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STUDY ON THE IMMUNO-MODULATORY EFFECT OF HERBAL EXTRACT OF ASPARAGUS RACEMOSUS WILLD. IN BROILER CHICKS

Kumari R.1, Tiwary B.K. 1, Prasad A.1 and Ganguly S.2

1.Department of Veterinary Microbiology, Faculty of Veterinary Science & Animal Husbandry, Birsa Agricultural University, Ranchi 834 006, India 2.AICRP-PHT (ICAR) [Kolkata Centre], Department of Fish Processing Technology, Faculty of Fishery Sciences, West Bengal University of Animal and Fishery Sciences, Kolkata 700 094, India Corresponding Author, e-mail: ganguly38@gmail.com

Received: 13/12/2011; Revised: 19/12/2011; Accepted 23/12/2011

Abstract The present study was carried out to determine the immuno-modulatory effects of Asparagus racemosus extract treated feed and to analyze the role of T and B cells in host defense against Newcastle disease in one week old normal and immuno-compromized broiler chicks. After the treatment significant (P<0.01) positive effects were observed in both humoral and cell mediated immune responses of the birds which was found to be evident by increased antibody titer after Hemagglutination inhibition (HI) test. The variation in skin thickness was significantly (P<0.01) more among the herbal treated groups rather than the non-treated groups which was a clear marker for immuno-stimulation among the birds.

Key words: Immuno-modulatory, Asparagus racemosus, host defense, humoral, cell mediated

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com Introduction

GJRMI, Volume I, Issue I, January 2012, 1-6

Poultry rearing is the fastest growing industry in livestock sector currently which is benefiting us from production and advantages in prices as well as providing with healthy food. In India, poultry industry is recognized as an important cottage as well as fast growing, large commercial agriculture industry. Poultry farming is always prone to a heavy risk of increased disease incidences leading to high mortality even if scheduled mass vaccination programmes are implemented. Certain diseases, pesticides and chemicals may lead to immunesuppression in the birds. Large numbers of report are available on outbreak of Newcastle disease (ND) resulting in alarming economic losses mainly due to ‘Vaccine failure state’ even after programmed vaccination schedules have been used (Chakraborty and Chatterjee 1998).

Many plant drugs have been categorized under ‘Rasayana Dravyas’ and are prescribed in Ayurveda to hasten host resistance (Thatte and Dahanukar 1986). Asparagus racemosus fall in this category along with many other useful plants.They exhibit immuno-modulatory activities. The root of Asparagus racemosus (commonly called ‘Satavar’) possesses antidiarrheal, anti-ulcerative, anti-spasmodic, aphrodisiac, galactogogue and other properties and has therefore gained its importance in Ayurveda, Siddha and Unani systems of medicine (Nadkarni 1954).

In the present study, judgment of the immuno-modulatory effects of Asparagus racemosus root extract has been attempted by monitoring their effects on various non-specific and specific humoral and cellular immunological response parameters.

Materials and Methods Fifty (50) days old broiler chicks were procured from a private hatchery and were maintained under standard hygienic conditions of feeding and housing. On the 7th day, they were divided into three groups (Group 1-3) comprising of fifteen (15) chicks in each group. They were

provided with ration as broiler starter (0-2 weeks), broiler grower (3-4 weeks) and broiler finisher (5-6 weeks). Group 1 consisted of treated chicks fed with A. racemosus extract treated feed, Group 2 was kept as vaccinated control comprising of chicks administered with Newcastle disease (ND) vaccine as per recommended schedule but without being fed with A. racemosus extract treated feed, Group 3 was the non-vaccinated control which consisted of untreated and unvaccinated chicks respectively.

Monitoring of humoral immune response Hemagglutination (HA) test and Hemagglutination inhibition (HI) tests were performed according to Buxton and Frazer (1977) with certain modifications. Fowls were screened for suitable RBCs required for HA test. Their blood was collected aseptically in Alsever’s solution and was centrifuged at 1500 g for 10 min. The solution was washed and centrifuged three times in phosphate buffer saline (PBS) solution. The supernatant was discarded and packed blood cells were resuspended in PBS solution, pH 7.2 to make 1% v/v RBC suspension. La Sota strain of Newcastle disease virus vaccine was procured and was used as virus stock solution for conducting HA test. The readings of antibody titer were converted into log10 values and the converted values were subjected to statistical analysis. For seeing the effect of different treatments on humoral immune response, analysis of variance (ANOVA) was done as per the method of Snedecor and Cochran (1967).

Monitoring of cellular immune response by contact sensitivity test Six chicks were randomly picked up from each experimental group on 28th day of experiment for standardization with 2,4 dinitrofluorobenzene (DFNB) by single percutaneous application of 0.25 ml DFNB @ 10 mg/ml in the vehicle consisting of acetone and olive oil (4:1) mixture as per the method described by Tiwary and Goel (1985).

Featherless area of about 20 cm2 was chosen on

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www.gjrmi.com GJRMI, Volume I, Issue I, January 2012, 1-6 left and right lateral abdomen for DFNB Dahanukar 1993). There have been studies on the immunomodulatory activities of A. application. racemosus in mice with myelo-suppression The virus challenge was done on 14th induced by cyclophosphamide, azathioprim or day post-application (DPA) with 0.25 ml DNFB prednisolone. This finding supports our result in 1 mg/ml solution on left marked area and the the current investigation that A. racemosus root right side was painted with vehicle only. The extracts stimulate immune response activities in skin reaction was measured with Vernier broiler chicks. Extracts of A. racemosus have Calipers before challenge and post-challenge on also shown immuno-potentiating effects in 0, 6, 24 and 48 days age of the birds. An cyclophasphamide treated mouse with ascetic average of three consecutive measurements was sarcoma (Diwanay et al. 2004). The findings in made to find out the mean skin thickness of the present study simulate with those reported individual chicks within the groups.

by Kalita and Dutta (1999) who also reported that maternal antibody was persistently found in All data obtained were subjected to sera samples tested against (ND) virus during statistical analysis as per standard methods and the first week of age. This was attributed to techniques laid down by Snedecor and Cochran natural passive immunity in young chicks as (1967). demonstrated by Hellar (1975). In the present study, the highest antibody level as detected by Results and Discussion HI test on 1st day of age decreasing up to 21st Mean hemagglutination inhibition day with no detectable antibody titer on 28th day (MHI) antibody titer of chick sera has been which is similar to the findings of Deka et al. presented in Table 1 and their analysis of (2002) and Kalita and Dutta (1999). In the variance indicated an overall significant effect present study, non-significant effect of of treatment with herbal preparation on HI treatment on skin thickness in contact antibody titer at all the days post-vaccination at sensitivity test with DNFB at 0 as well as 72 weekly intervals up to 42nd day posthour post-challenge. These results were similar vaccination. There was significant decrease in to Kumari (2005) than the treated group with mean antibody titer from Day 1 to Day 7 in somnifera which revealed Withania chicks. significantly higher results at 48 and 72 hour post-challenge with DNFB. Muruganadan et al.

The extent of cell mediated immune (2001) reported the effects of ethanol extracts of reaction was observed by increase in mean skin A. racemosus on humoral immune system thickness of sensitized broiler chicks (Table 2). which was assessed by humoral immune The cell mediated immune response showed by response and cell mediated immune response in the increase in skin thickness was more among mice. Kumari Rita et al. (2011) showed the chicks treated with herbal preparation than the determinative role of extracts of Ashwagandha other groups which did not differ significantly (W. somnifera) and Satavar (A. racemosus) as between themselves. herbal feed additives in obtaining higher humoral and cell mediated immune responses In the present study it was seen that providing better protection level against administration of A. racemosus dried root infections in protecting the immuno-deficient powder might have significantly stimulated broiler chicks against infections. The reports both humoral and cell mediated immune simulated to the findings in the present study responses. This finding simulates with the which showed the immuno-modulating effects findings of Kuttan and Kuttan (1992) who also of dried root powder of A. racemosus on reported the same observations in Swiss albino responses of humoral immune system assessed mice. The previous studies have demonstrated by rise in HI antibody titer and by cell mediated that A. racemosus extracts increase phagocytic immune responses in broiler chicks. activities of macropahages in vitro (Rege and Global Journal of Research on Medicinal Plants & Indigenous Medicine

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immunity against Newcastle disease.

Indian J. Comp. Microbiol. Immunol.

Infect. Dis. 23(1): 29-31.

