Effect of Arogyavardhini in Experimental Prostatic Hyperplasia and Inflammation in Rats

Dumbre R. K.¹*, Kale A. P.², Patil V. R.¹, Bhosale A. V.², Kamble M. B.³

¹TVES’s Loksevak Madhukarrao Chaudhari College of Pharmacy, Faizpur, Maharashtra, India

²PDEA’s Seth Govind Raghunath Sable College of Pharmacy, Saswad, Pune, Maharashtra, India

³Rajgad Dyanpeeth’s College of Pharmacy, Bhor, Pune, Maharashtra, India

ABSTRACT:

Arogyavardhini, an Ayurvedic herbomineral formulation, was studied for its effect in experimental prostatic hyperplasia induced by testosterone injection s.c for 21 days in rats and inflammation induced in rat hind paw by carrageenan injection s.c. The results showed that Arogyavardhini at dosage of 100, 200 and 400 mg/kg orally for 21 days showed significant reduction of prostatic hyperplasia as indicated by decreased prostate weight and volume and hispathological observations. It also significantly inhibited carrageenan induced hind paw oedema in dose dependant manner. These results suggest that Arogyavardhini is effective in prostate disorder like Benign Prostatic Hyperplasia (BPH) by inhibiting proliferative and inflammatory mechanisms in prostate

KEYWORDS: Arogyavardhini, herbomineral preparation, Ayurvedic formulation, prostatic hyperplasia, anti inflammatory.

INTRODUCTION:

Prostate disorder like Benign Prostatic Hyperplasia (BPH) is mostly elderly disorder affecting quality of life and interferes with day to day activities. BPH is a nonmalignant enlargement of the prostate due to excessive cellular growth of both the glandular and the stromal elements of the gland and usually associated with Lower Urinary Tract Symptoms (LUTS) which may present with filling symptoms (frequency, urgency, andurge incontinence) or voiding symptoms (hesitancy, poor urinarystream, straining, intermittent stream, and a feeling of incompletebladder emptying), or both including complaints of nocturia (night-time voiding urine). ^(1-3)

BPH one is usually associated with the enlargement and another inflammation. Prostate is an androgen dependant organ where testosterone and dihydrotestosterone play important role for maintaining size and functions of gland. Hormonal imbalance in prostate contributes in the development of BPH. Hence utilization of androgen deprivation drugs like gonadotropin-releasing hormone (GnRH) analogues, anti-androgens, and 5α-reductase inhibitors decrease the size of the prostate and the resistance to outflow through the prostatic urethra, and thus improves the ability of many patients to urinate.^{(4, 5)} Infectious and non infectious type of prostatitis of is associated with BPH. Inflammatory cells and mediators like leucotrines, prostaglandins, and throboxanes etc are found in prostatic secretion. Increase Urinary tract infections (UTIs) are frequently observed with BPH and may be related to the amount of urinary retention due to BPH conditions. Anti inflammatory activity can reduce various mechanisms of Prostatitis associated with BPH. ⁽⁶⁾

Ayurvedic formulation Arogyavardhini is a herbomineral preparation recommended for conditions associated with urinary tract disorders and inflammatory conditions of glands and organs of body; obesity; liver and skin disorders; hydrocele; digestive tract disorders; balancing the imbalance of endocrinal gland function.^{(9, 10)} Present study is done to understand the effect of Arogyavardhini in experimental prostatic hyperplasia and inflammation in rats.

MATERIALS AND METHODS:

Chemicals and Formulation

Formulation containing following content was purchased of Baidyanath Ayurved Bhawan Pvt. Ltd., India

Formula (As per the AFI (Ayurvedic Formulary of India)-I)

Sr. No	Name of Ingredient	Parts
1	Shuddh Parad (Purified Mercury)	1
2	Shuddh Gandhak (Purified Sulphur)	1
3	Lauha Bhasma (Calcined Iron)	1
4	Abhrak Bhasma (Calcined Mica)	1
5	Tamra Bhasma (Calcined Copper)	1
6	Hareetaki (Powder of fruit of Terminalia chebula)	2
7	Vibheetaki (Powder of fruit of Terminalia belerica)	2
8	Amalaki ((Powder of fruit of Emblica officinalis)	2
9	Shuddh Shilajatu (Purified Asphaltum)	3
10	Shuddh Guggulu (Purified resin obtained from Commiphora mukul)	4
11	Chitrakmool (Powder of roots of Plumbago zeylanica)	4
12	Katuka (Powder of dried rhizome Picrorrhiza kurroa)	22

All the ingredients are finely powdered individually and mixed and processed in juice obtained from fresh leaves of Neem (Azadirachta indica). Other chemicals were obtained as follows Testosterone Propionate (German Remedies, India-Testoviron); Finasteride (Dr. Reddy, India-Finax); Carrageenan and Indomethacin from Sigma Aldrich.

