4 (F) Physical and chemical properties of the substance of the present invention The physical and chemical properties of the non-dialysed part (Aa-50) after one week of dialysis are as follows: Optical rotation: Car, )50-s7. xo(cm0.65
, 0.3M NaC1) Akatsuki absorption spectrum (2ゝ”
, cm-1): 3300 (strong, -0H), 170
0 (strong, -COOH), 1650 (strong.

-Nl2) Ultraviolet absorption spectrum (V Hatake W+ nm): 2
8002) The average number including younger patients decreased to 33.396. Additionally, this extract was effective in 429% of endogenous patients, but only 16.7% of exogenous patients. Furthermore, when comparing the steroid-administered group and the non-steroid-administered group, it is important to note that although aloe extract was more than 40% effective against the latter, it was ineffective against the former. This poses an interesting question regarding the action of extracts.

Table-5 (+4)　　　　　　　　　　

Claims (1)

[Scope of Claims] (1) A method for producing a substance that enhances bacterial cell function, which comprises dialyzing the juice of fresh aloe leaves and freeze-drying the dialyzed solution. (2) The method according to claim (1), wherein the supernatant is obtained by centrifuging fresh aloe leaf juice prior to dialysis. (3) The manufacturing method according to claim (1) or (2), wherein the raw material aloe leaves are stored in a dark and cool place in advance. (4) A yellowish-white powder obtained by dialysis of fresh aloe leaf juice and freeze-drying the dialyzed fluid, which contains a substance (Aa
-50). KBr −1゜(i) IR(ν, cm), 330
0.1730.1650°ax 1540, 1050 0.3MNaC1 (ii) UV (λ, cm0.65):
280nmax (hl) CαII) -57,1° (cm0.5
, 0.3M NaCl) (1v) Constituent amino acid: As
p, Thr, Ser, Glu, Pro. Gly, Ala, Val, TIL Leu,
Try, Phe, Lys. His, Arg. (v1 elemental analysis value: 5 bands (coloring agent, 5 bands of Kumanzu Brilliant Blue and Periodate Schiff reagent)