Conclusion The use of A. racemosus dried root powder in a specific dose during the scheduled vaccination regimen may be helpful in obtaining higher protective antibody against different vaccinations including more effective cell mediated immune response for protection against various bacterial, viral and other diseases. Herbal formulations containing extracts of A. racemosus may be therefore recommended for use as positive immunomodulator in normal and immuno-compromized broiler chicks. The present study also indicated the determinative roles of herbal feed additives in effective augmentation of humoral and cell mediated immune responses providing better protection level against infections.

Diwanay, S., Chitre, D. and Patwardhan. 2004.

Immunoprotection by botanical drugs in cancer chemotherapy.

J. Ethnopharmacol. 90(1), 49-55.

Acknowledgement

Kumari, P. 2005. Comparative studies on immuno-modulatory effects of Curuma longa and Centella asiatica in broiler birds with special reference to micronutrients. MVSc thesis submitted to Birsa Agricultural Univ., Ranchi, India.

The authors are thankful to Honâ&#x20AC;&#x2122;ble Vice-Chancellor, Birsa Agricultural University and the Dean, Faculty of Veterinary Science & Animal Husbandry, Ranchi, India for providing all the necessary facilities to carry out this research work.

References

Buxton, A. and Fraser, G. 1977. Animal ed.

Blackwell Microbiology.

1st Scientific Publication Ltd. Victoria, Australia.

Hellar, E.D. 1975. Res. Vet. Sci. 18, 117 (Cited

by Rao et al. 1987). Resistance of maternal antibodies against Newcastle disease virus in chicks from immune parents and its effect on vaccination.

Indian J. Comp. Microbiol. Immunol.

Inf. Dis. 8(3), 106-110.

Kalita,

D.N and Dutta, G.N. 1999.

Immunomodulatory effect of levamisole upon Newcastle disease, pigeon pox and Markâ&#x20AC;&#x2122;s disease vaccination in broiler chicks. Indian Vet. J. 76, 490-492.

Kumari, R., Tiwary, B.K., Prasad, A. and Ganguly, S. 2011. Immuno-modulatory effect of herbal feed supplement in normal and immuno-compromised broiler chicks. Indian J. Anim. Sci, 81(2), 158-161.

Kuttan, G. and Kuttan, R. 1992. Immunomodulatory activity of a peptide isolated from Viscum album extract. Immunol.

Invest. 21, 285-296.

Chakraborty, D. and Chatterjee, A. 1998.

Studies on immunomodulatory effects of levamisole in New Castle disese vaccinated chicks. Indian J. Comp.

Microbiol. Immunol. Infect. Dis. 19, 8587.

Muruganandan, S., Garg, H., Lal, J., Chandra, S. and Kumar, D. 2001. Studies on the immuno-stimulant and anti-hepatotoxic activities of Asparagus racemosus root extract. J. Med. Arom. Pl. Sci. 22, 49-52.

Deka, D., Sarma, D.K. and Dutta, G.N. 2002.

Immunomodulatory effect of Stressroak and Levamisole on the maternal

Nadkarni, A.K. 1954. Indian Materia Medica, Bombay, Popular Book Depot, 3rd ed. 1, 153-155.

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com GJRMI, Volume I, Issue I, January 2012, 1-6 Rege, N.N. and Dahanukar, S.A. 1993.

Quantitation of microbicidal activity of mononuclear phagocytes : an in vitro technique. J. Postgrad. Med. 39(1), 2225.

Snedecor, G.W. and Cochran, W.G. 1967.

Statistical Methods. 7th edn. Oxford and IBH Publishing Co. New Delhi.

Thatte, U.M. and Dahanukar, S.A. 1986.

Ayurveda and contemporary scientific thought. Trends in Pharmacol. Sci. 17, 248-257.

Tiwary, B.K. and Goel, M.C. 1985. Contact sensitivity to DNCB in normal and cell mediated immunity deficient chickens, in vivo detection and correlation with lymphocyte transformation and graft versus host reaction. Vet. Immunol.

Immunopathol. 8, 329-339.

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www.gjrmi.com GJRMI, Volume I, Issue I, January 2012, 1-6 Table 1. Mean hemagglutination inhibition (MHI) antibody titer ((log10 values) in different groups of chick.

Age of chicks

Group 1

Group 2

Group 3

(in day)

ANOVAvalue

1

1.154 ± 0.050

1.154 ± 0.050

1.154 ± 0.050

NS

7

0.502 ± 0.100

0.502 ± 0.100

0.502 ± 0.100

NS

14

1.154 ± 0.092

1.003 ± 0.063

0.301 ± 0.134

0.771NS

21

1.355 ± .067a

1.153 ±0.092a

0.100 ± 0.100b

59.195*

28

1.304 ± .063a

1.054 ± .067b

0.000c

169.058*

35

1.606 ±0.126a

1.204 ± .077b

0.000c

95.181*

42

1.957 ± .128a

1.505 ± .109b

0.000c

110.47*

Values bearing different superscripts in a row differed significantly, Each value is the average of 6 observations ± SE, NS: Not significant, (*P<0.01)

Table 2. Average skin thickness (in mm) of broiler chicks at different hours post DNFB challenge.

Age of chicks

Group 1

Group 2

Group 3

(in hour)

ANOVAvalue

0

0.261 ±0.014a

0.244 ± .012a

0.216 ± 0.100a

3.48NS

6

0.418 ±0.020a

0.367±0.050ab 0.307 ± 0.019ab

7.5**

24

0.619 ±0.036a

0.586 ± .018a

0.478 ± 0.032b

6.00*

48

1.090 ± 0.061a 0.983 ±0.047a

0.815 ± 0.026b

8.846**

72

1.392 ± 0.168a 1.217 ±0.135a

0.920 ± 0.040a

3.55NS

Values bearing different superscripts in a row differed significantly, Each value is the average of 6 observations ± SE, NS: Not significant, (**P<0.01), (*P<0.05) Global Journal of Research on Medicinal Plants & Indigenous Medicine

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ANTIMICROBIAL ACTIVITIES OF ESSENTIAL OIL AND CRUDE EXTRACTS FROM ARTEMISIA HERBA-ALBA ASOO, GROWING WILD IN BECHAR, SOUTH WEST OF ALGERIA. Makhloufi A1, L.Benlarbi1, L.Mebarki2, Akermi M.M 1.

1

- Laboratory of valorization of biological resources and food security in semi arid area , south west of Algeria, BP417 University of Bechar, Algeria 2-Laboratoire des productions, valorisations végétales et microbiennes, Université des sciences et de la technologie d’Oran Mohamed Boudiaf, Algeria Corresponding Author E-mail: makhloufi_75@yahoo.fr

Received: 14/12/2011

Revised: 20/12/2011

Accepted: 30/01/2012

Abstract Crude extracts (Aqueous, ethanolic) and hydrodistilled essential oil from the aerial parts of Artemisia herba-alba Asoo, were investigated for their antimicrobial activities against six strains of bacteria and

three strains of fungi. The antimicrobial activity of the extracts and essential oil was

determined by disc diffusion method and the minimum inhibitory concentration (MIC) determination methods. Essential oil and extracts showed varied levels of antimicrobial activity against one or more test bacteria and fungi. The essential oil of A. herba-alba Asoo, have an antimicrobial activity against all the investigated strains except Bacillus cereus and Listeria monocytogenes. The diameters of growth inhibition zone ranging from 11mm - 20 mm for bacteria (including the diameter of the disc-6 mm) and from 13.5 mm to 26 mm for fungi. The highest inhibition zone values observed against Aspergillus niger (26 mm) and Staphylococcus aureus (20 mm).Aqueous and ethanolic extracts have greater antibacterial activity, but they did not show any activity against fungi. The MIC ranges from 0.35 - 3.5 mg/ml.

Keywords: Artemisia herba-alba Asoo, Essential oil, Crude extracts, Antimicrobial activity.

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Introduction Disease causing microorganisms have always been considered a major cause of morbidity and mortality in humans. The appearance of resistant microorganisms paved the way to the occurrence of infections that are only treated by a limited number of antimicrobial agents.

The emergence of resistant Gram negative bacteria presents a major challenge for the antimicrobial therapy of infectious diseases and increases the incidence of mortality and morbidity. Bacterial resistance to antimicrobial agents is a medical problem with public health, socioeconomic, and even political implications (Abdel massih et al., 2010).

Experimental Artemisia herba-alba Asoo specimen (Photo 01) was collected from Bechar region in February 2011, These biomasses were dried for fifteen days in the dark at ambient laboratory temperature (20 - 28 ° C).

Distillation of essential oil: The dried aerial parts were grounded before the operation, and then, 100 g of grounded A.herba-alba Asoo was submitted to hydrodistillation for 3h using a Clevenger apparatus (Amarti et al., 2008). The distilled essential oils were dried over anhydrous sodium sulfate, filtered and stored at +4 °C for further use (Ayoughi et al., 2011; Chanthaphon et al., 2008).

