STUDY OF HERBOCHOL- POULTRY FEED SUPPLEMENT ON ESTROGENIC ACTIVITY IN RATS.
Shivalinge Gowda KP*1Albin Jose2, Madhavapriya BN 3, Khader Shareef KS4, Natarajan Loganayaki5 , Venkateshwarlu K6.
1Asso. Prof and HOD, Department of Pharmacology, 2, 3M.Pharm Research Scholar, PES College of Pharmacy, Hanumanthanagar, Bengaluru, Karnataka, India.
4,5,6 M/S Suguna Foods Pvt Ltd, Herbal Division, Suguna Lifeherbs, Kottamangalam Village, Tiruppur main road , Tamil Nadu, India.
The estrogenic activity of Herbochol-poultry feed supplement was investigated using ovarectomized The Herbochol-poultry feed supplement was administered orally at a dose of 400 and 800 mg/kg bw po for 07 consecutive days to overectomized rats. The bilateral overectomy in rats was performed under ketamine and xylazine (50,10mg/kg ip) anaesthesia and semi-sterile conditions. Sham operation was performed in same manner but only exposing the ovaries. They were administered with prophylactic gentamicin (10 mg/kg, ip) for 3 days. Ethynyl estradiol (2mg/kg sc) was used as a standard estrogenic drug. After 24h of the last day treatment, blood was withdrawn by retro orbital puncture and serum calcium, phosphorus and ALP levels were determined.
The initial and final body weights of the rats were measured. The animals were sacrificed by over dose ketamine anaesthesia and the uteri were dissected out and weighed using digital electronic balance. The serum calcium, phosphorus levels were significantly decreased and ALP levels significantly increased in overiectomized rats when compared to sham control group. The OVX rats treated with Herbochol-poultry feed supplement showed significant increased levels of serum calcium and phosphorus and significant decreased levels of serum ALP levels. The OVX rats treated with Herbochol PFS showed significant increase in the uterus weight when compared to OVX rats. From the results of the present study, it is concluded that Herbochol poultry food supplement, developed by M/s Suguna Foods Pvt Ltd, Herbal Division, Suguna Lifeherbs Coimbatore, TN, possesses estrogenic properties.
Keywords– Serum calcium, sham control, alkaline phosphatase, ethynyl, estradiol.
Cholesterol is a sterol (steroid alcohol). Cholesterol is essential for the formation of cell membrane, vit D, steroid hormones and bile acids. Since cholesterol is insoluble in water, it is transported in the blood in the form of lipoproteins. Lipoproteins are classified on the basis of density into- VLDL, LDL, IDL and HDL. All lipoproteins carry cholesterol, but elevated levels of the lipoproteins other than HDL are associated with an increased risk of atherosclerosis and coronary heart disease. The higher levels of HDL cholesterol are protective.
Long term elevated levels of cholesterol results in atherosclerosis. This may lead to stenosis (narrowing) and occlusion of the arteries. A sudden occlusion of a coronary artery results in a myocardial infarction or heart attack. An occlusion of an artery supplying brain can cause a stroke. Hypercholesterolemia also leads to xanthelasma palpebrarum (yellowish patches near the eyelids, xanthomas (deposition of yellowish cholesterol rich material). Normal total cholesterol is 180-200mg/dL.
During the synthesis of cholesterol, the 3-hydroxy 3-methyl glutaryl Co-enzyme, A (HMG CoA) reductase which convert HMG CoA to mevalonic acid. The mevalonic acid is then (after 20steps) converted into cholesterol. Statins act by inhibiting the HMG-CoA Reductase, thus cholesterol synthesis decreases. This leads to increase expression of LDL receptors in hepatocytes. This increases the uptake of IDL and LDL into the liver 1.
Cholesterol biosynthesis occurs mainly during sleep. Hence statins should be given at bed time. Atorvastatin is a long acting drug; this can be given at any time. Combination with cholestyramine or nicotinic acid enhances LDL lowering effect. Statins also act by increasing the production of nitric oxide and this NO prevents the oxidation of LDL. Grape fruits interferes the metabolism of statins and hence the combination should be avoided.
Estrogen is the female sex hormone secreted by the corpus luteum of the ovary.During pregnancy it is secreted by the placenta. Some estrogens are also produced in smaller amounts by other tissues such as the liver, adrenal glands, and the breasts. These sources of estrogens are important in postmenopausal life of women 2.
The estrogens promote the development of female secondary sexual characteristics, such as breasts, and are also involved in the thickening of the endometrial and other aspects of regulating the menstrual cycle. In males, estrogen regulates certain functions of the reproductive system important to the maturation of sperm 3, 4, 5.
