ABSTRACT
In the present study the hydroalcoholic extract of Seeds of Carthamus tinctorius was investigated for its steroidal and metabolic activity in albino rats of either sex in two different tests. In both the tests, the animals were treated with the test drug (100 mg/kg/p.o.) twice a day for three days and were sacrificed subsequently on day 4. Thymus gland was dissected out and weighed in the test for steroidal activity while in the test designed for metabolic activity, liver was dissected out for glycogen estimation and blood was collected for the estimation of blood sugar, serum protein and serum cholesterol. The test drug reduced the thymus weight significantly (p<0.01) as compared to the plain control. It also induced hyperproteinemic, hypocholesteraemic and liver glycogen increasing effect and moderately increased the blood glucose level. The findings suggest that the hydroalcoholic extract of seeds of Carthamus tinctorius possesses marked steroidal and metabolic activity. Steroidal effect may be one of the bases for its use in kidney diseases especially nephrotic syndrome like condition. Keywords: Steroidal activity, Metabolic activity, Carthamus tinctorius. 

INTRODUCTION

The seeds of Carthamus tinctorius Linn (Fam. Compositeae) commonly known as Tukhm-e-Qurtum are in use in Unani Medicine (Tibb-e-Unani) since ancient times to ameliorate various renal diseases (Ghani 1920; Aawan, 1993). Carthamus tinctorius is a slender, glabrous or pubescent, much branched, annual herb (Chatterjee & Pakrashi, 1997) growing to a height of 45-60 cm (tall varieties 85-150 cm). It is native to Europe and Asia. The cultivated safflower is considered to have originated either from the saffron thistle (Carthamus lanata) or the wild safflower (Carthamus oxyacantha) in the two primary centres of origin i.e. the mountainous regions of Ethiopia and Afghanistan; and also from the plains of India and Mayanmar (Burma), which are considered to be its secondary centre of origin. India is the second largest producer of safflower in the world, Mexico producing the maximum (Anonymous, 1992). The plant is cultivated throughout a large part of India (Chopra et al., 1956; Hooker, 1882; Kirtikar & Basu, 1987) as an oil seed crop, particularly in Andhra Pradesh, Bihar, Gujrat, Karnataka, Madhya Pradesh, Maharashtra, Tamil Nadu, Uttar Pradesh and West Bengal (Chatterjee & Pakrashi, 1997). Numerous races of safflower are known under cultivation. Nearly 63 types have been recorded. The plant can be broadly classified under two distinct varieties; one with very spinous leaves and the other with spineless or moderately spinous leaves. The spinous varieties are considered particularly valuable for oil production and spineless forms for dye extraction (Anonymous, 1950). It’s attributed effects in Unani literature such as diuretic (Aawan, 1993; Anonymous 1950; Caius, 2003; Chopra et al., 1956; Farooq, 2005; Kirtikar and Basu, 1987; Nadkarni, 1954; Singh, 1974), lithotryptic (Ghani, 1920), nephroprotective (Huang, 1999), tonic (Anonymous, 1996; Caius, 2003; Chopra et al., 1956; Karim, 1888; Khory and Katrak, 1985; Kirtikar and Basu, 1987) and antiinflammatory (Anonymous 1950; Ayub, 1907; Ghani, 1920; Khan, 1313H; Kirtikar and Basu, 1987; Nabi, 1901) etc are considered instrumental for its efficacy in various renal diseases. Ethnobotanical reports suggest almost similar effects and indicate its therapeutic application in kidney diseases such as chronic nephritis (Huang, 1999). In a recent study it has been shown to possess significant nephroprotective effect against gentamicin induced nephrotoxicity in experimental animals. It also improved a condition which was simulating with the symptoms of nephrotic syndrome (Wasim et al., 2011). It was hypothesized therefore that the steroidal effect (immunological/anti-inflammatory) may be one of the reasons for its efficacy in such a condition. Therefore the hydroalcoholic (30:70) extract of Tukhm-e-Qurtum was studied for steroidal effect by Thymus Regression Test (Stephenson, 1954). Further since the steroidal agents induce certain metabolic effects therefore metabolic activity was also studied by observing its effect on liver glycogen, serum glucose, serum protein and serum cholesterol levels.

