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15PHARMACEUTICAL SCIENCES | ФАРМАЦЕВТИЧЕСКИЕ НАУКИ
ALLOCATION PHYTOECDYSTEROIDS FROM STACHYS HISSARICA AND STUDYING THEIR HEPATOPROTECTIVE ACTIVITY
Ramazonov N.SH.
Bobayev I.D.
Yusupova U.Y.
Mahmudova M.M.
Usmanov D.A.
Tursunova N.B.
Syrov V.N.
Intfitute of the Chemitfry of Plant substances, Academy of Sciences,of the Republic of Uzbekitfan
Tashkent, Uzbekistan
ABSTRACT
The plant of Stachys hissarica excretes phytoecdyfleroids such as ecdyflerone, polipodin B, 2 -deoxy -20- hydroxyecdysone, integrifleron A, and 2- desoxyedisone were isolated from Stachys hissarica plant. The biological activity of the plants extract was tefled on rats. It is shown that the indomethacin introduction (three times in doze 20 mg / kg) per os to male rats (180-200 g) gives a heavy damage of the hepatobiliary syflem. The ecdyfleroid sum from Stachys hissarica limits manifeflations of indomethacin hepa-totoxicity. Under its influence the symptoms of hepatocyte cytolysis, choleflasis phenomena and lipid peroxidation were eliminated, the protein synthesis and glycogen level by the liver as well as the formation of bile were recovered. The sum of ecdyfleroid from Stachys hissarica as hepatoprotector exceeded the Liv 52.
Keywords: phytoecdyfleroids, Stachys hissarica, hepatoprotective activity
Introduction
Phytoecdyfleroids have different biological activities , which, are firfl of all, characterized by expressive protein-anabolic activity .The medical preparation of ecdyflen was eflablished in the Inflitute, on the basis of one of the compounds of this class (ecdyflerone) which is excreted from Rhaponticum carthamoides, that is showing observable Simulating influence on regeneratative processes in organism [1-3].
However, considering, that Rhaponticum carthamoides practically is not met in Uzbekiflan, nowadays the topical issue is to find an alternative source of phytoecdyfleroids, oriented
r4 or5
ho, r2o'
r6
oh
o
on local raw materials. As the result of the search made on ecdyfleroid-containing plants among the local flora, some types of Stachys [4] are considerate to be perspective enough. Thus, Stachys hissarica (Fam. Labiatae) is frequently found on the territory of Central Asia, particularly in Tashkent and Fergana regions. The present report presents data on the availability, composition and quantity of phytoecdyfleroids in Stachys hissarica, and also the tefl results of the total ecdyfleroid preparation.
Results and Discussion
ri or2
h
oh
h
o
1. Ri — R2 — R3 — R5 — H; R4 — OH; R6 — CH3
2.Ri — R2 — R5 — H; R3 — R4 — OH; R6 — CH3 4.R2 — R3 — R5 — H; Ri — R4 — OH; R6 — CH3
3.R1 — OH; R2 — H 5.R1 — R2 — H
Figure i. The Sructures of phytoecdy^eroids inve^igated in this &udy.
A preliminary invefligation of S. hissarica plant is allowed to isolate and identify its main ecdyfleroids, such as 20 -hydroxyecdysone (1) (0.30% of dry plants weight), polipodin B (2)
(0.21%), 2- dezoxy -20- hydroxyecdysone (3) (0.2%) integrifleron A (4) (0.2%) and 2- dezoxyekdizon (5) (0.01%) [ 5-9 ] .
Physical and chemical properties
The isolated individual ecdyfleroids have been identified on the basis of the IR spectroscopy, 1H NMR- spectroscopy and mass- spectroscopy, and as well as by comparison with reference compounds. Table 1 provides the physico-chemical data for the individual subflances and ecdyfleroids yield from Stachys hissarica plant. The NMR 1H and 13C spectra are recorded by VN MRS 400 (Varian) NMR- spectrometer with an operating frequency of 400 MHz.
