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Inhibition of hypersensitivity reactions by soluble derivatives of baicalein.

Baicalein, a flavonoid, is anti-allergic but only slightly soluble in water. The soluble derivatives of baicalein, disodium baicalein-6-phosphate (BPS) and sodium baicalein-6-sulfate (BSS), were synthesized and examined regarding their effects on hypersensitivity reactions. These derivatives inhibited type I and II reactions as classified by Coombs and Gell. The Arthus reaction belonging to type III reaction, however, was hardly affected with either BPS or BSS. The experimental asthma caused by passive systemic anaphylaxis in guinea pigs was prevented with application of BPS. Thus even by the oral route, BPS appears to be clinically applicable to extensive allergy related diseases.

Baicalin is a flavone glycoside contained in the radix of Scutellaria baicalensis GEORG (Woogon), which has been used from ancient times in Chinese medicine as a remedy for allergic diathesis and inflammation. Hitherto, the authors (1-3) have investigated the pharmacological actions of baicalin and its aglycone, baicalein, and reported that each showed a potent protective action against anaphylaxis, i.e. systemic and cutaneous ana phylaxis were prevented in guinea pigs and/or mice, and release of histamine and SRS-A from sensitized guinea pig lung treated in vitro was also inhibited. The functional structure in baicalin may depend upon a part of baicalein, since the inhibitory potency of baicalin was much the same as that of baicalein in an equal molar concentration. and relaxed the tracheal muscle fairly well. Thus it would appear that baicalein may be useful for treating allergic disorders, in particular asthma. In the present paper, the anti allergic actions of soluble derivatives of baicalein, disodium baicalein-6-phosphate (BPS) and sodium baicalein-6-sulfate (BSS), were investigated and the structural formulas of these compounds as contrasted with that of disodium cromoglycate (DSCG), a specific inhibitor of reagin-mediated allergic reaction (4) are shown in Fig. 1.

MATERIALS AND METHODS
Animals: Hartley guinea pigs of both sexes weighing 260 to 350 g, male Wistar rats weighing 120 to 180 g, and male Albino rabbits weighing 2 to 3 kg were used.
Preparation of antisera: Rabbit anti-egg albumin sera (Rab anti-Ea) were prepared from rabbits which had been immunized by injecting 10 mg of egg albumin emulsified in complete Freund's adjuvant i.m. 4 times weekly. The serum was obtained 7 to 10 days after the last injection and lyophilized until use. Rat anti-dinitrophenylated ascaris extract sera (Rat anti-DNP-As) were prepared according to the method of Tada and Okumura (5). Rats were immunized by injecting 1 mg of DNP-As mixed with 1010 killed Bordetella pertussis into the four foot pads. Five days later, the animals were given a booster of 0.5 mg of DNP-As alone into the muscles of the back. Serum collected from each animal 3 to 4 days after the last injection was pooled and frozen until use. The antibody titre of this serum was 1:128 to 1:256 as estimated by 48 hr homologous passive cutaneous anaphylaxis (PCA). Rabbit anti-rat sera (Rab anti-rat) were prepared from rabbits which had been im munized by injecting 1.0 ml of rat serum i.v. every other day for a total of 10 doses. The serum was obtained on the 20th day after the last injection and lyophilized until use. Anti sheep erythrocyte sera (hemolysin) were prepared from rabbits which had been immunized by injecting 109 sheep erythrocytes i.v. every other day for a total of 10 doses. The serum was also obtained on the 10th day after the last injection and frozen until use.
Heterologous PCA: Heterologous PCA in guinea pigs was carried out by the method of Ovary (6). Guinea pigs whose left backs had been shaved one day before were given At 1, 2, 4, 6, 12 and 24 hr after the injection, the dimension of inflamed region was mea sured macroscopically. Forssman cutaneous vasculitis: Guinea pigs were injected with 0.1 ml of hemolysin diluted 8 fold with physiological saline i.d. into their shaved backs followed by an i.v. in jection of 1.0 ml of 1 % Evans blue. After 1 hr, the animals were sacrificed by exsangui nation and the skin was removed. The blueing spot caused by the Forssman cutaneous vasculitis was measured by the same method as was used for PCA.
Experimental asthma: Guinea pigs were sensitized with an intracardiac injection of 1.0 nil of Rab anti-Ea. After 24 hr, the animals were given 1/4 lethal dose of the antigen i.v. to provoke an anaphylactic reaction. The rate and depth of respiration were recorded on a smoked drum by the method described previously (3). The carotid blood pressure was also simultaneously recorded.