Preparation of ethanolic extract: Fresh plant was shade -dried at room temperature and ground in a coffee bean grinder. 15g of the dried plant material was soaked in 100 ml of ethanol for 24h with continuous shaking in a shaker at room temperature. The plant material was filtered and the filtrate was collected (Abbassi et al., 2005). This was repeated and the filtrates were combined and concentrated in a rotary evaporator at 30°C to obtain the crude extract and stored at 4°C for further use (Kassi et al., 2008; Iqbal & Arina, 2001).

Preparation of aqueous extract: Aqueous extract of A.herba-alba Asoo was prepared by boiling 25 g in 500 ml sterile distilled water for 15 -20 min. The flasks were then plugged and removed from heat and allowed to cool. After cooling, the contents of flasks were filtered (Sqalli et al., 2001; Loubaki, 1999).This was repeated and the filtrates were combined and concentrated to obtain the crude extract (Kassi et al., 2008).

Antimicrobial activity Microbial strains: The antimicrobial activity was evaluated by paper disc diffusion and dilution methods against six selected Gram-positive and Gramnegative species: Escherichia coli ATCC 2592, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC27853 ,Bacillus cereus ATCC 11778 , Enterococcus feacalis ATCC 29212 and Listeria monocytogenes ATCC19115, and three strains of fungi: Aspergillus niger, Aspergillus flavus and Penicillium escpansum.

Disc diffusion method The qualitative antimicrobial activity was carried out by disc diffusion method (Boussaada et al., 2008). It was performed using culture growth at 37 °C for 18 h and adjusted to approximately 108 colony forming unit per milliliter (CFU/ml) for bacteria (Gachkar et al., 2007; Joffin & Leyral, 2001) and 106 colony for fungi . The culture medium used for the bacteria was Mueller Hinton Agar (MHA), and Potato Dextrose Agar (PDA) for fungi (Ouraïni et al., 2007). Five hundred microliter of the inoculum was spread over plates containing MHA and a Whatmann paper disc (6 mm) impregnated with 5µl of the essential oil or crude extracts was placed on the surface of the media. The plates were left 30min at room temperature to allow the diffusion of oil. Then they were incubated 24h at 37°C (Bekhechi et al., 2008; Bourkhiss et al., 2007 and Shunying et al., 2005). After incubation period, the inhibition zone obtained

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around the disc was measured. Two controls were also included in the test, the first was involving the presence of microorganisms without test material and the second was two standard antibiotics: Ampicillin was used to control the sensitivity of the tested bacteria.

The experiments were run in triplicate, and the developing inhibition zones were compared with those of reference discs (Yesil Celiktas et al., 2007).

Dilution method (MIC): Essential oil Antimicrobial tests were performed according to the method reported by Remmal et al (1993), and Farah et al (2001). The essential oil was emulsified with an agar solution of 0.2% to disperse the compounds and improve their contact with the tested germs, then diluted to one tenth in the agar solution .Quantities of this dilution were added to test tubes containing MHA for bacteria and PDA for fungi. The final concentrations of essential oil are from 1/10, to 1/1000 (v/v). In parallel, Control assay containing only the culture medium and agar solution at 0.2% were also used.The MIC is the lowest concentration of essential oil giving no visible growth in the naked eye (Kaloustian et al., 2008).

Crude extracts Dry extracts were weighed and dissolved in sterile distilled water. The solutions were filtered through 0.22 ¾m sterile filter membranes and stored at 4°C for further use.

The concentration of the original solution of the plant extract/fraction corresponds to the concentration obtained after resuspension of the dried plant extracts. This was used as the stock solution and the most concentrated one from which the MIC series were prepared (Abdel massih et al., 2010).

Results and discussion Antimicrobial Activity Disc diffusion assay: Artemisia herba-alba Asoo has provided a yield of 1.92% essential oil. According to these results, this yield is lower than that quoted by

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Salido et al., (2004) (2.30%) and is higher to those obtained by Chebli et al., (2004) (0.3%), and Chami et al., (2008) (1.23%).this essential oil has an antimicrobial activity against the investigated strains except P.aeruginosa and B.cereus, these results are similar to those obtained in previous works (Mastasyoh, 2007; Imelouane et al., 2010) , They confirm that P. aeruginosa was resistant to the investigated oil .

The growth inhibition zones measured by disc diffusion method are presented in Fig 1.The diameters of growth inhibition zone ranged from 12-20 mm for bacteria (including the diameter of the disc mm), and from 13.5 - 26 mm for fungi. The highest inhibition zone values are observed against A.niger (26 mm) and S.aureus (20 mm). These values are lower than those found with Imelouane et al (2010).

On the other hand, the results indicated that the A.herba-alba Asoo extracts showed an antibacterial activity with the concentration of 75 mg/l (Fig 1), mainly against the Gram positive bacteria (E. feacalis 31mm and S.aureus 26 mm).

The extracts also exhibited an effect against the Gram-negative bacteria (E.coli 15.5 mm).

However, this effect was less efficient than that presented against the Gram positive bacteria, since a higher MIC value was obtained with the Gram negative bacteria. These results are different to those quoted by Abou El-Hamd et al (2010), which they report that only the essential oil was active against some Grampositive bacteria and Gram-negative bacteria.

(Insert Graph Fig No. 1) Dilution method (MIC): A.herba-alba Asoo EO exerted an inhibitory effect on E. coli, E. feacalis and S.aureus. The best MIC was observed against S.aureus 1/100, E. coli 1/75 and E. feacalis 1/50 (Table 1).

These effects were observed with the crude extracts (aqueous, EtOH), the best inhibitory activity represented by the lowest MIC was

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observed with EtOH extract at 1.5 and 2 mg/ml for S.aureus and E.coli respectively (Table 2).

of Chemistry of Globe Publications Rec. Nat. Prod. 4:1 1-25

The lowest MIC (0.35 and 0.45 mg/ml) was recorded for the aqueous extract with S.aureus and B.cereus (Table 2). The controls that were systematically run for all solvents did not exert any antibacterial activity. Bacterial growth was observed for the positive controls while no growth was observed for the negative controls.

(Insert Table No. 1 & 2)

Amarti F, Satrani B,Aafi A, Ghanmi M, Farah A, Aberchane M ,El Ajjouri M,El Antry S, Chaouch A (2008). Composition chimique et activité antimicrobienne des huiles essentielles de Thymus capitatus et de Thymus bleicherianus du Maroc : J Phytothérapie,6, 342–347 .

Ayoughi F,Barzegar M,Sahari MA, Naghdibadi H (2011). Chemical Compositions of Essential Oils of Artemisia dracunculus L. and Endemic Matricaria chamomilla L. and an Evaluation of their Antioxidative Effects: J. Agr. Sci. Tech, Vol. 13, 79-88

Conclusion Hydrodistillated-essential oils from Artemisia herba-alba Asoo had less inhibitory activity, compared to the crude extracts (Aqueous and EtOH) .The antibacterial activity of EO and crude extracts is due to the presence of a mixture of compounds and not a single compound.

The strong antibacterial activity of A.herbaalba Asoo against array of bacteria strains is an indication of the broad spectrum antibacterial potential of the oil and extracts. This could make the oil and the extracts a promising group of natural compounds for development of safer antibacterial agents.

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Abdel-Massih R, Abdou E, Baydoun E, Daoud Z (2010). Antibacterial Activity of the Extracts Obtained from Rosmarinus officinalis, Origanum majorana, and Trigonella foenumgraecum on Highly Drug-Resistant Gram Negative Bacilli .Journal of Botany .Article ID 464087, pp 8 Abou El-Hamd HM, Magdi AE, Mohamed EH,Soleiman E H, Abeer M E, Naglaa S M (2010). Chemical Constituents and Biological Activities of Artemisia herba-alba. Academy

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Chemical composition and antimicrobial activity of volatile components from capitula and aerial parts of Rhaponticum acaule DC growing wild in Tunisia: Microbiological Research 163 (2008) 87-95 Bourkhiss M, Hnach M,Bourkhiss B,Ouhssine M ,Chaouch A (2007). Composition chimique et propriétés antimicrobiennes de l’huile essentielle extraite des feuilles de Tetraclinis articulata (Vahl) du Maroc : J Afrique Science, 03(2) ,232 – 242 Chanthaphon S,Chanthachum S, Hongpattarakere T (2008). Antimicrobial activities of essential oils and crude extracts from tropical Citrus spp. against food-related microorganisms, Songklanakarin: J. Sci. Technol. 30 (Suppl.1), 125-131 Farah A, Satrani B, Fechtal M, Chaouch A, Talbi M (2001). Composition chimique et activités antibactérienne et antifongique des huiles essentielles d’Eucalyptus camaldulensis et son hybride naturel (clone 583) : Acta Bot. Gall, 148(3), 183-190.

