Annona squamosa seed extract in the regulation of hyperthyroidism and lipid-peroxidation in mice: possible involvement of quercetin.
Annona squamosa (Custard apple) seeds are generally thrown away as waste materials. The extract of these seeds was evaluated for its possible ameliorative effect in the regulation of hyperthyroidism in mouse model. Serum triiodothyronine ([T.sub.3]), thyroxine ([T.sbu.4]) concentrations, hepatic glucose-6-phospatase (G-6-Pase) and 5'-mono-deiodinase (5'DI) activity were considered as the end parameters of thyroid function. Simultaneously hepatic lipid peroxidation (LPO), superoxide dismutase (SOD) and catalase (CAT) activities were investigated to observe its hepatotoxic effect, if any.
L-[T.sub.4] administration (0.5 mg/kg/d for 12 days, i.p.) increased the levels of serum [T.sub.3] and [T.sub.4], activity of hepatic G-6-Pase, 5'DI and LPO with a parallel decrease in SOD and CAT activities. However, simultaneous administration of the Annona seed extract (200 mg/kg) or quercetin (10mg/kg) to [T.sub.4]-induced hyperthyroid animals for 10 days, reversed all these effects indicating their potential in the regulation of hyperthyroidism. Further, the seed extract did not increase, but decreased the hepatic LPO suggesting its safe and antiperoxidative nature. Quercetin also decreased hepatic LPO. When relative efficacy was compared with that of propyl thiouracil (PTU), a standard antithyroidic drug, experimental seed extract appeared to be more effective. Phytochemical analyses including HPLC revealed the presence of quercetin in the seed extract and the results on the effects of quercetin suggested the involvement of this phytochemical in the mediation of antithyroidal activity of Annona squamosa seed extract.
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Keywords: Annona squmosa seed extract; HPLC analyses; Hyperthyroidism; Lipid peroxidation; Quercetin
Thyroid hormones regulate almost all functional aspects of the body, including metabolic, respiratory, cardiovascular, nervous and reproductive functions, either directly or indirectly (Ganong, 1995). Alterations in the level of these hormones lead not only to altered basal metabolic rate but also to many health problems. Particularly, hyperthyroidism (chronic elevated circulatory thyroid hormones), if not treated properly, some times ends up with the common health problems such as diabetes mellitus and cardio-vascular diseases (Fry, 1993).
Despite the fact that plant-based drugs are gradually becoming the choice of the patients for their safe and economic nature, on herbal regulation of hyperthyroidism scientific investigations are meager (Tahiliani and Kar, 2003; Kar and Panda, 2004). Although Annona seeds are generally thrown away as waste materials, they are known to possess insecticidal, anti-ovulatory, abortifacient and anti-implantation properties (Vohora et al., 1975; Rao et al., 1979; Damasceno et al., 2002). In the present study an attempt has been made to reveal its hitherto unknown ameliorative efficacy, if any, in the regulation of hyperthyroidism using laboratory mouse as working model. Attempt has also been made to correlate the thyroid regulating activity of Annona squamosa seed with its major active component, quercetin by undertaking the phytochemical studies, including its quantification by HPLC and then comparing its effects with that of seed extract.
Materials and methods
Clean and good quality seeds of uniform size, from ripe A. squamosa (Annonacae) fruits were collected locally. After identification, the voucher specimen (No. AS/02) was deposited in the departmental herbarium for future reference.
Swiss colony bred albino mice (28-30 g each) were used in the experiments. As females are more prone to hyperthyroidism (Fry, 1993) only healthy female mice were used. They were kept in polypropylene cages and were acclimated for 7 days before using in the experiments in a temperature (27 [+ or -] [degrees]C) and light controlled (14h light: 10h dark) room with the provision of food (Gold Mohur mice feed, Hindustan Lever Ltd., Mumbai, India) and water ad libitum.
