KR960004025B1 - Anti-inflamatic, anti-histamine and anti-rheumatic agents comprising biflavonoids from ginko keaves - Google Patents

Abstract

The anti-inflamatic, anti-allergic and antirheumatic agent contains sciadopitysins [R1=R2=R4=CH3; R3=H , ginketins [R1=R2=CH3; R3=R4=H , isoginkgetins [R1=R3=H, R2=R4=CH3 , bilobetins [R1=R3=R4=H; R2=CH3 or anentoflavones [R1=R2=R3=R4=H of biflovones of formula(I). The biflavones are obtained by extracting gingko leaves with an methanol, adding a distilled water and a chlorofrom to the methanol extract, shaking and fractionating the mixture, adding an ethylacetate to the fractionated soln. and shaking the mixture, dehydrating the fractionated soln. with an anhydrous mirabilite to obtain a biflavone fractionated soln., and segregating the soln. with 0˜10% CHCl3-MeOH.

Description

Anti-inflammatory, anti-allergic and antirheumatic agents containing biflavonoids from ginkgo biloba

The present invention relates to a method for extracting and separating biflavonoids from ginkgo biloba leaves and the use of these substances.

Ginkgo biloba leaves are very diverse, but the main ingredients are flavonoids and terpene-based compounds, and these ingredients are known to have various physiological activities and pharmacological effects, and the current ginkgo leaf flavone glycoside is commercialized as a peripheral blood circulation improving agent. Commercially available.

Ginkgo biloba flavonoids generally refer to flavonoid glycosides and various chemical and pharmacological studies have been concentrated on these compounds.

However, biflavones contained in a large amount of ginkgo biloba have been reported to be separated and synthesized from six compounds so far, but the biological activity of these compounds is not known.

Researchers have discovered the most efficient method of extracting biflavones from ginkgo biloba and their potent anti-inflammatory, anti-allergic and anti-rheumatic effects and searched for various biological activities. It was.

The present invention takes the same method as in the following example for the efficient separation of the following five biflavone components from the ginkgo leaves.

Example 1

Methanol extract obtained by extracting dried ginkgo leaves (green leaves or yellow leaves) with methanol was taken in aliquots, distilled water was added, and the mixture was left to be shaken by adding almost the same amount of chloroform. This was obtained by dehydration with anhydrous forget-me-not and concentrated to obtain a ginkgo leaf biflavone fraction.

The biflavone fraction was suspended on a silica gel column and eluted with CHCl ₃ -MeOH from 0% to 10% to obtain five biflavones represented by the following structural formulas.

In the above structural formula

I. When R ₁ , R ₂ and R ₄ are CH 3 and R ₃ is H, sciadopitysin

Ⅱ. Ginggetin when R ₁ and R 2 are CH ₃ and R ₃ and R ₄ are H

III. When R ₁ and R ₃ are H and R ₂ and R ₄ are CH _3, isoginkgetin

Ⅳ. Bilobetin when R ₁ , R ₃ and R ₄ are H and R ₂ is CH ₃

Ⅴ. When R ₁ , R ₂ , R ₃ , R ₄ , is H, it is amentoflavone.

Example 2

Methanol extract (1.5g, obtained by extracting dried ginkgo leaf (green or yellow leaf) with methanol) was dissolved in 50ml of methanol, and 5ml of this was taken on a pre-prepared Amberlite XAD-2 column and 50% methanol (methol: water = 1: 1). After eluting with a solution, the solution obtained by eluting with methanol is concentrated under reduced pressure to obtain a fraction of biflavones.

Five biflavones can be obtained by hanging the biflavone fraction on a silica gel column in the same manner as in Example 1 and eluting with CHCl ₃ -MeOH from 0% to 10%.

That is, siadophyticin (I), gingzetin (II), isokingzetin (III), virobetaine (IV) and amentoflavones (V) can be obtained sequentially, and the respective physicochemical spectroscopic data In sum, it was confirmed that their chemical formulas were the above formulas.

