Novel biflavone diglycosides and biological activity of Jatropha multifida leaves

Chemistry of Natural Compounds, Vol. 48, No. 5, November, 2012 [Russian original No. 5, September–October, 2012]

NOVEL BIFLAVONE DIGLYCOSIDES AND BIOLOGICAL ACTIVITY OF Jatropha multifida LEAVES

M. S. Marzouk,1* F. A. Moharram,2 E. G. Haggag,3 S. M. El-Batran,4 I. I. Mahmoud,3 and R. R. Ibrahim3

UDC 547.972

Chromatographic separation of the 80% aqueous methanol extract (AME) of Jatropha multifida Linn. leaves has yielded three novel biapigenin di-C-glucosides, i.e., 6,6cc-di-C-E-glucopyranosyl-methylene-(8,8cc)biapigenin (7), 3,6 cc -di-C-E-glucopyranosyl-methylene-(6,8 cc)-biapigenin (8), and 6,6 cc -di-C-Eglucopyranosyl-methylene-(3,8cc)-biapigenin (9), named jatrophenols I–III. In addition, seven known polyphenolic metabolites were identified as apigenin 7-O-E-neohespredoside (1), ferulic acid (2), quercetrin (3), vicinin-II (4), isoorientin (5), vitexin (6), and luteolin (10) for the first time from this species. The AME of the plant was reported for the first time to have significant analgesic, anti-inflammatory, and antihypertensive effects. Keywords: Jatropha multifida, HR-ESI-MS, biapigenin di-C-glucoside, analgesic, anti-inflammatory, antihypertensive. The genus Jatropha (Euphorbiaceae) comprises about 175 species, some of which are used in folk medicine remedies [1, 2]. The principal compounds that have been isolated from Jatropha species were mainly diterpenes [3–6], lignans [7], triterpenes [8], coumarins [9], tannins [10], and flavonoids [11], which were studied for their different biological activities such as hypotensive [12], antitumor [13], antioxidant [14], antibacterial [15], anti-inflammatory [16], and analgesic [11], and as a hemostatic agent [17]. Our previous report on J. curcas has led to the identification of compound 7 and its immunomodulatory activity in chicks [18]. Additionally, two studies were reported on the constitutive polyphenols of J. multifida [10, 18]. The present phytochemical and biological study was carried out on this species to throw light on the importance of this species that grows in the Benha Region in Egypt. The AME was chromatographed on a polyamide column followed by successive separation on Sephadex LH-20 and cellulose columns, affording ten pure compounds. Their structures were established as apigenin 7-O-E-D-neohespredoside (1), ferulic acid (2), quercetrin (3), three flavone C-glucosides >vicinin-II (4), isoorientin (5), and vitexin (6)@ and luteolin (10) on the basis of their chromatographic properties, chemical and spectroscopic studies (UV, 1H, 13C NMR, and APT), and comparison with literature data of related compounds [19, 20]. In addition, we established the structures of the novel biflavone di-C-glucosides (jatrophenols I–III) as 6,6cc-di-C-E-glucopyranosyl-methylene-(8,8cc)-biapigenin (7), 3,6cc-di-C-E-glucopyranosyl-methylene-(6,8cc)biapigenin (8), and 6,6cc-di-C-E-glucopyranosyl-methylene-(3,8cc)-biapigenin (9) by extensive spectroscopic studies, particularly 2D NMR (1H–1H COSY, HMQC, and HMBC) and HR-ESI-MS. Compound 7 was identified before in our previous report on J. curcas, and full data on it (UV, 1D, 2D NMR, and HR-ESI-MS) were completely consistent with that published in [18], confirming its structure as 6,6cc-di-C-E-4C1-glucopyranosyl-methylene-(8,8cc)-biapigenin (jatrophenol I). Compound 8 exhibited more or less the same chromatographic properties and UV spectral data as 7, which is a bi-apigenin glycoside with free hydroxyls at C-5 and C-7 of A-rings and C-4c of B-rings [18]. Compound 8 was tentatively identified as bi-apigenin di-C-hexoside with an extra methylene group, depending on its resistance to the conditions of normal acid hydrolysis and negative HR-ESI-MS spectrum, which showed a molecular ion peak at m/z 875.21892 corresponding exactly to the same molecular weight and molecular formula of 7. 1) Department of Pharmacetical Chemistry, College of Pharmacy, King Saud University, P. O. Box 2457, 11451, Riyadh, Saudi Arabia, fax: +966 1 4676220, e-mail: [email protected]; 2) Department of Pharmaceutical Sciences, College of Clinical Pharmacy (Girls), King Faisal University, P. O. Box 400, 31982, Hofuf, Saudi Arabia; 3) Department of Pharmacognosy, Faculty of Pharmacy, Helwan University, Ain-Helwan, Cairo, Egypt; 4) Department of Pharmacology, Division of Medical Sciences, National Research Centre, Dokki, 12622, Cairo, Egypt. Published in Khimiya Prirodnykh Soedinenii, No. 5, September–October, 2012, pp. 685–689. Original article submitted October 11, 2011. 0009-3130/12/4805-0765 ”2012 Springer Science+Business Media New York