Diethylene triamine penta acetic acid, Tris, sodiumdodecyl sulfate, thiobarbituric acid, ethyelene-diamine-tetra-acetic acid (EDTA) and glucose-6-phosphate, dimethyl sulphoxide (DMSO) were supplied by E. Merck (Mumbai, India). Quercetin dihydrate, L-Thyroxine ([T.sub.4]), dithiothreitol (DTT), propyl thiouracil (PTU) were purchased from Sigma Chemical Co., USA. Radioimmunoassay (RIA) kits for the estimation of total serum triiodothyronine ([T.sub.3]) and [T.sub.4] were supplied by Bhabha Atomic Research Centre (BARC), Mumbai, India.
Preparation of the extract
Dry Annona seeds were pulverized in an electric grinder and the fine powder was defatted using hexane in a soxhlet apparatus. The defatted marc was taken out, dried and extracted with 90% methanol in a soxhlet apparatus for 6h. The extract, so obtained was freed from methanol under vacuum. Yield of the extract was approximately 3% of the seed powder. As it was not soluble in water, the seed extract suspension was prepared with gum acacia (1.00% w/v in distilled water) for experimental use.
The methanolic extract of the experimental seeds was analyzed by TLC and by HPLC/UV. TLC and UV-spectrum detected the presence of flavonoids. TLC was performed in silica gel 60[F.sub.254] (Merck) n-BuOH: glacial acetic acid:water (3:1:1) and the spot appeared to be bright yellow in color under UV with an Rf value 0.57 indicating the presence of quercetin. UV spectrum was taken in Shimadzu 2401PC, where UV (MeOH): [lambda].sub.max] 255 was observed, that further indicated the presence of quercetin in the extract.
The standard quercetin (dissolved in methanol) and the seed extract were chromatographed by HPLC following the method of Chang et al. (2003) using Jasco--PU-1580; HSS-1500 monitor system, LC-Net II ADC, equipped with a rheodyne injection valve (20 [micro]l) and UV-1570 diode array detector. The separation was performed on ODS C18 (5 [micro]m) column (250 x 4.6 mm). The final extract was filtered through 0.45 [micro]m (Acrodisc) filter and the filtrate (20 [micro]l) was injected into HPLC. Compounds were separated using MeOH-water (93:7) with a flow rate of 1 ml/min and UV detector set at 225 nm.
Standard stock solution of quercetin (500 [micro]g/ml) was prepared in methanol ([CH.sub.3]OH; HPLC grade) and a calibration curve was obtained by injecting 20 [micro]l/ml aliquots of solution prepared by serial dilutions up to 40 [micro]g/ml of the stock solution. The regression equations of the plot and its correlation coefficient were calculated as follows: Y: 76,540; r: 0.9988, where Y is the peak area given by data processor. The quantity of quercetin in extract was calculated by interpolation of the peak area obtained by the sample in the calibration curve.
Effects of seed extract
Seven groups, each with seven healthy female mice were established. Out of these, twenty-one mice in three groups (Gr. II-IV) were rendered hyperthyroidic by daily administration of L-thyroxine ([T.sub.4]) at a prestandardized dose of 0.5mg/kg for 12 consecutive days (Panda and Kar, 2001). While animals of Gr. II continued to receive [T.sub.4], that of Gr. III and IV were administered with 200mg/kg of seed extract (Vohora et al., 1975; Rao et al., 1979) and PTU (10mg/kg), respectively, along with equivalent dose of [T.sub.4]. Simultaneously animals of 3 other groups (V, VI and VII) received an equivalent amount of seed extract or PTU or suspending reagent (acacia gum and water), respectively. The remaining group (Gr. I) receiving 0.1ml distilled water served as control. While the seed extract was administered orally, PTU and L-[T.sub.4] were injected intraperitoneally for 10 consecutive days. All the doses were administered between 1000 and 1100 h of the day to avoid circadian variations.
On the last day, overnight fasted mice were sacrificed under ether anesthesia and blood was collected by cardiac puncture. Serum samples were stored at -20 [degrees]C until assayed for total [T.sub.3] and [T.sub.4] concentrations. After exsanguinations, the liver was removed, cleaned by phosphate buffered saline 0.1 M (pH 7.4) and immediately processed for the biochemical estimations. For the determination of hepatic LPO, superoxide dismutase (SOD), catalase (CAT), G-6 pase, 5' mono-deiodinase (5'DI) activities and for the estimations of thyroid hormones by RIA, routine methods were followed as mentioned elsewhere (Panda and Kar, 2001; Tahiliani and Kar, 2003).