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Gachkar L, Yadegari D, Bagher Rezaei M, Taghizadeh M, Alipoor Astaneh S, Rasooli I (2007). Chemical and biological characteristics of Cuminum cyminum and Rosmarinus officinalis essential oils: Food Chemistry, 102, 898–904

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Kassi Y, Aka K, Abo K J C, Méa A, Néné B S, Ehilé E E (2008).Effet anti hypertensive d'un extrait aqueux d'écorce de tronc de parkia biglobasa (mimosaceae) sur la pression artérielle de lapin : Sciences & Nature Vol.5 N°2: 133-143.

Loubaki BC, Outtara AS, Ouedraogo CAT, Traore AS (1999). Activités antimicrobiennes des extraits aqueux totaux de Detarium microcarpum Cesalpinaceae sur huit espèces bactériennes impliquées dans certaines maladies infectieuses au Burkina Faso : Science et Médecine, Rev. CAME-Série A, Vol.01:67.

Sqalli H, el ouarti A, ennabili A, Ibnsouda S, Farah A , Haggoud A, Houari A, Iraqui M Évaluation de l’effet (2007) .

Antimycobactérien de plantes du Centre-nord du maroc .bull. Soc. Pharm. Bordeaux, 2007, 146, 271-288 Thoresen MA, Hildebrand KS (2003).

Quantitative determination of phenolic diterpenes in rosemary extracts. Aspects of accurate quantification. J. Chromatogr.A., 119- 125.

Yesil Celiktas O, Hames Kocabas EE , Bedir E,Vardar Sukan F, Ozek T, Baser KHC (2007) .Antimicrobial activities of methanol extracts and essential oils of Rosmarinus officinalis, depending on location and seasonal variations :Food Chemistry ,100 , 553-559

Matasyoh JC,Kiplimo JJ, Karubiu NM, Hailstorks TP(2007). Chemical composition and antimicrobial activity of essential oil of Tarchonanthus camphorates, Food Chemistry, 101, 1183-1187 Ouraini D, Agoumi A, Ismaili-Alaoui M, Alaoui K, Cherrah Y, Alaoui MA and Belabbas Global Journal of Research on Medicinal Plants & Indigenous Medicine – Volume 1 Issue 1, January 2012

Inhibition zone(mm)

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35 30 25 20 15 10 5 0

Essential oil Aqueous extract ethanolic extract

Fig 1: Antimicrobial activity of EO,Aq and EtOH extract extract by disc diffusion assay

Concentration v/v

1/10

1/50

1/75

1/100

1/200

1/300

1/500

1/1000

Reference

E. faecalis

-

-

+

+

+

+

+

+

+

E. coli

-

-

-

+

+

+

+

+

+

S. aureus

-

-

-

-

+

+

+

+

+

P. aeruginosa

+

+

+

+

+

+

+

+

+

B.cereus

-

+

+

+

+

+

+

+

+

L. monocytogenes

-

+

+

+

+

+

+

+

+

A. flavus

-

+

+

+

+

+

+

+

+

A. niger

-

+

+

+

+

+

+

+

+

P.escpansum

-

+

+

+

+

+

+

+

+

1: Antimicrobial Activity of essential oil ( direct contact method) Table No.1:

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Bacteria

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EtOH extract

Aqueous extract

MIC mg/ml

MIC mg/ml

S.aureus

1.5

0.35

B.cereus

-

0.45

E. feacalis

3.5

-

L.monocytogenes

-

-

E.coli

2

-

P. aeruginosa

-

-

Table No. 2: MIC of crude extracts (mg/ml)

Photo 01: Artemisia herba-alba Asoo (Bechar Zone, Algeria)

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A REVIEW ON VAR YA DAŚEMĀNI WITH SPECIAL REFERENCE TO COSMECEUTICALS Ujjaliya Nitin. B. L1, Remadevi. R2 1. Dept of Dravyaguna, V.P.S.V. Ayurveda College, Kottakkal 2. Prof. & HOD. Dept of Dravyaguna, V.P.S.V. Ayurveda College, Kottakkal Corresponding author Email: drnujjaliya@gmail.com Received: 12/12/2011

Revised: 21/12/2011

Accepted: 05/01/2012

Abstract Cosmeceuticals refers to the combination of cosmetic and pharmaceuticals. These have ingredients that can influence biological function of skin and claims to improve skin care, texture, complexion, reducing wrinkles and anti-aging. Nowadays cosmeceuticals are the fastest rising part of natural personal care industry. It is observed that extensive uses of chemically derived cosmetic products harm the skin permanently. This awareness increases the demand of Āyurvedic or organic herbal cosmetics which itself have therapeutic effect. These herbals contain essential oils, flavonoids, alkaloids, proteins, vitamins and trace minerals which can exhibit skin care as well as anti-aging effect. Āyurveda, the science of life has talked about skin care, hair care and other aspect of cosmetics. Literary review of Caraka Sa hitā provides a unique combination, Var ya daśemāni for this purpose. It is being used in practice for treating skin ailments and to impart bright colour to the skin. Drugs of this daśemāni have been explored for various skin care functions. The outcomes of scientific studies show significance of this daśemāni in the diseases of skin, as chemo preventive and to maintain the youthfulness of the skin.

Present paper deals with concept of skin and anti-aging concept in Āyurveda with description of Var ya daśemāni on the basis of scientific findings and Āyurvedic approach.

Key words: Cosmeceuticals, concept of skin in Āyurveda, Anti aging, Var ya daśemāni.

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Introduction Cosmeceuticals represents the marriage of cosmetics and pharmaceuticals.1 Cosmeceuticals are cosmetic products with biologically active ingredients purporting to have medical or drug like benefits.1 Dermatological studies suggested that the bioactive ingredients used in cosmeceuticals have benefits beyond the traditional moisturizers.

These have ingredients to enhance the appearance as well as have a positive effect at cellular level. Nowadays herbal cosmetics are occupying more popularity than synthetic ones. Long term use of synthetic cosmetics may exhibit adverse reaction and withdrawal like symptoms on the skin. This awareness has increased demand of herbal cosmetics. Herbs contain some active ingredients which have a potential effect on skin. The uses of herbal ingredients in a cosmetic product have dual advantage. They act as a cosmetic product along with being nutritive to the skin.

Literary review of Āyurvedic Sa hitās provide unique combinations for this purpose. These combinations can help in reversing the signs of aging. Classical texts have talked about skin care and its importance on a healthy individual.3 Ācarya Caraka has mentioned Var ya daśemāni; a group of ten drugs having a broad range of skin care property.2 Daily skin care in the context of Svasthav tta (regimen for a healthy person) also has been described.4,5 Today, once again it is a revival of the concept of cosmeceuticals aspect in Āyurveda with a scientific insight. The review compresses scientific findings, which have been done on Var ya daśemāni related to its benefits in skin care.

Concept of Tvacā (skin) In Āyurveda the word Tvak (Tvacā) is used for skin. The meaning of Tvacā is to cover the body. There are seven layers mentioned in Tvacā as well as different diseases according to these layers. It is said that the formation of Tvacā take place by assimilation of rakta dhātu or it is the supernatant part of incorporation of

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Śuk a and So ita (sperm and ovum). The difference in all types of skin for its color, texture, glow, thickness, hairs etc. are due to the specific body constitution of an individual. Āyurveda has mentioned a particular type of skin for a particular type of Prak uti (body constitution). The skin care principles are applicable according to the type of Prak uti of an individual.

Concept of anti-aging Aging is a normal phenomenon which occurs at cellular level. It can be controlled by a healthy life style. Ācarya Sārangadhara divided the 120 years of life into 10 decades with ten qualities and mentioned that one quality of life will be lost after each decade.8 After 30 years; complexion of the skin will be reduced gradually. Similarly strength decreases and texture of the skin starts changing after 50 years. The equilibrium of Do as and proper nourishment of all seven dhātus delay the process of aging. Many other factors are responsible in maintaining the physical condition and youthfulness of skin. For example, a proper moisture balance, hormonal and heat regulation and effective circulation of blood and nutrients are some of the factors and they are controlled by tri-do as. Dhātus like rasa, rakta, medas and ojas play an important role in skin care. Rasāyanās (rejuvenatives) are advised according to the age and requirement of the body to improve strength, vigor and complexion.