In addition, about 2.6% of the green ginkgo biloba leaves (collected between the end of June and the end of September) and about 2.8% of the biflavonoids were obtained from the yellow ginkgo biloba leaves (collected between the end of October and early November). Could.

In addition, the activity and effectiveness of these extracts were found by the following experiment.

[Bioactivity Search]

1. Phospholipase A (PLA) hypothermic experiment

Phospholipase A ₂ (PLA ₂ ) is an enzyme involved in various inflammations, allergies, and rheumatism. Drugs that inhibit the activity of the enzymes can be developed as therapeutic agents that can treat inflammation, allergies, and rheumatism. Therefore, the PLA ₂ inhibitory experiment was performed on biflavonoids isolated from ginkgo biloba as follows.

1-acyl-2 [l- ¹⁴ C] -linoleoyl-3n-glycero-3-phos-phoethanolamine (PE) as substrate (1,000 cpm / nmol), 100 mM Tris-HC (pH 9.0), 6 mM A solution (200 μl) containing CaCl ₂ , a substrate of 10 nmole and 5 to 20 mg of purified enzyme was allowed to react at 37 ° C. for a suitable time.

At this time, when the inhibitor was added, when not added (100% residual activity) was compared as the residual activity or inhibitory activity.

After the reaction was completed, PLA2 activity was calculated by measuring radiolabeled glass radioactivity using a radiometric meter using Dole's method.

As a result, the results as shown in the following table were obtained.

TABLE 1 Inhibitory activity against rat platelet phospholipase A ₂ (PLA ₂ )

As can be seen in Table 1, the activity of 40 g / ml concentrations of ginkgo biloba biflavonoids obtained through Example 1 or 2, respectively, wasogingzetin (III), cyadopitisin (I) and virobe It was shown to inhibit PLA ₂ activity in the order of tin (IV) and amentoflavones (V). Among the most potent inhibitors of isogingzetin (III), do they show dose-dependent inhibitory activity against various PLA ₂ enzyme sources such as rat platelet, indocobratox, salsapox, porcine pancreas, human pleural fluid and rabbit platelet polymer? Experimental results at concentrations of 6.25 µg / ml, 12.5 µg / ml, 25 µg / ml and 40 µg / ml showed dose-dependent inhibition of these enzymes, as shown in Table 2 below. Since it shows effective activity at 6.25µg / ml or more, it can be used as an anti-inflammatory, anti-allergic and antirheumatic agent.

TABLE 2 Various PLA Inhibitory Activities of Isogging Zetin (III)

2. Histamine Free Reaction in Mast Cells

Next, the anti-allergic effect of these biflavonoids could be demonstrated by the free reaction using mast cells purified from the abdominal cells of rats.

In other words, 0.1% BSA-HBSS was added to the abdominal cavity of rats (300-350g), and then the peritoneal cells were separated, and mast cells were purified by 40% BSA gradient method and sensitized with IgE. HNP was released from mast cells by adding DNP-Ascaris and lyso-PS to antigens in sensitized mast cells.

At this time, the inhibitor is added to measure the degree of histamine free reaction. The amount of histamine was methylated in [ ³ H] -SAM using histamine methyltransferase, extracted with an organic solvent, and measured by radiometer.

TABLE 3 Histamine free inhibition by antigen (DNP-Ascaris) -antibody (IgE) from mast cells of biflavonoids

As can be seen in Table 3, the biflavonoids were amentoflavone (V), viroventin (IV) and cyadophyticin (I), isogingzetin (III), and the like at a concentration of 20 µg / ml. It was confirmed that only 4%, 33%, and 70% of histamine were freed.

Therefore, it was confirmed that these biflavonoids have a strong anti-allergic effect even in this experiment because they inhibit 96%, 67% and 30% of histamine release, respectively, compared to the control group.