MATERIALS AND METHODS

Preparation of ethanol extract
The seeds of Carthamus tinctorius, Linn were procured from Dawakhana Tibbiya College, Aligarh Muslim University (AMU), Aligarh, India. Prof S.H. Afaq and Dr. M. Inamuddin (Pharmacognosists), Department of Ilmul Advia, Faculty of Unani Medicine, AMU, Aligarh confirmed the identity of the drug. A voucher specimen (No. WA/2005/1) has been deposited in the museum of the department of Ilmul Advia, Faculty of Unani Medicine, AMU, Aligarh, India for future reference. The seeds were dried at room temperature and reduced to coarse powder by grinding. Powdered drug was macerated in 70% ethanol and left for 12 h at room temperature. It was then extracted for 6 h in a Soxhlet apparatus at 82 + 2 °C. 100 g of powder was extracted in 400 ml of solvent. The extract was filtered using Whatman filter paper and the filtrate was concentrated over a water bath. The yield of the extract was found to be 7.0% of crude drug (w/w). The extract was reconstituted a fresh in distilled water whenever it was intended to be administrated to the animals.
Experimental Animals
Wistar Albino rats of either sex weighing 40-50 g (Thymus regression test) and 100-150 g (Metabolic test), divided into three groups of six animals each were used. They were maintained on standard diet and water ad libitum and housed in clean polypropylene cages at room temperature (25-30 oC) with a 12 h light: 12 h dark cycle.
Treatment Schedule
The dose of Tukhm-e-Qurtum for albino rats was calculated by multiplying the human therapeutic dose, described and practiced in Unani Medicine (Aawan, 1993; Ghani, 1920; Nabi, 1901; Singh, 1974) by conversion factor of 7 (Freidrich et al., 1966). The dose thus calculated was found to be 100 mg/kg. The test drug suspended in distilled water was administered to the animals intragastrically with the help of a gastric canula twice a day.
Test for steroidal activity
The test drug was studied for steroidal effect by the method of Stephenson (1954) and Amin et al. (1994). Albino rats of either sex, weighing 40-50 gm were divided into 3 groups of 6 animals each having equal distribution of sexes and such that the total weight of animals in various groups were approximately the same. The animals in Group I served as plain control and received 3 ml of distilled water by oral route, twice a day, for 3 days. The animals in Group II serving as standard control were treated with Hydrocortisone 33.33 microgram/100 g, twice a day, for 3 days, by subcutaneous injection. While the animals in Group III were treated with the hydroalcoholic extract of Tukhm-e-Qurtum at a dose of 100 mg/kg, twice a day, for 3 days, by oral route and served as test group. The concentrated extract was reconstituted in suspension form with distilled water (100 mg/3 ml, w/v) and 2% gum acacia, before the administration. On the 4th day all the animals were sacrificed by overdosing of anaesthetic ether, administered by inhalation and the thymus gland was dissected out. The body weight and the weight of the thymus gland were recorded. The results were expressed as mg of thymus gland/100 gm of body weight.
Test for metabolic activity
The metabolic effect of the test drug was studied on liver glycogen by the method of Montgomery (1957), serum glucose by the method of Hultman (1959), serum protein by the method of Dumas (1971) and serum cholesterol by the method of Wybenga (1970) in albino rats. Albino rats of either sex, weighing 100-150 gm were divided into 3 groups of 6 animals and treated in the same way as in the previous test. On the 4th day all the animals were sacrificed by overdosing of anaesthetic ether, administered by inhalation, and blood sample was collected by cutting the throat for the estimation of blood sugar, serum protein and serum cholesterol, while the liver was dissected out for glycogen estimation.
Statistical Analysis
The results were given as mean ? S.E.M. Significance was determined by using the Student’s ‘t’ test. P-value equal to or less than 0.05 showed significance.

RESULTS

Steroidal effect
In plain control group the mean thymus weight was found to be 231.40 ± 0.314 mg/100 gm of body weight while in the standard group treated with hydrocortisone, 33.33 µg/100 g, it decreased to 146.54±0.355 mg/100 gm of body weight (p<0.001). The weight of thymus gland in the animals treated with the extract of seeds was found to be 168.90±0.579 mg/100 gm (p<0.01). The results are presented in Table-1.

Table 1. Effect of Tukhm-e-Qurtum on the weight of thymus gland. Click here to view full image

 