Heterologous PCA
The effects of BPS and BSS on heterologous PCA in guinea pigs were compared with those of certain drugs including aminophylline, isoproterenol, chlorpheniramine and pred nisolone in a dose of 5 mg/kg i.v. at 10 min prior to challenge. As shown in Table 1, all of the substances tested, except for aminophylline, showed an inhibition of the dye leakage caused by PCA, with BPS showing the most potent activity, that is a 69.0% inhibition, while about 30 % inhibition was seen in the others. An increase in capillary permeability after an i.d. injection of histamine was also inhibited with all of the substances. The in hibitory activities of BPS and BSS, however, were only 17.1 % and 14.3 % respectively those are fairly weak compared to those of isoproterenol and chlorpheniramine.
Thus, the inhibition of PCA by BPS and BSS did not appear to depend upon their antihistaminic activities.
Homologous PCA Regarding homologous PCA, the effect of substances given in a similar manner to those in the case of heterologous PCA is shown in phylline, in the following decreasing order; isoproterenol>chlorpheniramine>BPS> prednisolone. On the other hand, the amount of the dye leaked was inhibited by the sub stances, except for BSS, in the following order; chlorpheniramine>isoproterenol>BPS> aminophylline% prednisolone.

RCA
The effect of the substances was tested on RCA in rats following an i.d. injection of Rab anti-rat. Rats were administered 5 mg/kg of the substances i.v. at 10 min prior to the antiserum. As shown in Table 3, the manifestation of edema was decreased with all sub stances. Isoproterenol showed the most potent activity with a 38.7 inhibition, followed by BPS with 29.3 %. The others were in a range from 19.6 to 27.4%. -I-

Schultz-Dale reaction
Inhibitory activity of substances tested in a concentration of 10-4 g/1nl on the Schultz Dale reaction is shown in Table 4. A fairly large difference was observed in their activities in the ileum and tracheal muscle. In the ileum, chlorpheniramine and isoproterenol in hibited the reaction completely. BSS and aminophylline, however, showed little or no effect. BPS gave a 53.5 % inhibition which corresponded to about one half that of chlor pheniramine. On the tracheal muscle, the effect of BPS was 75.6 % inhibition, while amino phylline and/or isoproterenol alone relaxed the muscle below the base line and showed more

Experimental asthma
To guinea pigs sensitized passively with Rab anti-Ea , an i.v. injection of the antigen caused a transient increase followed by a persistent decrease in the rate of respiration, and a persistent decrease in the depth of respiration continued for more than 30 min. A con siderable fall in blood pressure was also observed, the maximum being about 60% . BPS and BSS were given in 3 different doses i.v. at 15 min before challenge. Five mg/kg of BPS prevented a decrease in the depth and there was a slight decrease in the rate of respi ration. A fall in blood pressure was also prevented by BPS . The same dose of BSS, how ever, failed to prevent the respiratory disorder or a fall in blood pressure. In doses of 10 to 20 mg/kg, BPS and BSS inhibited the hypotension and also the anaphylactic changes in respiration, especially the decrease in depth (Fig. 6).

DISCUSSION
In previous papers, the authors reported the inhibitory effects of baicalin and baicalein on active and passive anaphylaxis as well as results on experimental asthma.
In the present