Var ya daśemāni In the context of Mahāka āyās, Ācarya Caraka has mentioned Var ya daśemāni.9 Var ya means gives colour and the meaning of daśemāni is a group of ten drugs. These drugs together posses a wide range of effect but when used individually they have different aspect of skin care. In this daśemāni, Caraka has mentioned drugs which have different properties. The specialty of this type of groups is that they have synergistic action in combination irrespective to number.10 As per the name Var ya; it is specially meant to improve complexion and impart bright colour

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to the skin. Recent researches show that these drugs have properties beyond it. External application has narrow range of therapeutic effect than internal administration. It is a combination of drugs which pacify vitiated do as, gives complexion and also nourish the tissues. The brief description of this daśemāni on the basis of Āyurvedic and scientific views is given below: (Table no.1)

Pterocarpus santalinus Linn.

According to Ācarya Sārangadhara, for Lepam (external application) and decoction red variety of Candana should be taken but in other uses and preparations white variety is suggested.11 So, in the present study Rakta candana has been taken for review. The principle chemical constituents of Rakta candana (Pterocarpus santalinus Linn.) are liquiritigenin and isoliquiritigenin.12 These are potent anti-oxidants and blood detoxifiers. It is being used with curcuma for skin care, since times immemorial. According to Āyurveda it has the property of blood purification, when used both internally and externally. In-vitro studies had shown anti-fungal and anti-bacterial properties of its aqueous extract.13 Researchers have proved it’s anti-viral (against HSV-I &II), anti-oxidant and chemo preventive effects.14 Calophyllum inophyllum Linn. Ācarya Cakrapā ī while commenting on Var ya daśemāni has advocated Punnāga (Calophyllum inophyllum Linn.) as Tunāa.15 This plant is found in seacoasts of southern and south eastern India, the flowers of which has a good fragrance. Its oil has a unique ability to promote the formation of new cells thereby accelerate wound healing and growth of healthy skin.16 The appearance of scars had reduced when its oil was used for nine weeks in a clinical study.17 Its 4-phenylcoumarins have been proved as chemo preventive agents.18 Prunus cerasoides D. Don. Due to its cool and bitter property, Padmaka (Prunus cerasoides D. Don.) pacify vitiated pitta, blood born disorders, burning sensation, skin discoloration and eczema.19 It contains a

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flavanone glycoside, naringenin4-methyle ether 7-xyloside.20 It is a frequent ingredient of several Āyurvedic preparations. It can be applied as lepam for treating skin disorders.

Vetiveria zizanioides Linn.

Uśīra (Vetiveria zizanioides Linn.) is śīta (cooling property) in nature. It posses calming and stabilizing effect. Due to this it is a widely used medicine for the cure of vitiated pitta and rakta diseases.21 It is used in scrubs and face packs that are applied on face and whole body to remove dead cells. Uśīra has 97% sesquiterpenes which regenerate growth of new cells and gives a young look. In-vitro antioxidant activity22 of its methanolic and water extracts along with Ailanthus malabarica and Schinus terbinthifolius leaves has been proved.

Glycyrrhiza glabra Linn.

Yasthimadhu (Glycyrrhiza glabra Linn.) is famous for its natural anti-bacterial, antiinflammatory properties. It brightens and clarifies skin as well act as a blood purifier. It has also got properties as a rejuvenative (Rasāyana) drug.23 Ācarya Caraka has mentioned it several times in the context of Mahāka āyas.2 It has a rich nutritive value also. Its methanolic extract cream has been proved clinically for reducing melanin.24 National cancer research institute has recognized its chemo preventive property.

Rubia cordifolia Linn.

Manji thā (Rubia cordifolia Linn.) is considered as one of the best blood purifying herbs in Āyurveda. The principal constituents are purpurin, munjistin, xanthopurpurin, and pseudopurpurin. Clinical studies reveal that this herb possesses anti-oxidant, antiinflammatory, anti-platelet and hepatoprotective properties.25 Research also shows that the biologically active constituents of the extract facilitate blood circulation by removing congestion and improving blood quality through enhanced hemoglobin production. Its in-vitro free radical activity has been established.26

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Hemidesmus indicus Linn.

Sārivā (Hemidesmus indicus Linn.) is said to be tonic, diuretic and alterative. It has sweet and cooling property. It is being used in Āyurveda for treating various types of diseases related to vitiated rakta dhātu.27 The do a pitta causes disease mainly on skin and blood. The śīta gu a of Sārivā pacifies both the factors. Its anti-microbial property has been proved in recent years.28 Ipomoea mauritiana Jacq.

Vidārī (Ipomoea mauritiana Jacq.) is added to Var ya daśemāni for its nutritional value. It has been proved as an anti-oxidant and antibacterial also.

Its phyto-constituent Umbelliferone is a phenolic compound of plant origin, for which many biological activities against chronic disease have been proved.

Umbelliferone is used as an immuno-modulator in diabetes and cancer.29 Cynodon dactylon Linn.

Dūrvā (Cynodon dactylon Linn.) is one of the ten auspicious herbs that constitute a group Daśapu pā in Āyurveda particularly in Kerala tradition.30 Due to its cooling nature it pacifies excess heat of the body and cures dermatitis, eczema and burn. Inhibitory effect of C. dactylon on inflammation and oxidative stress in adjuvant treated rats has been recognized.

Free radical scavenging activity by DPPH method and anti-stress effect on diabetic rats has been illustrated.31 While commenting on Caraka Sa hitā; Ācarya Cakrapā ī advocated dūrvā for Sītā and Latā herbs as two varieties of dūrvā (śveta and śyāma dūrvā).32 Safety measure Herbal cosmetics are considered as free from side effects but these may act as allergen to skin of a particular person. They can cause itching, sneezing etc. if they are not properly purified or free from adulterants. Long term use of any cosmetic preparation should be done under prescription of an Āyurvedic physician.

One, who needs better skin forever, has to take internal medications. An attention should be

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paid to the gross changes or any other abnormal changes to the skin because skin is the diagnosis point for several diseases.

Discussion Ācarya Caraka mentioned Var ya daśemāni on the basis of pharmacological action. It is a comprehensive formulation for cosmeceutical purpose. Herbs of this daśemāni have dual advantage; these serve cosmetic as well as therapeutic benefits. Some herbs used in diseases like dermatitis, eczema and some has property of blood purification and imparting bright colour to the skin. Most of the herbs are being attempted for their chemo preventive property. The drugs Madhuka, Vidārī and Sārivā have rich nutritive values, which provide strength and nourishment to the skin. Var ya daśemāni offers external cosmetic benefits as well as uphold the youthfulness of the skin for a long.

Conclusion The above conceptual study unveils that: Āyurveda stated different aspects of cosmeceuticals and has mentioned the ways to achieve that effect and maintain and improve it.

Skin reflects the physical condition and nutrition of body. It cannot seem healthy and beautiful unless it is not appropriately nourished.

Var ya daśemāni of Caraka Sa hitā is a unique combination for skin care. It has got property to balance trido as and nourish the body tissues.

Drugs of Var ya daśemāni can be utilized individually as well as in a combination to have a wide therapeutic outcome.

Evidence based studies have shown magnitude of Var ya daśemāni in a variety of diseases as well as in regular care of skin.

The natural or the organic products contain amino acids, essential and fatty oils. They also contain adequate amount

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of vitamins and minerals which help in nourishing the skin and give an antiaging effect.

References 1. Jain et. al. (2010), Potential of Herbs as Cosmeceuticals / Indian Journal of Research in Āyurveda and Pharmacol, 1 (1), pp. 71-77.

2. Sastri Kashinath and Chaturvedi Gorakhnath (2001), Agnive a ’Caraka Samhita’, revised by Caraka and D u habala with ‘Ayurveda Dīpikā’ Cakrapā īdatta, commentary, by Chaukhamba Vishvabharti, Varanasi (India),Sutra Sthāna 4/8/8.

3. Caraka Samhita, SutraSthāna 5/88-89, Ibidem 4. Caraka Samhita, SutraSthāna 5/85-86, Ibidem 5. Caraka Samhita, SutraSthāna 5/93, Ibidem 6. http://en.wikipedia.org/wiki/ physiology of skin (Downloaded on 27/09/2011) 7. http://en.wikipedia.org/wiki/skin (Downloaded on 27/09/2011) 8. Sarangdhara’s Sarangadhara Samhita, translated by Prof. Murthy Srikantha K.R. (2006), Chaukhambha Orientalia, Purvakhanda 6/20.

9. Caraka Samhita, SutraSthāna 4/8/8, Ibidem 10. Caraka Samhita, SutraSthāna 4/25-29, Ibidem 11. Sarangdhara’s Sarangadhara Samhita, translated by Prof. Murthy Srikantha K.R. (2006), Chaukhambha Orientalia, Purvakhanda 1/50.