Animals

Male rats of Sprague-Dawley strain weighing between 240-300 gm were used for the experiments. All the animals were obtained from Animal House of S.G.R.S College of Pharmacy, Saswad. The animals were fed ad libitum with standard pellet (Chakan Oil Mills, Pune, India) diet and had free access to water. All the protocols of animal experiments were approved by the Institutional Animal Ethics Committee. Animals were maintained at a temperature of 25±1°C and relative humidity of 45 to 55% under 12-h light:12-h dark cycle. They were housed individually in the polypropylene cages after surgery.

Effect of Arogyavardhini in experimentally induced BPH

Rats were castrated and their testes removed. 6 days after castration animals were divided into following groups of negative control, test groups and positive control. Normal non castrated rats are taken as normal control. Negative control received testosterone propionate 0.5mg/0.1ml by s.c route. Test group Arogyavardhini was divided in dose range of 100mg/kg, 200mg/kg and 400mg/kg and dosage was given orally. Positive control Finasteride was given at dose of 5mg/kg orally. Half hour after oral dosing the test groups and positive control group received testosterone propionate 0.5 mg/0.1ml by s.c route. Dosing was done for 3 weeks. 24 hours after last dosing animals were sacrificed and prostate removed. Prostate weight and volume was measured. The volume was measured (by the formula: 1/2(a x b²), where a and b refer to longer and shorter dimension, respectively) Prostate was fixed in 10% formalin. The formalin fixed tissues were embedded in paraffin and thin sections taken were stained by H & E staining process. ^{(11, 12)}

Anti-inflammatory activity of Arogyavardhini

The rats were divided into normal vehicle control, test groups, and positive control. Acute inflammation was produced subplantar administration of 0.1 ml of 1% carrageenan in normal saline in the right paw of rats. Arogyavardhini was given in dose of 100, 200 and 400 mg/kg orally, Positive control received Indomethacin 10mg/kg orally. The paw volume is measured at 0, 1, 2, 3 hours after carrageenan administration by plethysmometer (Ugo Basile). Dosing was done 1 hour before carrageenan administration. The amount of inflammation was compared with the 0 hour reading of the same rat and percentage anti-inflammatory activity calculated by comparing difference in percentage inflammation with control group. ⁽¹²⁾

Statistical Analysis

The data was expressed as mean ± SEM. The results were analyzed statistically using ANOVA and student’s t test. The minimum level significance was considered as P<0.05

RESULT:

Testosterone at dose of 0.5mg/0.1ml significantly increased prostate weight of castrated animals compared to normal animals. Arogyavardhini showed effect on volume and weight of prostate after 21 days treatment. Arogyavardhini at doses of 100, 200, and 400mg/kg and positive control Finasteride at 5mg/kg significantly decreased prostate weight and volume when compared to testosterone treated (negative control) animals (Table 1).

Table 1: The effect of Arogyavardhini on the volume and weight Index of Prostate Gland of Castrated Rat

Group	Dose (mg/kg)	B.W. (g)	Volume of prostate (cm3)	Wet weight index of prostate (mg/100 g B.W.)
Control	--	282.33±22.26	0.43±0.4	131.67±2.35
Negative	Water	285.33±25.41	0.74±0.06	316.44±8.80
Finasteride	5	283.67±22.18	0.47±0.04*	157.99±4.33**
Arogyavardhini (AV)	100	281.50±20.44	0.60±0.02*	185.93±9.11**
	200	289.83±27.16	0.56±0.03*	191.87±3.86**
	400	282.67±24.43	0.55±0.03*	172.55±5.72**

n=6, mean± S.E.M. * p, <0.05, ** p, <0.01 vs. Group II. B.W. represents body weight.

Table 2: Histopathology result of drug treated & control

Label	Gland crowding	Epithelial hyperplasia	Stromal hyperplasia	Stromal inflammation
Normal Control	---	---	---	---
Testosterone treated control	++	+++	+++	---
Finasteride +testosterone	+	+	+	---
100 mg/kg AV+ testosterone	+	++	+	---
200 mg/kg AV + testosterone	+	++	++	+
400mg/kg AV + testosterone	+	+	+	--

+ :- Mild Hyperplasia ++ :- Moderate Hyperplasia

+++ :- Severe Hyperplasia --- :- Absent Hyperplasia

Table 3: Anti-inflammatory activity of Arogyavardhini

Group	Dose mg/kg orally	Mean of % inflammation and increase in paw volume
Group	Dose mg/kg orally	1hr	2hr	3hr
Control		27.6±3.3	45.2±4.0	59.0±2.6
Arogyavardhini (AV)	100	33.5±1.7	45.8±1.4	48.5±2.1*
	200	22.8±5.8	41.9±5.7	36.9±7.8*
	400	14.0±4.3*	27.9±5.6*	28.2 ±5.5**
Indomethacin	10	3.7±1.3**	22.2±3.2**	20.6±1.7**

n=6, Values are expressed as Mean of % inflammation ± S.E.M, ( ) % inhibition; * p, <0.05, ** p, <0.001 when compared with control group

Histopathology of prostates of negative control show epithelial hyperplasia compared to normal control and decreased acini diameter. Fibrovascular stromal proliferation was also significantly observed. Significant reduction in gland proliferation, epithelial and cellular hyperplasia and stromal proliferation at dose of 100, 200, 400 mg/kg for Arogyavardhini vati compared to negative control was observed. The effect was more pronounced at 400mg/kg dose that was comparable with positive control Finasteride (Table 2).