Table 1.
icdyfleroids Isolated from Stachys hissarica
Compound No Compound name Composition T, 0C [a] (CH30H) Yield, %
1 20-hydroxyecdysone C27H4407 241-242 +63.2±2 0.35
2 Polipodin B C27H4408 252-254 +94.2±2 0.25
3 2-desoxy-20-hydroxyecdyson C27H4406 235-237 +82.2±2 0.2
4 Integristeron A C27H4408 246-248 +36.1±2 0.2
5 2-desoxy-a-ecdyson C27H4405 234-235 +93.2±2 0.01
It was discovered hepatoprotective properties of the sum of ecdyfleroids from Stachys hissarica under conditions of liver damage by indomethacin. It is known that among hepatobiliary syflem diseases are widespread hepatitis developing after high doses of some drugs, for example no fleroidal anti-inflammatory preparations (phenylbutazone, paracetamol, acetylsalicylic acid) [10]. As was shown on the Table 2, indomethacin had adverse effects on the liver in the experiments. Activity of enzymes alanine aminotransferase and aspartate aminotransferase as markers of hepatocytes cytolysis increased to 141.6 and 50.0 percent respectively. The activity of the alkaline phosphatase as a marker of choleflasis increased to 138.4 percent. The functions of hepatocytes protein synthesis markedly diflurbed and total
protein content in the serum was reduced to 29.7 percent. In the liver tissue revealed a significant decrease of glycogen content (to 64.8 percent) and increased lipid peroxidation as evidenced to the magnification in the content of conjugated diens to 31.2 percent and malondialdehyde to 92.5 percent. The level of reduced glutathione markedly decreased (to 38.4 percent).
It was shown impairment of bile secretion in animals after the adminiflration of indomethacin. The total bile of control animals during 4 hours of experiments decreased to 36.4 percent in comparison with total bile in intact animals. The negative shifts were identified in the synthesis of bile acids, choleflerol metabolism and excretion of bilirubin (Table 2).
Table 2.
Effect of ecdyfleroid sum from Stachys hissarica and Liv 52 on metabolism and function of liver on damage by indomethacin
Experimental conditions Intact animals Control (indomethacin) Indomethacin + ecdysteroid sum Indomethacin + Liv 52
Serum
Alanine aminotransferase mmol / ml / hr 0,96±0,06 2,32±0,42* 0,98±0,06** 1,36±0,008*,**,1
Aspartate aminotransferase mmol / ml / hr 1,16±0,14 1,74±0,20* 1,18±0,16** 1,24±0,18
Alkaline phosphatase, U /L 172±14,2 410±26,2* 180±15,6** 210±18,4**
Total protein, g% 7,4±0,18 5,2±0,14* 7,2±0,16** 6,4±0,13*,**,1
Liver
Glycogen, mg% 1972±126 694±84,2* 1870±102** 1560±98,4*,**,1
Conjugated diens mkmol / g protein 0,160±0,02 0,210±0,04* 0,172±0,03** 0,180±0,03**
Malondialdehyde, nmol / mg protein 0,478±0,04 0,920±0,07* 0,500±0,04** 0,660±0,05*,**,1
Reduced glutathione, micromoles / g 6,82±0,26 4,20±0,18* 6,42±0,22** 5,38±0,20*,**,1
Total bile for 4 hours,mg / 100 g 1056±52,6 672±28,4* 988±34,2** 824±30,6*,**,1
Bile acids, mg% 642±26,2 410±21,4* 592±24,4** 510±18,2*,**,1
Cholesterol, mg% 8,82±0,24 7,10±0,18* 8,74±0,22** 7,82±0,20*,**,1
Bilirubin, mg% 11,2±0,50 6,98±0,32* 10,4±0,48** 8,42±0,34*,**,1
Cholate-cholesterol ratio 72,8±2,6 57,7±1,8* 67,7±2,6** 65,2±2,2*,**,1
Note. Values are mean±SD, n = 6-8, * - p <0.05 for the comparison with the intact animals, ** - p <0.05 for the comparison with the control, 1 - p <0.05 for the comparison between the two experimental groups
The ecdyfleroids sum adminiflration prevented the development of cytolysis syndromes of hepatocytes and intrahepatic choleflasis: alanine aminotransferase, aspartat aminotransferase and alkaline phosphatase were below for 57.8; 32.2 and 56.1 percent respectively than in the control animals. The total protein content in the serum was increased to 38.5 percent relatively to a control group. The ecdyfleroid sum normalized of glycogen-synthesizing function of liver and decreased the content of conjugated diens and malondialdehyde. In the same time the reduced glutathione level in the liver became almofl the same as intact animals (Table 2). The biliary excretion improved in animals treated by fludy drug. Thus, the secretion of bile in this group was higher to 47 percent, the secretion of bile acids was higher to 44.4 percent, the contents of choleflerol and bilirubin was higher to 23,1 and 48.9 percent respectably than the control values. The cholate-choleflerol ratio improved. Status of the liver in conditions of damage by indomethacin and treated of the ecdyfleroid sum from S. hissarica was better than after treatment by hepatoprotector Liv 52.