12. http://www.himalyahealthcare.com (Downloaded on 26/12/2011) 13. Chaumont J.P. et. al (1989) Activities Antifrngques Invitro De Sept Huiles Essential, Fitoterrapia, Lx (3), pp. 263266.

14. Scartezzini P. et. al (2000), Review on some plants of Indian traditional Medicine with Antioxidant activity, J. Ethnopharmacol 71 (1-2), pp. 23-43.

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15. Caraka Samhita, ‘Ayurveda Dīpikā’ Cakrapā īdatta, commentary, by SutraSthāna 4/8/8, Ibidem 16. Dweck, A. C. (2002), Natural ingredients for colouring and styling.

International Journal of Cosmetic Science, 24: pp. 300.

17. Dweck, A. C. (2002), Natural ingredients for colouring and styling.

International Journal of Cosmetic Science, 24: pp. 288.

18. Masataka itoigawa et. al (2001), Cancer chemopreventive agents, 4phenylcoumarins from Calophyllum inophyllum, cancer letters, Science Direct. Com, vol.169, Issue I, pp. 15-19.

19. Sharma P.V. (2005), Dravyagna Vijnan, Chaukhambha Bharti Academy, Vol. II, Padmaka, pp. 43-45.

20. Srivastava S.P. (1982), A flavonone glycoside from Prunus cerasoides, Phytochemistry, Vol. 21, Issue 6, pp.

1464-1465.

21. Sharma P.V. (2005), Dravyagna Vijnan, Chaukhambha Bharti Academy, Vol. II, Usira, pp. 114-116.

22. V. Subhadradevi et. al (2010), Invitro antioxidant activity of Vetiveria zizonioidis root extract, Tanjania Journal of Health Research, Vol. 12, No.2.

23. Sastri Kashinath and Chaturvedi Gorakhnath (2001), Agnive a ’Caraka Samhita’, revised by Caraka and D u habala with ‘Ayurveda Dīpikā’ Cakrapā īdatta, commentary, by Chaukhamba Vishvabharti, Varanasi (India),Chikitsa Sthāna 1-3/30.

24. Akhtar Naveen et. al (2011), Glycyrrhiza Glabra Extract Cream: Effects On Skin Pigment Melanin, International conference on bioscience, Biochemistry and Bioinformatics IPCBEE, Vol.5, IPCBEE press, Singapore.

25. Tripathi Y.B., Singh Ajitha Vikram (2007), Role of Rubia cordifolia Linn. in Radiation Protection , Indian Journal of Exp. Biology, Vol. 45, pp. 620-625.

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26. Kaur Prabhjit et. al (2008), In vitro evaluation of free radical scavenging activity of Rubia cordifolia L., Journal of Chinese clinical medicine, 2008,5, Vol.3, No. 5.

27. Sharma P.V. (2005), Dravyagna Vijnan, Chaukhambha Bharti Academy, Vol. II, Sariva, pp. 789-800.

28. Gayathri M., Kannabiran K. (2009), Antimicrobial activity of Hemidesmus indicus, Ficus bengalensis and Pterocarpus marsupium roxb, Indian Journal of Pharma Science, Sep.-Oct. 71(5); pp. 578-581.

29. Adhyapaka Shreeda shreesh et. al (2011), Comparison of HPLC and HPTLC techniques for determination of umbelliferone from dried tuber powder of ipomoea mauritiana jacq., International Journal of pharmaceutical science and Research, Vol.2, issue 11, pp. 2894-2900.

30. Nitu Shree and Remadevi R. (2011), Preliminary Pharmacognostical and Phytochemical Analysis of Dashapushpa, Thesis submitted to Calicut University, Kerala, pp. 47-58 31. Helen A. et. al (2010), Inhibitory effect of C. dactylon on inflammation and oxidative stress in adjuvant treated rats, Inform health care.com/doi/abs/10.3109.

32. Caraka Samhita, ‘Ayurveda Dīpikā’ commentary, by Cakrapā īdatta, Sutra Sthāna 4/8/8, Ibidem.

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Table no. 1 Var ya daśemāni drugs Latin name

No.

Drugs

1

Candana (Rakta)

Pterocarpus santalinus Linn.

2

Tunāa (Punnāga*)

Calophyllum inophyllum Linn.

3

Padmaka

Prunus cerasoides D. Don

4

Uśīra

Vetiveria zizanioides Linn.

5

Madhuka

Glycyrrhiza glabra Linn.

6

Manji thā

Rubia cordifolia Linn.

7

Sārivā

Hemidesmus indicus Linn.

8

Payasyā

Ipomoea mauritiana Jacq.

9

Sītā (śveta dūrvā*)

Cynodon dactylon Linn.

10

Latā (śyāma dūrvā*)

Cynodon dactylon Linn.

* Cakrapā ī commentary on Caraka Sa hitā.

Fig. no. 1 - Pterocarpus santalinus Linn.

Source: http://beauty.ebest.in

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Fig. no. 2 - Calophyllum inophyllum Linn.

Source: http://2.bp.blospot.com Fig. no . 3 â&#x20AC;&#x201C; Prunus cerasoides D. Don

Fig. no. 4 - Vetiveria zizanioides Linn.

Source: http://motherherbs.com Fig. no. 5 - Glycyrrhiza glabra Linn.

Source: http://yasthimadhu.com

Source: http://neeroga.com

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Fig. no. 6 - Rubia cordifolia Linn.

Fig. no. 7 - Hemidesmus indicus Linn.

Fig. no. 8 - Ipomoea mauritiana Jacq.

Fig. no. 9 - Cynodon dactylon Linn.

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A CLINICAL STUDY ON GOKSHURADI CHURNA IN THE MANAGEMENT OF OLIGOSPERMIA Sudev. C1, Suresh.R.D2 1.P. G. Department of Kayachikitsa 2.Reader and assistant professor, P. G. Department of Kayachikitsa Corresponding Author: sudevc@gmail.com,

Received: 02/12/11

Revised: 16/12/11

Accepted: 31/12/11

ABSTRACT Oligospermia is one among the prime factors for male infertility and it is defined as a subnormal concentration of spermatozoa in the penile ejaculate i.e. less than 20 million/ml. The quantity and quality of sperms are decreasing day by day, which may be due to altered life styles, rapid industrialization etc. However, till date no satisfactory medical management has been developed for this problem. Hence an attempt has been made to evaluate the Effect of Gokshuradi Churna in Oligospermia. In this standard single blind clinical study, 20 patients fulfilling the inclusion criteria were selected, randomly categorized into single group and administered with Gokshuradi Churna along with milk, 6 gms twice daily, morning at empty stomach and night at bedtime, for 90 days followed by dietary measures. Severity of the disease was assessed by objective parameters. Follow up study was under taken for 30 days after 90 days of treatment. After the therapy, significant result was observed with respect to the objective parameters sperm count and motility, which marks the trial drug Gokshuradi Churna along with milk as adjuvant, helps in the management of Oligospermia.

Keywords: Oligospermia; Gokshuradi Churna; Sperm count; Sperm motility.

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INTRODUCTION Ayurveda is entitled as a “Divine science” due to its origin as well as its unimaginable potency in curing the diseases and protecting the health of a healthy person, physically and mentally. Even centuries after its golden period, Ayurveda continues to play a major role in maintaining the health status of majority of Indians and some sections of people of many other countries. The world is recognizing Ayurveda as a system which can become a universal health care system in treating a number of diseases which has minimal treatment options in the contemporary sciences.

Infertility is one such condition with Global incidence of about 13 – 18%. Male infertility is found to have a role in approximately 50% of infertile couples1. As the sperm count decreases, there is a corresponding decrease in the likelihood of conception.

Modern medical science has reached to its peak in MART (Medically Assisted Reproductive Technology), artificial insemination, invitrofertilization, and intra-cytoplasmic sperm injection. These are considered as advanced technologies, but not accepted widely in India where poverty is rampant. So it is unable to provide service to all class of people in the society and also, it may be hardly successful many a times. Hence by keeping the prevalence of Oligospermia in view, an attempt has been made to evaluate & establish the Therapeutic efficacy of a formulation Gokshuradi Churna2 (A medicated powder of plant drugs starting with Tribulus terrestris) along with milk in the management of Oligospermia.