765

TABLE 1. 13C NMR Spectral Data of 7–9 (DMSO-d6, G, ppm) C atom

7

2, 2cc 3, 3cc 4, 4cc 5, 5cc 6, 6cc 7, 7cc 8, 8cc 9, 9cc 10, 10cc 1c, 1ccc 2c/6c, 2ccc/6ccc 3c/5c, 3ccc/5ccc 4c, 4ccc 1cccc, 1ccccc 2cccc, 2ccccc 3cccc, 3ccccc 4cccc, 4ccccc 5cccc, 5ccccc 6cccc, 6ccccc

163.9 100.9 181.3 160.2 109.6 162.7 105.0 154.3 103.6 122.1 127.7 115.6 159.3 73.9 71.0 79.5 70.1 81.4 61.8

______

8

9

162.6 111.3 181.0 160.6 108.4 160.7 94.0 154.3 106.1 122.2 128.5 115.6 158.1 74.0 70.9 79.0 70.1 81.4 61.7

163.6 101.9 181.4 160.7 109.3 162.3 106.8 154.3 106.1 122.3 128.7 115.7 159.4 74.0 71.1 79.5 70.1 81.4 61.7

164.0 112.4 181.2 160.8 110.4 162.6 93.8 154.3 107.6 121.9 128.7 115.8 159.2 74.1 71.7 79.3 70.8 81.5 61.5

162.6 102.0 181.4 160.8 109.1 160.8 104.8 154.3 106.1 122.0 128.6 115.7 159.2 74.1 71.1 78.9 70.3 81.3 61.5

The resonances of CH2-bridge was expected to be masked by DMSO-d6 signal in case of 7 and 9, while it was assigned at 37.6 in case of 8. OH

OH HO HO HO

OH

O

5'' 10''

O HO

9''

7''

1'''

O

HO

[6-C-8 '']

OH

5''' 5'

O

OH OH

8''

OH

9

O

6''

HO

3'

1'

O

OH

3 5

OH OH

O OH

7

HO HO

OH

O

10

OH

HO

OH

O O

1

HO 7 O OH

3

6

3'''

1''

[8-C-8 '']

HO HO HO

O

HO

3''

HO OH

HO

OH

8

O

O 3

6

OH HO HO HO

OH

O

[3-C-8'']

HO

8''

O

OH

O

OH

O 6''

OH 9

The duplication of signals all over the total 1H and 13C NMR spectra reflected the probability of an asymmetric connection between the two apigenin moieties through a methylene bridge and the similar position of the two hexoside moieties and their type. In the aromatic region, the 1H NMR spectrum showed a pair of two AAcXXc spin coupling systems, each of the two ortho doublets, assignable to H-2c/6c-H-3c/5c and H-2ccc/6ccc-H-3ccc/5ccc of four pairs of equivalent protons in two 4-hydroxy B-rings. This was supported also by the duplication of their four 13C-resonances at 122.3/122.2, 128.7/128.5, 115.7/115.6, and 159.4/158.1 for C-1c/1ccc, C-2c/6c-2ccc/6ccc, C-3c/5c-3ccc/5ccc, and C-4c/4ccc (Table 1). Observation of two singlets at G 6.61 and 6.52 for one H-3 proton and one H-8 proton was evidence of the location of the methylene bridge between C-6 and C-8cc and the two C-glycoside on C-3 and C-6cc. On the application of 13C NMR substituent additive rules and comparison with previously reported data [19, 20], it was found that the downfield location of both C-6 and C-8cc at G 108.4 and 106.8 (# 10 ppm), respectively, is evidence of the C6-CH2-C8cc bridge between the two apigenin moieties. Also, the downfield shift of both C-3 and C-6cc resonances to 111.3 and 109.3 ppm, respectively, confirms the C-glycosidation on these carbons of both aglycones. 766