Thus, the obtained results show that the using of ecdyfleroid-containing preparations for eliminate the negative impact on the liver of some medications is promising.
EXPERIMENTAL PART
Chemical evaluation
General comments. UV - spectra were measured on Lambda -16 spectrophotometer (Perkin - Elmer). The temperature of fusion of subflances was defined on device Boetius (Germany) and Mel-Temp (USA). IR- spectrum was removed with Fourier spectrometer by Perkin - Elmer, model 2000 (KBr), nuclear magnetic resonance spectra 1H and 13C were regiflered with spectrometer VNMRS 400 (Varian), with working frequency of 400 MHz. The samples were removed in solution Py-d5, internal flandard HMDS , 5 scale in dissolve Py-d5, for TLC - chromatographic plates Silufol UV 254, was used for display of plates - 3 % a vanillin and ethanol solution was used in syflems of solvents chloroform-metanol 1, 9:1; 2, 4:1; (syflem), chloroform-methanol-water 1, 4:1:0,1; 2, 70:23:3 (syflem B). In thin-layer and column chromatography the silica gel marks « KSK « and «L « ( Czechoslovakia) , alumina (active IV c.) were used. Thin layer chromatography ( TLC) was carried out on the fixed bed of silica gel , sifted through a sieve of 0.08 mm, containing 7 % of gypsum , and on the plates «Silufol».
For column chromatography the silica gel with a particle size of 0,1-0,16 mm and 0.08-0.1 was used .
20-hydroxyecdysone (1), C27H4407, m.p. 241-242°C (from acetone), [a] D +63.2 ±2° (from 6.30; methanol). UV - a spectrum (C2H50H, Xmax> nm): 245 nm (Ig e 4.01). IR - a spectrum (KBr, v, sm-1): 3435 (OH), 1665 (7-en-6-ketogrouping).
Mass spectrum, m/z (%): 480 (M; 0.03), 462 (1), 444 (2), 446 (14), 11 (4), 408 (10), 393 (5), 363 (10), 345 (33), 327 (20), 301 (16), 300 (12), 161 (5), 143 (10), 125 (8), 107 (6), 99 (100), 81 (34), 69 (21).
The comparison of the resulted conflants and spectral characteriflics [7] with literary data has allowed to identify ecdyfleroid with ecdyfleroid.
Polipodin B (2), C27H44O8, after recryflallization from acetone had m.p. 252-254 °C, [a] D +94.2±2 ° (from 0.50; methanol). The IR -spectrum (KBr, v, sm-1): 3400 (OH), 1673 (7-en-6-ketogrouping). The UV-spectrum (C2H50H, Xmax> nm): 244 nm (Ig e 3.9).