MATERIALS AND METHODS: Materials Here for the clinical study, Gokshuradi Churna and Goksheera (cow’s milk) as Anupana (Adjuvant) was taken for the analysis of the efficacy of the drug in Oligospermia. The trial drug “Gokshuradi Churna” contains

Gokshura, Kokilaksha, Satavari, Kapikachu, Naagabala, Atibala. (Insert Table No. I) All the above mentioned drugs were taken in the mentioned quantity and powdered separately after proper drying3, except Asteracantha longifolia seed which is fried in Ghee first, then powdered & filtered (with mesh size 85) and mixed with the other finely powdered ingredients. The reason for frying Asteracantha longifolia in Ghee is that, it will become mucilaginous when it is pounded otherwise. Frying in Ghee therefore facilitates the powdering process. Finally the fine powders of all the drugs were mixed and were packed in airtight polythene covers (360 g. in one packet), and stored in airtight plastic containers. To take uniform quantity of medicine, a 6g. spoon had been supplied with the packet of medicine.

For the Physico-chemical analysis, methods were adopted as given in Ayurvedic Pharmacopeia of India. viz. Loss on drying, total Ash, water soluble ash, acid insoluble ash, water soluble extractives, alcohol soluble extractives for physico chemical tests and qualitative test parameters like reducing sugar, protein, anthraquinone glycoside, cardiac glycoside, steroids, tannin, alkaloid and flavonoid were done.

Thin layer chromatography of Gokshuradi Churna has been done using; Solvent System - Chloroform: Methanol - 90:10 (v/v), Spraying Agent: Anisaldehyde Sulphuric acid.

Methods Clinical source: Patients of male sex attending OPD and IPD of A. L. N. Rao M.A.M. College Hospital, Koppa and its associated Hospitals, who were diagnosed as Oligospermia, were selected for study.

Study Design - Single blind clinical study.

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Methods of collection of data (including sampling procedures) 20 patients diagnosed as Oligospermia, from OPD and IPD of A. L. N. R. M .A .M .College and its associated hospitals were selected for the study after following the inclusion and exclusion criteria. The duration of the treatment was 90 days. The objective parameters were graded and the patients were assessed before scheduled treatment. The changes were observed for every 30 days of treatment and it was documented based on grade of chronicity.

Inclusion criteria:

Assessment response: The improvements of patients were assessed on the basis of relief in objective parameters. According to severity, the grading for the parameters was given as below;

Grading for objective parameter 1. Sperm count a) b) c) d)

Normal ( 20 million / ml or more) – Mild ( >14 – <20 million / ml ) – Moderate ( 7 – 14 million / ml ) – Severe ( >0 – 7 million / ml ) –

0 1 2 3

2. Sperm motility

1. Patient diagnosed as Oligospermia.

2. Male patients of age ranging from 21 to 45 years.

Exclusion Criteria:

a) b) c) d)

Normal (motility Above 75 %) Mild (motility - 50% - 75%) Moderate(motility - 25% - 50%) Severe (motility - Below 25%)

–0 –1 –2 –3

1. Known cases of genetic abnormalities.

2. Patients associated with systemic disorders like Diabetes, Hypertension, Tuberculosis and HIV infection.

3. Patients of Sexually transmitted diseases.

4. Post traumatic Oligospermia.

5. Patients diagnosed as Azoospermia.

6. Patients with the conditions were surgical interventions needed.

Collection of semen for Analysis

Method:

Masturbation method was preferred for the sample collection.

Koshta Shuddhi (purification of alimentary tract)

Container:

The Laboratory supplied, dried and wide mouthed bottle was used.

Place:

A private room adjacent to the laboratory was used for semen

The Selected patients had been given Triphala Churna, 10 – 15 gms, at bed time, on the day prior to the first day of treatment schedule.

Treatment intervention Sample size

:

20 patients

Medicine

:

Gokshuradi Churna

Dose

:

6 gm, twice a day

Adjuvant

:

Milk

Abstinence: Before collection of the ejaculate minimum of 3 days and maximum of 5 days abstinence was followed.

collection Time:

Semen collection time was restricted between 9.30 am to 11.30 am

Laboratory investigations 1. Sperm count and motility 2. ESR – to rule out the infections 3. Hb% - to rule out Anemia

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4. RBS – to rule out Diabetes

Statistical Analysis: Descriptive data that included Mean, Standard Deviation (S.D), Standard Error (S.E),‘t’ value and p value were calculated for all the variables in trial group. Post treatment changes were assessed by paired ‘t’ test.

(Significance level: P>0.05)

Assessment of the overall effect: To assess the overall effect of the therapies, net result obtained on various parameters of assessment, both before and after treatment were taken in to consideration. Then it was graded in terms of percentage of relief in symptoms.

Grading of Remarks or Criteria for assessment of overall effects of the therapy Complete cure

- 100% relief

Marked improvement - >66% to <100%

a) Effect of therapy on Sperm count (Insert Table No. II) The trial drug Gokshuradi Churna provided high significant result on increase in the sperm count after 30 days of treatment with P>0.001. After 60 days, 90 days and after follow up also, the trial drug shown high significant results with P<0.001. There was increase in the t value gradually till 90 days of treatment and it reduced gradually after the follow up. But the same significance P<0.001 persisted after follow up. b) Effect of therapy on Sperm motility (Insert Table No. III) The trial drug Gokshuradi Churna provided high significant result in increase in sperm motility after 30 days of treatment with P>0.001. Also after 60 days, 90 days and after follow up, the trial drug shown the same high significant results with P<0.001. There was increase in the ‘t’ value gradually till 90 days of treatment and it reduced gradually after the follow up. But the same significance P<0.001 persisted after follow up.

Moderate improvement- >33 to <66% of relief Mild Improvement

- >1 to <33 % of relief

Overall result of the treatment

Unchanged

- 0% of relief

(Insert Table No.IV)

Follow up study Follow up study was undertaken for 30 days after the treatment schedule to assess the result.

RESULTS Among 24 patients included for this clinical trial, 21 patients completed the treatment and 3 patients left the treatment due to various reasons. Among the 21 patients 20 patient’s data has been included for the statistical analysis.

Two patients got complete relief and marked improvement after the treatment schedule. One patient got complete and marked improvement after the follow up. 12 patients got moderate improvement after the treatment and 6 patients had got moderate improvement after the follow up. There were 3 patients who got mild improvement after the treatment and 9 patients after follow up. There was no change in one patient after the treatment and 3 patients after follow up.

Overall result on the basis of conception in the partner (Insert Table No. V)

Effect on the objective parameters

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After the treatment of 90 days, 2 patients reported that their partner got conceived with an incidence of 10%.

Results of phytochemical Gokshuradi Churna

Analysis

of

1. Physico-chemical Tests Loss on Drying

:

10.00%

Total Ash

:

6.76%

Water soluble ash

:

3.65%

Acid insoluble ash

:

0.75%

Water soluble extractives

:

6.51%

Alcohol soluble extractives

:

5.21%

: : : : : : : :

++ + ++ ++++ +++ ++++ + +++

2. Qualitative Tests Reducing sugars Proteins Anthraquinone glycosides Cardiac glycosides Steroids Tannins Alkaloids Flavonoids

(+ - Present in very low quantity, ++ - Present in low quantity, +++ - Present in moderate quantity, ++++ - Present in abundance)

3. Thin Layer Chromatography Rf Values: 0.05, 0.31, 0.49, 0.61, 0.64 DISCUSSION Discussion on observations related to disease In this clinical study family history was not so prominent. Only 15% of the patients had revealed that they were having the family history of infertility or delayed conception in mother or abortion in mother, or similar incidences from any of the family members with blood relation. 85% doesnâ&#x20AC;&#x2122;t have any type of history from their family. This may be due to

the lack of their knowledge regarding such incidences in their family.

People who follow vegetarian diets are more prone to get the vitamin deficiencies like B12. Vitamin A, Vitamin B, Vitamin C, Vitamin E, Omega-3 Fatty Acids, Selenium and Zinc which may further hamper the process of spermatogenesis. In this clinical study 50% of the patients were taking Vegetarian diet. So among them these deficiencies may be a precipitating factor. The rest of 50% of patients who are Non- Vegetarians might have become Oligospermic due to a high spicy diet or may be due to the Hormonal injections used on cattle & Poultries to get a better yield of meat.

It is proved that the incidence of Oligospermia is more in the persons who are addicted to smoking and drinking (Alcohol), both of which are considered to be testicular toxins or Leyding toxins4. This clinical study also proved that, it is more prominent in patients who are addicted to smoking (45%) and drinking (Alcohol) (40%). Tobacco chewing had been observed in 20% of the patients and only in 5% of the patients, other addictions like Gutka/tea/coffee were observed.

It indicates that other addictions apart from Alcohol and cigarette smoking are less dominant in causing Oligospermia.