Metabolic effect
Effect of test drug on liver glycogen
The liver glycogen was found to be 14.12?0.43 mg/gm in plain control group while it increased to 28.89?0.23 mg/gm (p<0.001) in the standard group treated with hydrocortisone, 33.33 µg/100 gm of body weight. In the animals treated with the test drug it increased to 18.10?0.87 mg/gm (p<0.01). Effect of test drug on serum glucose Serum glucose was found to be 76.32±0.45 mg/dl in the plain control group. It increased to 114.74±0.86 mg/dl (p<0.001) and 88.46±0.54 mg/dl (p<0.05) in standard and test groups, respectively. Glucose level was significantly lower (p<0.01) in test group as compared to standard group. Effect of test drug on serum protein Serum protein was found to be 5.74±0.49 gm/100 ml of serum in plain control group while in hydrocortisone treated group it amounted to 9.76±0.79 gm/100 ml of serum (p<0.01). In the animals treated with the extract of test drug, it was found to be 6.66±0.31 gm/100 ml of serum showing a significant increase as compared to plain control (p<0.05). Effect of test drug on serum cholesterol Serum cholesterol was found to be 173.54±0.52 mg/dl in plain control group. It increased to 201.33±0.42 mg/dl (p<0.05) in hydrocortisone treated group. However it decreased slightly to 162.50±0.24 mg/dl in the group treated with the test drug. The results of metabolic effects are presented in Table-2.

Table 2. Effect of Tukhm-e-qurtum on metabolic parameters. Click here to view full image

 

DISCUSSION

The thymolytic activity of hydrocortisone and its analogues particularly in immature animals is well documented (Stephenson, 1960). In an in vitro study it has been shown that basophilic cells normally found in 12 and 13-day embryonic thymus glands disappeared after steroid treatment (Younan, 1968). Thymus regression effect of steroids was also evident from the findings of the present study where the weight of thymus gland was found to be decreased significantly (p<0.001) under the influence of hydrocortisone. Similarly, the test drug Carthamus tinctorius by decreasing the weight of thymus gland significantly (p< 0.01), which was only moderately lesser than the regression produced by hydrocortisone, indicated having thymolytic and thereby steroidal effect (Table.1). Since, the steroids have an immunosuppressant effect, which is the basis of their therapeutic application in nephrotic syndrome therefore the efficacy of test drug in nephrotic syndrome like condition for which it has been recommended in Unani literature and validated in an experimental study (Wasim et al., 2011), may be attributed at least partially to its steroidal effect. This finding is also suggestive of its potential to alleviate other diseases where steroids may have a role. Other effects reported to the test drug such as diuretic (Wasim et al., 2011) and anti inflammatory (Jun et al., 2011) etc may have a direct bearing on nephrotic syndrome and related conditions and/or act as adjuvant to the principal drug. It is interesting to mention that steroids in addition to their immunosuppressive effect also possess anti-inflammatory effect and may modify the body’s immune response to diverse stimuli (Waldman et al., 2007). Thus the combined nephroprotective, anti inflammatory, diuretic and steroid like effect the test drug is attributed with, appears to be in direct commensuration with the physiopathology of nephrotic syndrome. The findings are also suggestive that the diseases with diverse physiopathological appearance can be treated even with a single drug of Unani medicine because they commonly have multiple and related, even synergistic effects. This is one of the many advantages that crude drugs have over the isolated compounds. Steroidal agents play important part in controlling salt and water balance in the body, and regulating carbohydrate, fat, and protein metabolism. They are responsible for certain metabolic effects although when they are used in immunosuppressive and anti-inflammatory therapy their metabolic and other effects are taken as unwanted side effect (Rhen & Cidlowski, 2005). Therefore, different metabolic effects induced by the steroids are mostly not desirable therapeutically. The present study showed that the test drug has significant hyperproteinaemic and liver glycogen increasing effects. It also produced moderate hyperglycaemia which was significantly less than the findings of standard group, but did not alter the cholesterol level. These findings are more or less in consonance with steroidal activity (Stephenson, 1960). Thus the metabolic effects produced by Carthamus tinctorius further confirmed that it possessed steroidal effect. Hyperglycaemia and hyperchesterolaemia are not desirable effects of steroids at all whereas hyperproteinemic and glycogenic effects at occasions are used therapeutically. By demonstrating moderate effect on glucose level and not modifying the cholesterol level, the test drug exhibited that it has relatively lesser chances of producing unwanted side effects and is therefore safer than the common steroidal agents.

CONCLUSION

The findings of the present study have shown that the seed of Carthamus tinctorius possesses marked steroidal and metabolic activity. The steroidal effect may be the basis of its wide therapeutic application in various renal disorders including nephrotic syndrome like condition. It has a definite edge over the pure steroids on account of having minimum chances of producing side effects that are common to the steroidal drugs.

ACKNOWLEDGEMENT
We thank Prof. K.M.Y. Amin (Pharmacologist) Department of Ilmul Advia, Faculty of Unani medicine, Aligarh Muslim University, Aligarh for guiding us to formulate the study design. We are also grateful to Prof. S.H. Afaq and Dr. M. Inamuddin (Pharmacognosists) for helping us to undertake the necessary pharmacognostical studies to confirm the identity of the test drug.

CONFLICT OF INTEREST
None declared.

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