TABLE 2. Analgesic Effect of AME on Induced Writhing Mice and Using Hot Plate Method on Mice Potency latency, s Group

Control IMTCN AME AME

Dose, mg/100 g by weight

Number of writhings

Inhibition, %

– 1.8 20 40

82.1 r 8.0 32.2 r 3.8* 54.6 r 3.0* 15.0 r 1.6*

– 60.3 32.8 81.5

1h Change, % potency

Xc r SE 16.9 r 1.4 24.3 r 2.6** 21.6 r 2.2* 20.3 r 1.9*

43.8 27.8 20.1

Xc r SE

Xc r SE

1.00 0.63 0.46

26.3 r 2*** 21.6 r 2.5* 23.2 r 2.5*

2h Change, % potency 42.9 17.4 26.1

Xc r SE

1.0 0.1 0.6

______ Values are Xc r SE, n = 10; *p < 0.05 vs. control; **p < 0.01 and ***p < 0.001 vs. zero time z potency is considered as the percent of change of the different treatment to the percent of change of IMTCN. The % change denotes change from control group. IMTCN: indomethacin. TABLE 3. Anti-inflammatory Effect of AME on Carrageenan-Induced Paw Edema in Rats Group Control IMTCN AME AME

Dose, mg/100 g by weight – 1.8 20 40

Percentage of change from the +ve control value in the same group

Inhibition, %

1h

2h

3h

4h

after 4 h

50.8 r 5.5 21.6 r 2.5*** 24.3 r 2.2*** 15.0 r 1.2***

65.8 r 6.3 25.9 r 2.6*** 46.6 r 6.6* 31.4 r 5.0***

84.5 r 8.1 36.2 r 3.6*** 58.2 r 5.1* 41.9 r 6.3***

120.3 r 11.4 45.8 r 5.3*** 69.5 r 5.9** 47.1 r 4.7***

– 75.2 50.8 73.2

______ *p < 0.05, **p < 0.01, and ***p < 0.001 vs. control. The determination of the type of both C-glycoside moieties as two E-glucopyranosides in the 4C1-conformation was confirmed by the intrinsic G and J-values of the two anomeric protons and the diagnostic and duplicated six 13C-resonances for the two sugar moieties in the range of 82–60 ppm. All the remaining 13C-resonances were assigned by comparison with the previously reported data for similar structures [19, 20] and of 7 [18]. The resonances of the CH2-bridge were expected to be masked by the DMSO-d6 signal in the case of 7 and 9, while it was assigned at 37.6 in the case of 8. The structure of 8 was confirmed by the cross-peaks of the proton-proton correlation (1H–1H COSY) and proton-carbon correlations in direct (HMQC) and long-range (HMBC) 2D NMR. Therefore, compound 8 was finally identified as 3,6cc-di-CE-4C1-glucopyranosyl-methylene-(6,8cc)-biapigenin, which was empirically named jatrophenol II. Compound 9 exhibited more or less the same chromatographic properties and UV spectral data of the previous two compounds 7 and 8. Referring to the negative HR-ESI-MS, it displayed a molecular ion at 875.21796 [M – H]–, corresponding to the same molecular weight of 7 and 8. Thus, it was expected to have a bi-apigenin di-C-hexosylmethane-like structure with free 5-OH and 7-OH on the basis of UV and MS data. As in the case of 8, compound 9 was expected to have an asymmetric dimeric structure due to the duplication of all 1H and 13C signals in both NMR spectra. The two singlets at 6.64 and 6.56 were interpreted for H-3cc and H-8; this document was proved by observation of their own carbons at G 102.0 and 93.8. Moreover, the characteristic downfield location of both C-3 and C-8cc at 112.4 and 104.8 confirms the C3-CH2-C8cc bridge forming the biflavonoid structure. The two C-glycosyl moieties were found to be on the two C-6 carbons because of their downfield location at G 110.4 and 109.1 (' + ~10 ppm) (Table 1, [19]), as in the case of the previous two biflavonoids (7, 8), the two hexoside moieties were determined to be of the C-E-4C1-glucopyranoside type on the basis of G and J-values of their 1H and 13C signals. The complete assignment of all 13C signals for 9 was based on a comparison of the previously reported data of similar structures [19, 20] and those of compounds 7 [18] and 8. The 2D NMR correlations in 1H–1H COSY, HMQC, and HMBC confirmed the assignment of all 1H and 13C signals in the 1D NMR spectra. Hence, compound 9 was identified as 6,6cc-di-C-E-D-4C1-glucopyranosyl-methylene-(3,8cc)-biapigenin, named jatrophenol III. It was found that the AME is nontoxic up to the maximum soluble dose 4 g/kg by weight. Moreover, it inhibits writhing much more significantly than the positive control indomethacin at a dose 40 mg/100 g by weight (Table 2). Also both dose levels of AME (20 and 40 mg/100 g by weight) produced a significant prolongation of reaction time to thermal stimulus after 1 and 2 h administration, confirming the analgesic activity of the extract. 767