In addition, the results of comparing the antihistamines, disodium chromoglycate (DSCG) and traniest, which are currently clinically used, with amentoflavone (V), which shows the strongest histamine release, are shown in Table 4 below. As can be seen, amentoflavone (V) inhibits histamine glass about 100 times stronger than disodium chromoglycate (DSCG) or tranist at 1.2 × 10 ^-4 M (= 64.56 µg / ml) concentration. It was confirmed that there is a much stronger anti-allergic action than drugs, this inhibitory effect appeared to be dose-dependent.

Table 4 Comparison of histamine free inhibitory reactions from mast cells of DSCG (Tisolast Chromoglycate), Tranranilast, and Amentoflavone (V)

3. Mouse ear edema inhibitory effect

Next, virobetaine (IV), isogingzetin (III), and cyadophyticin (I) in male mice were measured in order to determine the anti-inflammatory effects of these biflavonoids. After 1 hour of oral administration of 2 mg, arachidonic acid was applied to both ears, and then, an hour later, the thickness of the ear was measured using a dial thickness gauge to calculate the inhibitory effect.

Table 5 Inhibitory Effects of Biflavonoids on Arachidonic Acid-induced Mouse Ear Edema

As can be seen in Table 5, mouse ear edema was inhibited by 24% of virobetaine (IV), 27% of isogingzetin (III), and 22% of cyadophyticin (I) compared to the control group. This biflavonoids have been shown to have strong anti-inflammatory activity.

4. Inhibition of Proliferation of Mouse Spleen Cells

In order to confirm the antirheumatic activity of the biflavonoids, the experiment of inhibiting the proliferation of mouse splenocytes was performed as follows.

In other words, C57BL / 6 mouse spleens were extracted and incubated for 4 days by inducing proliferation with Concanavalin A at 0.5 µg / well, and 0.5 µCi / well concentration [ ³ H]-18 hours before the end of the culture. Tmidine was added and collected by a cell collector to measure radioactivity to obtain a stimulation index.

TABLE 6 Effect of Biflavonoids on Inhibition of Proliferation of Mouse Spleen Cells Induced by Concanavalin A (con A)

As can be seen in Table 6, the stimulation index is 0.30, 0.02, and 0.27, respectively, compared to the control at 10 ^-5 M concentrations of virobetin (IV), isogingzetin (III), and cyadophyticin (I), respectively. It is confirmed that isogingzetin (III) has the strongest antirheumatic effect. As shown in Table 6, the anti-rheumatic effect can be seen that the dilution of biflavonoids to 10 ^-6 M and 10 ^-7 M increases the stimulation index, indicating an anti-rheumatic effect in a concentration-dependent manner. .

Biflavonoids isolated from Ginkgo biloba as shown in the above example showed strong activity against various PLA ₂ , in particular, it inhibited histamine release from mast cells and was about 100 times stronger than DSCG or traniest, which is used as an allergy treatment in hospitals. It was found to have activity and exhibit anti-inflammatory and immunosuppressive properties.

Thus, inflammation, allergy and rheumatism in humans can be treated when biflavonoids isolated from these ginkgo biloba are administered orally or parenterally, either alone or in combination with a pharmaceutically acceptable carrier.

Claims (3)

Biflavonoids represented by the following structural formula Wherein R ₁ to R ₄ are R ₁ , R ₂ , R ₄ are CH ₃ , R ₃ is H, R ₁ , R ₂ is CH ₃ , R ₃ , R ₄ is H, R ₁ , R ₃ is H, R ₂ , R ₄ is H, R ₂ is CH _3, or R ₁ , R ₂ , R 3, R ₄ are H. Or an anti-inflammatory agent containing one or two or more of methyl derivatives and disodium carboxyl derivative acetate derivatives of these biflavonoids. An antiallergic agent containing the compound of claim 1. An antirheumatic agent containing the compound of claim 1.