Estrogen increases the HDL and triglyceride level and Decrease the LDL, fat deposition.
MATERIALS AND METHODS
Chemicals –Calcium, Phosphorous, Alkaline phosphatase, were procured from Anjan Distributors, Bengaluru, authorized supplier for Erba diagnostic kits. The semi auto-analyzer available in the PG research lab was used for the biochemical estimations.
Drugs – The drugs ketamine, gentamicin, ethynyl estradiol were procured from the medical store and used for the study. The rats were provided with rat pellets procured from Krish Scientific Co, Bengaluru.
Animals: Wistar rats weighing between 150-200 g of female wistar rat were were procured from Sri Venkateshwara Enterprises Bengaluru. The animals were kept under standard conditions of light and dark cycle with food and water. Animals acclimatized to laboratory condition before the test. The animals were acclimatized for 7 days under standard husbandry conditions, i.e., room temperature of (26+10) o C, relative humidity of 45-55% and light:dark cycle of 12:12 h. The experimental protocol was presented in the Institutional Animal Ethics Committee of PES College of Pharmacy (CPCSEA Reg No-66/PO/Ere/S/02/CPCSEA dated 20-3-2015),Bengaluru meeting held on 16-6-2015 and IAEC approval was obtained (PESCP/IAEC/18/2015). The animal experiments were conducted according to the Committee for the Purpose of the Control and Supervision and Experiments on Animals (CPCSEA) guidelines.
Study of estrogenic activity in rats-
Ovariectomy Procedure: Female albino Wistar rats (150-200g) were used in the study. The bilateral overectomy in rats was performed under under ketamine and xylazine (50,10mg/kg ip) anaesthesia and semi-sterile conditions by making two dorsolateral incisions using sharp dissecting scissors.
Figure1: showing ovariectomy in rat
The skin and dorsal muscles were cut and the peritoneal cavity was opened. The uterine horn was picked out and the fatty tissue around the ovary was removed. The connection between the fallopian tube and the uterine horn was clamped by artery forceps and a cut was made under the clamped area to remove the ovary. Skin was closed bilaterally with one simple catgut suture. Tincture iodine solution (antiseptic) was applied locally on the skin at both sites of the operation8. Sham operation was performed in same manner but only exposing the ovaries. They were administered with prophylactic gentamicin (10 mg/kg, ip) for 3 days. Rat cages were arranged randomly to limit variation based on temperature and light. They were maintained in barrier rooms under 12:12 h light: dark cycle, with a temperature of 22+ 1oC and relative humidity of 50%.
The animals were divided into five groups of six rats in each group.
Gp I (sham operated control) received 0.2ml of vehicle ( 1%Tween-80) orally for 7 days.
Gp II (ovariectomized control) received 0.2ml of vehicle ( 1%Tween-80) orally for 7 days.
Gp III ovariectomized) received Herbochol-PFS (400mg/kg) orally for 7 days.
Gp IV (ovariectomized) received Herbochol-PFS (800mg/kg) orally for 7 days.
Gp V (ovariectomized) received Std ethinyl estradiol in olive oil (0.2mg/kg) by SC route for 7 days.
All the above treatments were given on 8th to 14th day (seven days) after overectomy. After last day (14th day) treatment, all the rats were deprived of food for one night (12h). The final body weight of each rat was measured. The animal was scruffed with thumb and forefinger and the skin around the eye is pulled taut. A capillary tube was inserted into the medial canthus under the nictitating membrane of the eye. Slight thumb pressure with rotation motion was applied to puncture the tissue and to enter the capillary tube tip into the plexus/sinus. As soon as the sinus was punctured, blood entered the tubing by capillary action. When the desired amount of blood was collected, the tube was withdrawn and a clean gauze pad was applied on the eye to ensure hemostasis. The blood collected in eppendorf (centrifuge) tube was kept for incubation in an upright position for a period of 30-45 min to allow clotting. Then it was centrifuged using a cold centrifuge for a period of 15min at 1000-2000rpm. Using a clean pipette the supernatant serum was aspirated and poured into another eppendorf tube. This was used for further estimation of serum parameters such as calcium, phosphorus and alkaline phosphatise7.
Then the rats were sacrificed by over dose ketamine (150mg/kg bw ip) and the blood was collected by retro orbital puncture. The uteri were dissected out and separated from the adherent tissues and weighed using digital electronic balance.