TABLE 4. Hypotensive Effect of AME on Systolic Blood Pressure (SBP) on DOCA-salt Hypotensive Rats SBP (mm Hg) Xc r SE

Inhibition, %

Group

Dose, mg/100 g by weight

Normal

DOCA

Normal

DOCA

Control AME AME

– 20 40

126.2 r 5.5 118.5 r 5.0 103.2 r 7.4*

195.7 r 7.4 173.0 r 10.5 154.8 r 7.6**

– 6.1 18.2

– 11.6 20.9

______ *p < 0.05, **p < 0.01 vs. control. Table 3 shows that the induction of inflammation (swelling and erythema) by carrageenan is time-dependent and the paw volume reaches the maximal after 4 h. The two dose levels of AME resulted in significant anti-inflammatory activity after 1, 2, 3, and 4 h, and the percentage inhibition reaches the maximum after 4 h in comparison with indomethacin. Table 4 shows that the oral administration of AME at 40 mg/100 g by weight leads to a significant decrease in SBP after 2 weeks (percentage inhibition 18.2%). In case of DOCA-salt hypertensive rats, AME (40 mg/100 g by weight) exhibits a significant hypotensive effect with a percentage inhibition of 20.9%. Taken together, this species, growing in Egypt, could be considered as an available natural source for flavonoid glycosides, especially of the C-type, and some novel biflavonoids, mainly di-C-glycosyl apigenin. Moreover, its aqueous methanol extract can be used for drug formulation as an analgesic, anti-inflammatory, and hypotensive if it is proved to be nontoxic.

EXPERIMENTAL General. The NMR (1H and 13C NMR) spectra were recorded at 300, 600 (1H) and 75, 150 (13C) MHz, on Varian Mercury 300 and Inova-600 spectrometers. The G-values are reported as ppm relative to TMS in DMSO-d6 and the J-values are in Hz. ESI-MS were measured on a double-focusing sector field MAT 90 mass spectrometer connected to an ESI-II ion source (Finnigan, Bremen, Germany), in negative and positive modes. The UV spectra for pure samples were recorded separately as solutions in MeOH and with different diagnostic UV shift reagents on a Shimadzu UV 240 spectrophotometer. All chemicals and reagents used for the pharmacological activity were purchased from Sigma-Aldrich Co. (St. Louise, MO, USA), and other chemicals, solvents, and reagents used in chromatography were of analytical grade. Authentic reference phenolic compounds were obtained from Phytochemistry Laboratory, Department of Molecular and cell Biology, University of Texas at Austin (Austin, TX, USA). Plant Material. Leaves of J. multifida Linn. were collected from plants growing in the Benha Region, Egypt in July 2007 during the flowering and fruiting stage. Identification of the plant was confirmed by Dr. Wafaa M. Amer, assistant professor of “Flora and Taxonomy”, Botany Department, Faculty of Science, Cairo University, Giza, Egypt. Voucher specimens (Reg. No. J-1) are kept in the Herbarium, Pharmacognosy Department, Faculty of Pharmacy, Helwan University, Cairo, Egypt. Extraction and Isolation. Powdered, air-dried leaves of J. multifida (1 kg) were exhaustively extracted with hot 80% MeOH (4 u 5 L, 2 h, 70qC) under reflux. The dry residue obtained (250 g) was defatted with petroleum ether (60–80qC) under reflux (3 u 1 L, 1 h, 70qC) to give 200 g of dry extract. Preliminary purification of this extract was carried out by precipitation of its concentrated aqueous solution twice by the addition of excess EtOH. The dry filtrate (AME, 100 g) prepared in water was fractionated on a polyamide 6S (Riedel de Hean AG, Seelze, Hannover, Germany) column (‡ 7 u 140 cm, 300 g) and was eluted with H2O followed by increasing MeOH gradients with decreasing polarity. Elution with H2O and 10–20% MeOH afforded fraction A (5 L), elution with 30–40% MeOH afforded fraction B (5 L), elution with 40–50% MeOH afforded fraction C (8 L), elution with 55% MeOH afforded fraction D (6 L), elution with 65–85% MeOH afforded fraction E (9 L), and, finally, elution with MeOH gave rise to fraction F (4 L). Elution and fractionation processes (collections: 250 mL portions) were controlled by the detection of compounds with UV-light and spray reagents on comparative paper chromatography (CoPC) and TLC. Fraction A was found to be free from polyphenols. Compound 1 (25 mg) was obtained by exhaustive extraction of the concentrated solution of fraction B using n-BuOH and by repeated fractionation (twice) on Sephadex LH-20 (Pharmacia, Uppsala, Sweden) using MeOH (95%) for elution. Separation of fraction C on Sephadex LH-20 and elution by EtOH (95%) gave two major subfractions i and ii. Repeated CC (twice) on Sephadex LH-20 of subfraction i using MeOH afforded compound 2 (20 mg), while fractionation of ii on cellulose (Merck) using n-BuOH saturated with H2O for elution yielded two subfractions iia and iib. Subfractions iia afforded compound 3 (30 mg) when were fractionated on Sephadex LH-20 768