Mass spectrum, m/z (%): 478 (M-H20; 0.03), 463 (0.1), 460 (0.2), 445 (0.3), 427 (0.4), 424 (0.4), 409 (0.7), 379 (6), 361 (100), 360 (86), 344 (4), 343 (4), 325 (5), 316 (10), 99 (12), 81 (13), 69 (15).
The flructure of compound 2 was confirmed by spectral data and by comparison with original sample on thin layer chromatography[9].
2-desoxy-20- hydroxyecdysone (3), C27H4406, m.p. 254256 0C (from water ethanol),
[a] D23+82.0+3o (with 1,3; methanol). XC2H50Hmax:246 nm (lg e 4,10); the IR -spectrum (KBr, v, sm-1): 3370 (OH), 1645 (7-en-6-ketogroup) [6].
Mass spectrum; m/z (%): 464 (M +, 1); 446 (3), 428 (10), 413 (8), 410 (14), 395 (6), 392 (6), 377 (5), 347 (35), 329 (90), 312 (33), 311 (28), 303 (9), 302 (11), 297 (13), 295 (14), 285 (30), 284 (35), 234 (15), 233 (15), 99 (100), 81 (73).
Integrifleron A (4), C27H44O8, with m.p. 246-248 °C (from mixture of ethyl acetate- methanol), [a] D20 +36.1±2 ° (from 0.43; methanol). UV - a spectrum (C2H50H, Xmax> nm): 245 nm(Ig e 4.00). IR - a spectrum (KBr, v, sm-1): 3400 (OH), 1660 (7-en-6-keto grouping).
Mass spectrum, m/z (%): 478 (M-H20; 3), 460 (4), 445 (10), 442 (18), 427 (14), 409 (7), 391 (4), 379 (73), 374 (5), 368 (17), 361 (74), 343 (100), 325 (43), 316 (17), 309 (7), 283 (41), 143 (60), 135 (61), 99 (35), 81 (34).
1H NMR (C5D5N; 5, ppm; 0-TMC ): 1.19 (3H at C-18,); 1.40 (3H at C-19,); 1.39 (6H at C-26/27,); 1.58 (3H at C-21,); 3.57 (1H at C-9,); 3.77 (1H at C-22,); 4.30 (3H at C-1,2,3,); 6.17 (1H at C-7, br.s).
The spectral data and comparison with the original sample (by TLC) have allowed to identify the integrifleron A.
2-desoxy - a -ecdysone (5). C27H44O5, m.p. 235-236 °C (from water ethanol), the IR-spectrum (KBr; v, sm-1): 3429 (OH), 1662 (7-en-6 keto group).
Mass spectrum; m/z (%): 448 (M 2), 430 (13), 415 (14), 412 (11), 402 (16), 285 (12), 284 (12), 235 (27), 234 (50), 233 (32), 99 (100), 81 (63).
Biological evaluation
In experiments used male rats weighing 180-200 g with liver damage by indomethacin was adminiflered intragaflrically at a dose of 20 mg / kg daily for 3 days [10]. The ecdyfleroid sum from Stachys hissarica was adminiflered for 5 days per os as an aqueous emulsion with a gum arabic (the firfl 3 days together with indomethacin) at a dose of 10 mg / kg. The reference drug was Liv 52 (The Himalaya drug Co., India) was adminiflered to rats in a similar manner in a dose of 50 mg / kg. Control animals obtained indomethacin and aqueous emulsion of gum arabic an adequate amount per os. All animals were kept under flandard conditions of vivarium with free access to feed and water. On the 6th day part of rats from each group were decapitated under
light ether aneflhesia. Thereafter, serum enzyme activities of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein content was determined by using La Chema reagents (Czech Republic) and content of glycogen, conjugated diens, malondialdehyde and reduced glutation was determined by the methods described in [11,12,13]. The other part of the animals was aneflhetized (sodium thiopental 45 mg / kg i.p.) and was collected of bile for 4 hours through a catheter inserted into the common bile duct. The concentration of bile acids, bilirubin and choleflerol was determined in bile. The data are processed by using variation flatiflics with Student's t-criterion.
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