Arogyavardhini showed significant anti-inflammatory activity by inhibiting carrageenan induced rat paw inflammation in dose response manner. The effect at dose of 400mg/kg was comparable with standard Indomethacin (Table 3).

DISCUSSION:

Benign Prostatic hyperplasia is a complex disease where various mechanisms are leading to enlargement of prostatic tissue and retention of urine. Age related hormonal imbalance of testosterone/estrogen and activity of testosterone and dihydrotestosterone after binding to cellular receptors set complex secondary reactions that signal the cell to produce growth factors resulting in hyper plastic growth of prostate.⁽^1-5) Acute and chronic prostatitis can trigger BPH where proinflammatory cytokines and chemokines in stromal cells can lead to prostate growth. ⁽¹³⁾

Result shown by Ayurvedic formulation Arogyavardhini is indicative of blockage of processes of enlargement of prostate. Inhibition of testosterone induced proliferation in castrated rat signify anti proliferative activity by effecting all process initiated by androgen responsible growth of epithelial and stromal elements of prostate. Secondly anti-inflammatory activity shown in carrageenan induced paw oedema in rat paw is indicative of inhibition of Cox and Prostaglandin mechanisms which are responsible factors in growth of prostate. This finding has initiated studies in exploration of many other Ayurvedic formulations and further mechanisms in benign prostatic hyperplasia.

ACKNOWLEDGEMENT:

Authors are thankful to Dr. Sujit Joshi, MD (Path) for carryout this histopathological study of samples.

REFERENCES:

1. Abrams P. New words for old: lower urinary tract symptoms for "prostatism." British Medical Journal. 308; 1994: 929-30.

2. Abrams P, Cardozo L, Fall M, Griffiths D, Rosier P, Ulmsten U, van Kerrenboeck P, Victor A, and Wein A. The standardization of terminology in lower urinary tract function: report from the standardization sub-committee of the International Continence Society. Urology. 61; 2003: 37–49.

3. Thorpe A and Neal D. Benign prostatic hyperplasia. Lancet. 361; 2003:1359–1367.

4. White JW. The results of double castration in hypertrophy of the prostate. Ann Surg. 22; 1895:1-80.

5. Huggins C, Clark PJ. Quantitative studies of prostatic secretion. II. The effect of castration and of estrogen injection on the normal and on the hyperplastic prostate glands of dogs. J Exp Med. 72; 1940:747-61.

6. Steiner GE, Djavan Bob, Kramer Gero, Handisurya Alessandra, Newman Martin, Lee Chung, Marberger Michael. The Picture of the Prostatic Lymphokine Network Is Becoming Increasingly Complex. Reviews in Urology. 4(2); 2002:171-177.

7. Egon Koch. Extracts from fruits of Saw Palmetto (Sabal serrulata) and roots of Stinging Nettle (Urtica dioica): viable alternatives in the medical treatment of Benign Prostatic Hyperplasia and Associated Lower Urinary Tract Symptoms. Planta Medica. 67; 2001:489-500.

8. Yablonsky F, Nicolas V, Riffaud JP, Bellamy F. Antiproliferative effects of Pygeum africanum extracts on rat prostatic fibroblasts. J Urol. 157(6); 1997: 2381-2387.

9. Rasaratnasamuccaya, Visarpadicikitsa, Chapter 20; 106-108

10. In: Ayurvedic Formulary of India, Part I, published by Chaukhamba Surbharati Prakashan, 2^nd Edition, Varanasi; 2003;

11. Qing-long Guo, Qi-Long Ding, and Zhao-Qiu Wu. Effect of baicalein on experimental prostatic hyperplasia in rats and mice. Biol Pharm Bull 27(3); 2004: 333—337

12. Chapter H: Analgesic, anti-inflammatory, and anti-pyretic activity. In: H. Gerhard Vogel, eds, Drug Discovery and Evaluation Pharmacological Assays. published by Springer-Verlag Berlin Heidelberg, New York. 2002; 2^nd ed: pp.759-760

13. Chughtai Bilal, Lee Richard, Te Alexis, Kaplan Steven. Role of inflammation in Benign Prostatic Hyperplasia. Reviews In Urology 13(3); 2011: 47-150

Received on 16.10.2012

Modified on 21.10.2012

Accepted on 25.10.2012

Research Journal of Pharmacognosy and Phytochemistry. 4(6): November –December 2012, 314-317