using EtOH (95%) for elution, while compound 4 (33 mg) was obtained by repeated fractionation of subfraction iib on Sephadex LH-20 using MeOH (95%) for elution. Pure samples of compounds 5 (20 mg) and 6 (38 mg) were obtained by repeated CC fractionation of fraction D on cellulose (40% aqueous MeOH) and Sephadex LH-20 using 50% EtOH. Fraction E was concentrated in hot 50% aqueous MeOH and, by repeated fractionation (twice) on cellulose column using 50% MeOH, gave two major subfractions i and ii. Successive CC of subfraction i on Sephadex using n-BuOH-iso-propanol–H2 O (BIW 4:1:5, top layer) for elution afforded 7 (27 mg). Repeated CC of subfraction ii on cellulose and Sephadex LH-20 using MeOH and BIW for elution gave 8 (35 mg) and 9 (42 mg), respectively. Part of fraction F (40 mg) was successively fractionated on Sephadex LH-20 using MeOH (95%) as an eluent to yield a pure sample of compound 10 (16 mg). All separation processes were followed up by 2D PC and CoPC using Whatman No. 1 paper with (S1) n-BuOH–AcOH–H2O (4:1:5, top layer) and (S2) 15% aqueous AcOH eluents. cccc-Di-C-E-glucopyranosyl-methylene-(8,8cc cccc)-biapigenin (7). Full experimental data and description of 7 appears 6,6cc in our previous report [18]. cccc-Di-C-E-glucopyranosyl-methylene-(6,8cc cccc)-biapigenin (8). Dark yellow amorphous powder. It gave a deep 3,6cc green and greenish yellow color with FeCl3 and Naturstoff spray reagents, respectively. Rf 0.35 (S1), 0.37 (S2) on PC. UV/Vis (MeOH, Omax, nm): 278, 298 (sh), 336; (+ NaOMe): 276, 392; (+ NaOAc): 278, 300 (sh), 362; (+ NaOAc + H3BO3): 278, 306 (sh), 350; (+ AlCl3): 280, 300 (sh), 350, 380 (sh); (+ AlCl3 + HCl): 280, 302 (sh), 350, 380 (sh). 1H NMR (600 MHz, DMSO-d6, G, ppm, J/Hz): 13.70 (5-OH) and 13.75 (5cc-OH), 8.23 and 7.93 (4H, d, J = 8.7, H-2c/6c, 2ccc/6ccc), 6.93 and 6.86 (4H, d, J = 8.7, H-3c/5c, 3ccc/5ccc), 6.61 (1H, s, H-3cc), 6.52 (1H, s, H-8), 4.94 (2H, d, J = 9.2, 2 u H-1G), 4.29 (m, 2 uH-2G), 3.88–3.14 (m, remaining sugar protons and CH2-bridge). 13C NMR (150 MHz, DMSO-d6, G, ppm), see Table 1. Negative HR-ESI-MS m/z 875.21892 [M – H]– (calcd 875.22920). cccc-Di-C-E-glucopyranosyl-methylene-(3,8cc cccc)-biapigenin (9). Yellow amorphous powder. It gave a deep green 6,6cc and greenish yellow color with FeCl3 and Naturstoff spray reagents, respectively. Rf 0.33 (S1), 0.39 (S2) on PC. UV/Vis (MeOH, Omax, nm): 275, 300 (sh), 332; (+ NaOMe): 276, 316 (sh), 392; (+ NaOAc): 276, 310 (sh), 348; (+ NaOAc + H3BO3): 276, 310 (sh), 360; (+ AlCl3): 279, 304 (sh), 350, 380 (sh); (+ AlCl3 + HCl): 280, 300 (sh), 345, 372 (sh). 1H NMR (600 MHz, DMSO-d6, G, ppm, J/Hz): 13.96 (5-OH) and 13.60 (5cc-OH), 7.94 (4H, d, J = 8.4, H-2c/6c, 2ccc/6ccc), 6.94 and 6.87 (4H, d, J = 8.4 and 9, H-3c/5c, 3ccc/5ccc), 6.64 (1H, s, H-3cc), 6.56 (1H, s, H-8), 4.92 and 4.70 (2H, d, J = 9.2, 2 u H-1G), 4.23 (m, 2 u H-2G), 3.73–3.17 (m, remaining sugar protons and CH2-bridge). 