Effects of quercetin
As done in earlier experiment thirty-five Swiss albino female mice were divided into five groups (n = 7). Gr. II and III were intraperitoneally injected daily with 0.5 mg/kg/d L-[T.sub.4] for 12 consecutive days to induce hyperthyroidism. Two additional groups of female mice were also established. Gr. I animals receiving 0.1 ml of distilled water served as control. While animals of Gr. II continued to receive equivalent amount of [T.sub.4] only, animals of Gr. III were treated with equivalent dose of [T.sub.4] along with 10mg/kg/d of quercetin (p.o.) (Janbaz et al., 2004). Animals of Gr. IV received only 10.00 mg/kg/d of test compound, quercetin and that of Gr. V were administered with 0.1 ml of 0.1% DMSO (the vehicle). Everyday the drug or vehicle was administered between 1000 and 1100h of the day to avoid circadian interference. In this experiment also the test drug administration was continued for 10 consecutive days.
Data were subjected to analysis of variance (ANOVA), followed by Student's t-test. Differences between the values of two groups were considered significant at p<0.05.
The methanolic extract of the dried seed powder, subjected to TLC and UV analyses revealed the presence of quercetin, which was latter identified in HPLC chromatogram (Fig. 1a) with a retention time Rt, 8.41 min against the pure standard (Fig. 1b, Rt, 8.45 min). The concentration of quercetin in the extract was calculated out to be 5.27 [micro]g/ml.
Effects of the seed extract
While in [T.sub.4] treated animals a significant increase in serum [T.sub.3], [T.sub.4] concentrations, hepatic 5'DI and glucose-6-phospatase (G-6-Pase) activity was observed as compared to that of control animals (Fig. 2 and Table 1), a decrease in all these parameters was exhibited in the hyperthyroid animals, treated with A. squamosa seed extract and thyroxine together. In animals, receiving seed extract alone, a significant decrease in [T.sub.3] and 5'DI activity was also observed. When PTU was administered to euthyroid or hyperthyroid animals, thyroid hormone concentrations, hepatic 5'DI and G-6-Pase activities also decreased as compared to the values of control or hyperthyroid groups.
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
Hepatic LPO (Table 1) increased significantly with a concomitant decrease in SOD and CAT activities following [T.sub.4] administration. On the other hand, LPO was decreased and CAT activity increased when [T.sub.4]-induced hyperthyroid animals received the seed extract simultaneously. When comparisons were made between the different values of PTU treated and PTU + [T.sub.4] treated groups, no significant differences were observed with respect to hepatic LPO, SOD and CAT activities.
Effects of quercetin
While the administration of L-[T.sub.4] to the euthyroid animals significantly increased serum thyroid hormone concentrations and hepatic 5'DI activity, simultaneous treatment of quercetin (10 mg/kg) reversed all these effects. In euthyroid animals also a significant decrease was observed in all three parameters such as serum [T.sub.3] and [T.sub.4] concentrations and hepatic 5'DI activity (Fig. 3). The increase in hepatic glucose-6-phosphatase activity, a thyroid-dependent parameter in hyperthyroidic animals was also decreased by the administration of quercetin.
Results of quercetin on hepatic lipid peroxidation (LPO) in hyperthyroid mice (Table 2) indicated a significant decrease in the hepatic LPO with a concomitant increase in SOD and CAT activities (Table 2). In euthyroid animals also quercetin exhibited similar effects.