Serum calcium estimation– Serum calcium was estimated using Erba diagnostic kit and semiautoanalyzer. Calcium functions as a structural component of bones and teeth. It is also important in the release of neurotransmitters at the nerve ending. Calcium is also essential for muscle contractions. Calcium functions as second messenger in signal transduction across a cell. Calcium ions act as factor in clotting process of the blood. Hypercalcaemia may develop in patients with Paget’s disease of bone and hyperparathyroidism. In Rickets, Coeliac diseases, idiopathic steatorrhea, osteomalacia and following surgical resection of the small intestine, serum calcium is often moderately reduced. Reference values in humans 8.6 to 10.2 mg/dL and in rats- 5.3 to 13 mg/dL. The serum calcium was measure using the formula
Serum Phosphorus estimation- Serum phosphorus was estimated using Erba diagnostic kit and semi auto analyzer. Phosphorus levels may be used in the diagnosis and management of a variety of disorders including bone, parathyroid and renal disorders. More than 80% of the body’s phosphorus is present in bones as calcium phosphate. The remainder is found intracellular as organic phosphates such as phospholipids, nucleic acids and ATP or extracellular as inorganic phosphorus. Increased levels of serum phosphorus are seen in renal diseases, hypoparathyroidism and excessive vitamin D intake. Decreased levels of phosphorus are seen in rickets, osteomalacia (adult rickets),hyperparathyroidism and in diabetic coma. Reference values in humans Adult 2.5 – 4.5 mg/dL, in rats- 3.11-11 mg/dL. The serum Phosphorus was measure using the formula
Serum ALP (alkaline phophatase) estimation– Human ALP consists of enzymes which hydrolyse phosphates at an alkaline pH. The osteoblasts of bone, liver, placenta, kidney, intestinal wall and lactating mammary glands consist of ALP in higher concentration. In children serum ALP level is higher due to growth of bone. In pregnancy also raises the normal values of ALP. In bone or liver diseases also ALP level increases. Increased ALP is seen in Osteomalacia and Rickets, primary hyperthyroidism, Paget’s disease, bone cancer, hepatitis, hepatitis, and cirrhosis. Low levels of ALP are observed in conditions which arrest gone growth or hypophospahtase. Reference values in adult humans 42 – 98 U/L, in children- 42 – 98 U/L. In rats 56.8-128 U/L. The serum ALP was measure using the formula
Table 1: Effect of Herbochol- poultry feed supplement on serum calcium, phosphorus and ALP in ovariectomized rats
|Sham control||6.07 + 0.75||2.99±0.28||82.74±7.81|
|OVX-control||2.36 + 0.19*||1.38±0.26*||174.98±13.59*|
|OVX + Herbochol-PFS-400mg/kg po||3.95±0.15**||2.15±0.13**||77.31±6.58**|
|OVX + Herbochol-PFS-800mg/kg po||3.83±0.27**||2.10±0.17**||109.42±7.21**|
|OVX + Std EE 0.2mg/kg sc||4.20±0.28**||2.23±0.16**||98.63±6.45**|
Statistical analysis is carried one way ANOVA followed by Tukey-Kramar multiple comparison test. Values are represented as mean +SEM (n=6) *P < 0.01 vs sham control: **P < 0.01 vs. OVX.
The mean values of serum calcium, phosphorus and ALP levels of sham control and ovariectomized and treated animals are shown in table No- 1
The OVX rats showed significant decrease in the serum calcium (2.36 + 0.19 mg/dL) when compared to sham control group (6.07 + 0.75mg/dL). OVX rats administered with Herbochol PFS (400 and 800mg/kg bw po) showed significant increase (3.95±0.15, 3.83±0.27 mg/dL) in serum calcium level when compared to OVX control group. The OVX rats treated with standard ethynyl estradiol also showed significant increase in the serum calcium level when compared to OVX group.
The OVX rats showed significant decrease in the serum phosphorus (1.38±0.26 mg/dL) when compared to sham control group 2.99±0.28 mg/dL). OVX rats administered with Herbochol PFS (400 and 800mg/kg bw po) and standard EE (2mg/kg bw sc) showed significant increase (2.15±0.13, 2.10±0.17, 2.23± 0.16 ) in serum phosphorus level when compared to OVX control group.
The OVX rats showed significant increase in the serum phosphorus (174.98±13.59IU/L) when compared to sham control (82.74±7.81 IU/L). OVX rats administered with Herbochol PFS (400 and 800mg/kg bw po) and standard EE (2mg/kg bw sc) showed significant decrease (77.31±6.58, 109.42 ±7.21, 98.63±6.45 IU/L ) in serum ALP level when compared to OVX control group.