13C NMR (150 MHz, DMSO-d6, G, ppm), see Table 1. Negative HR-ESI-MS m/z 875.21796 [M – H]– (calcd 875.22920). Animals. Rats of both sexes (150–200 g) were used for anti-inflammatory and hypotensive activity and mice (18–20 g) for LD50 and analgesic activity. Animals were housed under standard conditions of light and temperature and received standard rat chow and tap water ad lib. Each group was kept in a separate cage. All animal procedures were performed after approval from the Ethics Committee of the National Research Centre, Cairo and in accordance with the recommendations for the proper care and use of laboratory animals (Canadian Council on Animal Care Guidelines, 1984). Median Lethal Dose (LD50). For the determination of LD50 of AME, it was dissolved in distilled H2O using Tween 80 and was given orally to mice in graded doses. The control group received the same volume of distilled H2O. The percentage mortality was recorded 24 h later [21]. Analgesic Activity. The analgesic activity was determined by measuring the responses of animals to chemical stimulus (acetic acid method, [22]) and thermal stimulus (hot plate method, [23]). The animals were divided into four groups of ten animals each. The first group received saline (control), the second group received indomethacin (1.8 mg/100 g by weight sc., + ve control), and the last two groups received orally the AME in two dose levels (20 and 40 mg/100 g by weight). After 0.5 h interval, the mice received 0.6% acetic acid ip. and the number of writhes in 20 min period was counted. In the hot-plate test, an electronically controlled hot-plate was adjusted at 52 r 0.1qC. The four groups of mice received saline, indomethacin (+ ve control), and two dose levels of AME, respectively, 0.5 h prior to testing. The time elapsed until either hind paw licking or jumping occurred was recorded just before and after 1 and 2 h from drug administration (Table 2). Anti-inflammatory Activity. The acute anti-inflammatory effect of AME was tested by dividing the rats into four groups, which were treated in the same previous manner as the analgesic effect [24]. After 0.5 h, paw edema was induced by subplanter injection of 0.1 mL of 1% sterile carrageenan solution in saline into the pad of the right hind paw; the left paw was injected with 0.5 mL saline. The paw volume was determined immediately before carrageenan injection and after 1, 2, 3, and 4 h using a plethysmometer, and the edema was expressed as the percentage change from the control value (Table 3).

769

Hypotensive Activity. The rats received a suspension of deoxycorticosterone acetate (DOCA) (50 mg/kg in 0.9 NaCl sc.) as a hypertensive for 6 weeks in addition to 2% glucose (to increase the intake of NaCl used in drinking water and KCl to reduce the severity of the hypokalemia which resulted as a response to DOCA administration, [25]). Of six groups, ten male rats each, three were considered as normal (+ ve control) and the other three were pretreated with DOCA. The first group in each case was considered as control (received saline) and the other three groups received the AME (20 and 40 mg/100 g by weight). The systolic blood pressure (SBP) was measured twice weekly from the tail of pre-warmed unanaesthetized rats by the tail-cuff technique [26] for all pre-trained animals. An average of three readings was recorded for each animal. After a basal period of 7 days, the treated groups received, by gavages (0.5 mL/100g), a daily dose of AME (20 or 40 mg/100 g by weight) for two weeks (Table 4). Statistical Analysis. The group means were compared by the ANOVA test, followed by Tukeycs multiple range test [21]. The values were considered to be significantly different when p was less than 0.05.

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