[FIGURE 3 OMITTED]
From the results it was evident that administration of L-[T.sub.4] increased the serum [T.sub.3] and [T.sub.4] concentrations as well as hepatic 5'DI and G-6-Pase activities, as observed in our earlier investigations (Panda and Kar, 2000; Kar and Panda, 2004). However, when 200mg/kg of seed extract was administered along with an equivalent amount of [T.sub.4], it reversed the effects as evidenced by a marked decrease in the concentration of both the thyroid hormones, G-6-Pase and hepatic 5'DI activity, indicating the antithyroidic nature of the extract. Although an inhibition in [T.sub.3] concentration was also observed in vehicle (acacia) treated animals it was only 22.35% as compared to 58.82% in seed extract. Obviously the marked decline in [T.sub.3] concentration in the latter was primarily because of the seed extract. The decrease in [T.sub.4], [T.sub.3] concentrations and 5'DI activity by the seed extract was 64%, 31% and 68%, respectively, as compared to that of PTU, that decreased by 57%, 44% and 64%, respectively. These observations do suggest that the Annona seed extract has a similar effect to the standard antithyroid drug and may prove to be an alternative to PTU. Interestingly when [T.sub.4]-induced hyperthyroidic animals were treated with quercetin, the major constituent of Annona extract, it also decreased thyroid hormone concentrations as well as 5'DI activity, suggesting that the antithyroid effects of the test seed extract might have been mediated through quercetin.
Earlier some plant extracts have been reported to inhibit thyroid function in animal models (Kar and Panda, 2004). However, on the thyroidal regulation by seed extracts, scientific investigations are meager (Schone et al., 1986; Panda et al., 1999; Tahiliani and Kar, 2003). Moreover, none of these studies looked into the changes in the activity of 5'DI, the principal enzyme involved in peripheral conversion of [T.sub.4] to [T.sub.3], the major process of [T.sub.3] generation. In the present investigation this important enzyme of thyroid function has also been evaluated. Therefore the present study on Annona seed extract appears to be a significant one, also because of the fact that Annona seeds are easily available as waste materials and may be utilized for therapeutic purpose. When the effects of the principal active compound, quercetin was studied, almost similar effects on the thyroid functions were observed suggesting the possible involvement of quercetin in the mediation of the antithyroidal effects of the crude seed extract.
Out of the two major circulating thyroid hormones, [T.sub.4] is considered as a precursor for the [T.sub.3]. While the former is primarily produced by the thyroid gland itself, the major amount of the latter one is generated by the peripheral monodeiodination of [T.sub.4] (Chopra et al., 1978) with the help of the enzyme 5'DI. In the present investigation both thyroid hormones as well as 5'DI activity were decreased by the seed extract suggesting that the seed extract might be inhibiting the synthesis and/or the release of thyroid hormones at the glandular level as well as at the level of peripheral inhibition of 5'DI activity.
Flavonoids are known to inhibit thyroid function (Divi and Doerge, 1996). Quercetin too is reported to decrease a thyroid hormone-dependent function (Davis et al., 1983). These findings do corroborate our findings on the antithyroid role of quercetin and the test seed extract. As TLC, UV spectra and HPLC analyses also revealed the presence of quercetin in the test sample, it is presumed that the antithyroidal role of A. squamosa seed extract might have been mediated through the quercetin which was at a concentration of about 5.27 [micro]g/ml.
LPO of polyunsaturated fatty acids leads to the formation of malondialdehyde and causes cellular injury that results in several pathological conditions (Halliwell and Gutteridge, 1989). In this investigation, while LPO was increased, SOD and CAT activities were decreased following exogenous [T.sub.4] administration, as observed by Pereira et al. (1995), on seed extract administration, either alone or with [T.sub.4], LPO was reduced and CAT activity was increased in liver, the primary target organ of drug metabolism. These results suggest that the seed extract may not cause hepatotoxicity; but acts as antiperoxidative/protective agent.
In conclusion it is emphasized that seeds of A. squamosa may prove to be a potential source of drug for the treatment of hyperthyroidism/thyrotoxicosis. However, further investigations are required before it is used for human therapy.
Financial assistance to Dr. S. Panda (Senior Research Associate) from Council of Scientific Industrial Research (CSIR), New Delhi, India is gratefully acknowledged.
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S. Panda, A. Kar*
Thyroid Research Laboratory, School of Life Sciences (Annex), Devi Ahilya University, Vigyan Bhawan, Takhshila Campus, Khandwa Road, Indore 452017, Madhya Pradesh, India
*Corresponding author. Tel.: +917312477166, +917312467029; fax: +917312360026.
E-mail address: firstname.lastname@example.org (A. Kar).