Table 2: Effect of Herbochol- poultry feed supplement on body weight and uterus in ovariectomized rats
|Body weight in gm||Wt of uterus in gm||Average uterine weight ration in %
|OVX-control||175±38.85||225± 30.82||28.57 *||0.31±0.05*||0.13*
|OVX + Herbochol-PFS-400mg/kg po||193.3± 18.61||245± 8.36||26.94||0.38±0.04**||0.15|
|OVX + Herbochol-PFS-800mg/kg po||195±13.78||228.3±11.69||16.92**||0.30±0.04ns||0.13|
|OVX + Std (EE 0.2mg/kg sc||193.3±27.3||228.3±21.36||18.10**||0.37±0.03**||0.16|
Statistical analysis is carried one way ANOVA followed by Tukey-Kramar multiple comparison test. Values are represented as mean +SEM (n=6) *P < 0.01 vs. sham control: **P < 0.01 vs OVX.
Body weight and organ weight– The mean values of body weight and uterine weight of sham control and ovariectomized and treated animals are shown in table No- 2 The mean % weigh gain was 28.57 in OVX group and is significantly greater than sham control group (21.18). However OVX+ Herbochol PFS (800mg/kg po) and standard ethynyl estradiol administered rats showed significant decrease in the weight gain when compared to OVX. However the low dose of test drug did not shown reduction in the weight gain when compared to OVX group.
The OVX rats showed significant decrease of uterus weight (0.31±0.05g) when compared to sham control group. The OVX rats treated with Herbochol PFS (400mg/kg bw po)/standard ethynyl estradiol showed significant increase in the uterus weight 0.38±0.04 g, 0.37±0.03g when compared to OVX rats. However OVX rats treated with Herbochol PFS (800mg/kg po) did not shown reduction in the weight gain when compared to OVX group.
Estrogenic deficiency is the main factor for the development of osteoporosis. The present study provides the scientific evidence for the usefulness and beneficial effects of Herbochol poultry food supplement in the prevention of osteoporosis. Phytoestrogens are plant derived polyphenolic compounds, which are widely used for treatment and protection against various health problems, including bone diseases. Phytoestrogens are thought to protect from bone loss mainly through estrogen dependent mechanisms. It is reported that estrogen has high affinity toward estrogen receptor Era and ERb on osteoblasts and activation of ER complex is vital in maintaining bone remodelling processes. Because phytoestrogens have a stable structure and low molecular weight, they can pass through cell membranes and interact with estrogen receptors (ER). In the present study OVX rats administered with Herbochol PFS (400 and 800mg/kg bw po) showed significant increase (3.95±0.15, 3.83±0.27 mg/dL) in serum calcium level when compared to OVX control group. It is reported that osteoporotic women have decreased calcium absorption. The Herbochol PFS induced increased serum calcium is may be due to its estrogenic activity that increases the serum calcium level. This gives the basis for claiming the estrogenic activity of Herbochol PFS.
The present study report reveals OVX rats showed significant decrease in the serum phosphorus (1.38±0.26 mg/dL) when compared to sham control group 2.99±0.28 mg/dL). The OVX rats administered with Herbochol PFS (400 and 800mg/kg bw po) showed significant increase (2.15±0.13, 2.10±0.17, 2.23±0.16) in serum phosphorus level when compared to OVX control group.
The present study revealed that the raised serum alkaline phosphatise (ALP) level occurring with overiectomy could contribute to high bone turnover rate, being characterized by an increase in bone resorption. The excess bone resorption leads to bone loss. OVX rats administered with Herbochol PFS (400 and 800mg/kg bw po) showed significant decrease (77.31±6.58, 109.42 ±7.21, 98.63±6.45 IU/L ) in serum ALP level when compared to OVX control group. This confirms the protection against bone resorption by the Herbochol PFS. This effect is may be due to its estrogenic and inhibition of osteoclast activity. The OVX rats treated with Herbochol PFS (400mg/kg bw po)/standard ethynyl estradiol showed significant increase in the uterus weight 0.38±0.04 g, 0.37±0.03g when compared to OVX rats.
From the results of the present study, it is concluded that Herbochol poultry food supplement, developed by M/s Suguna Foods Pvt Ltd, Hebal Division, Suguna Lifeherbs Coimbatore, TN, possesses estrogenic properties. Thus Herbochol poultry food supplement developed by Suguna Lifeherbs can be used as a natural approach to help in preventing bone loss associate with states of estrogen deficiency.
Acknowledgement– The authors are thankful to PES College of Pharmacy Bengaluru, Karnataka and Suguna Foods Pvt Ltd, Coimbatore Tamil Nadu for providing necessary facilities and financial support to carry out this research.
Conflict of interest– Nil
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