Table 1. Effects of Annona squamosa seed extract and of PTU in the alterations in hepatic LPO (nM MDA formed/h/mg protein), SOD (units/mg protein), CAT ([micro]M of [H.sub.2][O.sub.2] decomposed/min/mg protein) and G-6-Pase ([micro]M of inorganic phosphate liberated/h/mg protein) activities in euthyroidic and hyperthyroidic mice LPO SOD CTRL 0.96 [+ or -] 0.13 4.99 [+ or -] 0.42 [T.sub.4], 0.5mg/kg b. 1.37 [+ or -] 0.09 (a) 3.70 [+ or -] 0.13 (c) wt. [T.sub.4] + S. extract, 0.63 [+ or -] 0.07 (x) 4.38 [+ or -] 0.32 200 mg/kg PTU + [T.sub.4] 0.91 [+ or -] 0.08 (y) 4.23 [+ or -] 0.43 S. extract 0.76 [+ or -] 0.13 5.93 [+ or -] 0.50 PTU, 10mg/kg 0.80 [+ or -] 0.03 4.83 [+ or -] 0.08 Acacia 0.62 [+ or -] 0.04 (c) 4.14 [+ or -] 0.37 CAT G-6-Pase CTRL 52.58 [+ or -] 3.29 0.23 [+ or -] 0.02 [T.sub.4], 0.5mg/kg b. 34.93 [+ or -] 3.12 (b) 0.38 [+ or -] 0.02 (a) wt. [T.sub.4] + S. extract, 67.54 [+ or -] 4.95 (x) 0.10 [+ or -] 0.09 (x) 200 mg/kg PTU + [T.sub.4] 56.24 [+ or -] 2.73 (x) 0.17 [+ or -] 0.01 (x) S. extract 57.51 [+ or -] 4.68 0.21 [+ or -] 0.02 PTU, 10mg/kg 54.24 [+ or -] 0.98 0.12 [+ or -] 0.01 (a) Acacia 65.74 [+ or -] 4.18 0.16 [+ or -] 0.01 (y) Data are mean [+ or -] SEM (n = 7). (x) p < 0.001, (y) p < 0.01 as compared to the respective values of [T.sub.4] treated animals. (a) p < 0.001, (b) p < 001, (c) p < 0.05 as compared to the respective control values. Table 2. Effects of quercetin (Qr) in the alterations in hepatic LPO (nM MDA formed/h/mg protein), SOD (units/mg protein), CAT ([micro]M of [H.sub.2][O.sub.2] decomposed/min/mg protein) and G-6-Pase ([micro]M of inorganic phosphate liberated/h/mg protein) activities in euthyroidic and hyperthyroidic mice GROUPS LPO SOD CTRL 0.062 [+ or -] 0.006 4.56 [+ or -] 0.20 [T.sub.4], 0.5mg/kg 0.108 [+ or -] 0.005 (a) 2.9 [+ or -] 0.23 (a) [T.sub.4] + Qr, 0.050 [+ or -] 0.001 (x) 4.80 [+ or -] 0.24 (x) 10mg/kg Qr 0.048 [+ or -] 0.002 (c) 5.58 [+ or -] 0.34 (c) DMSO 0.059 [+ or -] 0.005 4.33 [+ or -] 0.23 GROUPS CAT G-6-Pase CTRL 41.19 [+ or -] 3.06 0.218 [+ or -] 0.01 [T.sub.4], 0.5mg/kg 26.50 [+ or -] 1.35 (a) 0.446 [+ or -] 0.07 (a) [T.sub.4] + Qr, 50.82 [+ or -] 2.37 (x) 0.215 [+ or -] 0.009 (x) 10mg/kg Qr 58.45 [+ or -] 3.08 (a) 0.104 [+ or -] 0.003 (a) DMSO 37.07 [+ or -] 1.80 0.197 [+ or -] 0.010 Data are mean [+ or -] SEM (n = 7). (x) p < 0.001 as compared to the respective values of [T.sub.4] treated animals. (a) p < 0.001 and (c) p < 0.05 as compared to the respective control values.
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|Author:||Panda, S.; Kar, A.|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Dec 1, 2007|
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