Excessive masturbation may lead to Oligospermia. As such general parameters are not there to assess this incidence; hence it is very difficult to understand the normal frequency of masturbation. In this clinical study, the history of masturbation was found in 55% of the patients, out of which, 35% of the patients had occasional masturbation and 20% of the patients had regular masturbation. The history of masturbation was absent in 45% of the patients.

It was observed in this clinical study that, 50% of the patients were having physical stress. It indicates the history of hectic work load in the past. Frequency of coitus cannot be graded due to the absence of the standard and

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specific grading in the modern sciences. It may be assumed that the number of coitus for a speculated time is according to their strength only, in such a way that it is not going to hamper their strength and sperm. In this clinical study majority of the patients had a frequency of coitus, 3 – 4 times/week with an incidence of 45% followed by a frequency of 2 – 3 times/week with an incidence of 35% and 5% of incidence with a frequency of coitus, 1 – 2 times/week. So it seems to be probable that with a frequency of coitus 1 – 2 times in a week, patients have got a less chance in the production of Oligospermia compared to the other groups with a higher frequency of coitus.

Wearing tight under garments is found to be one cause for the increase in the testicular temperature. This increase in temperature, in turn disturbs the spermatogenesis as it needs 1 degree Celsius less than the body temperature for normal production of sperms. In this clinical study it was found that 30% of the patients were wearing undergarments tightly.

Hot water bath had been observed in 60% of the patients, which might have resulted in increased testicular temperature.

Discussion related to the results Effect of Gokshuradi Churna on Sperm count and motility The trial drug Gokshuradi Churna provided high significant result in the increase of sperm count after 30 days of treatment with P>0.001 (24.44% improvement). After 60 days, 90 days (62.22%) and after follow up (46.66%) also, the trial drug shown high significant results with P<0.001. There was increase in the ‘t’ value gradually till 90 days of treatment and it reduced gradually after the follow up. But the same significance P<0.001 persisted after follow up.

The trial drug Gokshuradi Churna provided high significant result in the increase of sperm motility after 30 days of treatment with P>0.001 (23.40%). Also after 60 days (36.17%), 90 days (55.32%) and after follow

up (38.29%), the trial drug shown the same high significant results with P<0.001. There was increase in the ‘t’ value gradually till 90 days of treatment and it reduced gradually after the follow up. But the same significance P<0.001 persisted after follow up.

The trial drug contains Shukra Vardhaka drugs and the drugs have got property of Brimhana also. By these actions, it might have resulted in the proper production of Sperms and their nourishment. TLC report of the trial drug showed that, it contains Reducing sugar, proteins and plant steroids, which are essential for the production and maturation of the sperm that might have helped the patients to get an improvement in the Sperm count. Due to the vitiated Apana Vata the motility of the sperms decreases, it might be rectified by doing Vata Shamana (alleviating) property and Brimhana (nourishing) property of the trial drug.

Tribulus terrestris leads to a rise in the production of luteinizing hormone. When luteinizing hormone levels are increased, the natural production of testosterone also increases. The saponin in T. terrestris thought to be responsible for its effect on testosterone levels and on libido5. This drug might have increased the testosterone level and there by the production of sperms increased. T. terrestris is found to be effective for men with Anti-sperm antibodies6. This action might have helped the patients with anti-sperm antibodies, to recover from it. Astercantha longifolia is proved to be a best tonic for increasing reproductive organ weights. Increased spermatogenesis by this medicine extracts was also witnessed7. So the trial drug might have increased the spermatogenesis and thereby resulted in increased sperm count in this study. Asparagus racemosus has been proved to be a drug, which is increases the spermatogenesis and volume of semen8&9. So this action might have helped to increase the sperm count in this study. Mucuna pruriens might have improved Testosterone, LH, dopamine, adrenaline, and Noradrenaline levels in infertile men and there by the sperm count increased10. Steroidogenetic property and its action on the hypothalamus-pituitary-

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gonadal axis of M. Pruriens might have improved semen quality and production in the patients11. M. pruriens might have ameliorated psychological stress. Seminal plasma lipid peroxide levels along with improved sperm count, motility and reactivated anti-oxidant defense system of patients, might have helped in the management of stress, enhanced mental alertness and improved coordination and semen quality.12&13 TLC report of the trial drug showed that it contains low quantity of the reducing sugar.

Spermatogenesis is a precisely regulated process in which the germ cells closely interact with Sertoli cells. The molecular basis of this cell-cell adhesion is unknown. The researchers identified an N-glycan (asparagine-linked carbohydrates) structure that plays a key role in germ cellâ&#x20AC;&#x201C;sertoli cell adhesion and showed that a specific carbohydrate was required for In this way the spermatogenesis.14 carbohydrates present in this formulation might have helped in this study for increasing the sperm count. Proteins are needed for the activation as well as the structural formation of sperm during spermatogenesis. Activated mitochondrial proteins are the cause for the activity of the sperm. Both nuclear and cytoplasmic proteins are affected in Oligospermia and Azoospermia.15,16&17. This protein might be of further help in increasing the motility of sperms.

TLC report of the trial drug also showed the presence of moderate quantity of Flavonoids, which have got Anti-oxidative effects on spermatogenesis and may be promising in enhancing sperm healthy parameters18. TLC report of the trial drug showed presence of steroids (plant origin) in moderate quantity. Steroids include oestrogen, cortisol, progesterone, testosterone etc.

Testosterone induces and helps to complete the process of spermatogenesis. The plant steroids in the trial drug might have helped in the spermatogenesis by acting as a precursor for testosterone. TLC report of the trial drug also showed the presence of abundant amount of Tannins. Tannins have shown potential

antiviral, antibacterial and anti-parasitic effects19. This might have helped in counteracting with any infections occurred in the patients or these actions might have increased the immunity to resist against the infections.

Gokshura contains sterols. Sterols got meiosis inducing effect, which will help in the meiosis during the Spermatogenesis. Meiosis Activating Sterols (MAS) are found only in Gonads20. Thus it helps in the process of spermatogenesis. Sterols may act as precursors of other steroids as hormones and as membrane components. This might have triggered or acted as a catalyst in the production of Testosterone and there by initiated the Spermatogenesis in the patients. Kokilaksha contains protease (also termed peptidase or proteinase). Protease is an enzyme that conducts proteolysis, i.e. begins protein catabolism by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain forming the protein21. This process might have helped in the spermatogenesis. Kokilaksha also contains Diastase, which is a group of enzymes which catalyses the breakdown of starch into maltose22. This mechanism might have helped in the process of spermatogenesis as the sperm contains more amounts of carbohydrates as well as the proteins. Kapikachu contains LDOPA, which crosses the protective bloodbrain barrier, whereas dopamine itself cannot.

Thus, L-DOPA is used to increase dopamine concentrations. An Antiserotonin effect upon the limbic site of action is claimed for L-dopa and for Parachlorphenylalanine, both hailed as great aphrodisiacs. This Antiserotonin effect as well as increase dopamine concentrations might be stimulating the production of Androgens and thereby increase of Testosterone23. This testosterone further helps in the spermatogenesis.

CONCLUSION Wearing tight undergarments and bathing in hot water were found to be significant causes for Oligospermia along with other known and unknown causes.

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In this clinical study majority of the patients complained of premature ejaculation, for which performance anxiety is the main cause. Couple can be advised the new techniques with guidelines laid down by Master and Johnson.

The trial drug Gokshuradi Churna did not show any intolerance or reactions during the course of treatment in any of the patients except the nausea felt by 7 patients initially and overcame by taking milk after the intake of medicine.

The trial drug Gokshuradi Churna is proved to be an effective oral formulation, which helps in the management of Oligospermia, when the disease is not too advanced and when correctly used by the patient as per instructions.

Proper sex education and counselling are also needed for effective treatment. That is Physic and psych should be treated at the same time.

The Phytochemicals like Phyto-Sterols, Tannins, Cardiac glycosides, Flavonoids Carbohydrates & Proteins might have helped to get a significant result in this study.

ACKNOWLEDGEMENTS On the completion of this work, I render my immense and heartfelt thanks to Dr. Sanjaya K.S M.D.(Ay), Principal A. L. N. Rao Memorial Ayurvedic Medical College, Koppa for his constant help and support for completing this work. My sincere gratitude to Dr. D. Bhattacharya, H.O.D and Professor, Dept. of Kayachikitsa for the constant supports.

REFERENCES 1. http://www.medindia.net/patients/patienti nfo/geneticsmaleinfertility.htm#ixzz1hLu vMPcL. on 15/12/2011

Chakradutta with 2. Chakrapanidatta; Vaidayaprabha Hindi commentary by Tripathi Indradeva, 1997, Chaukambha Sanskrit Sansthan, 1997, Varanasi.

3. Sharangadhara; Sharangadhara Samhita Madhyamama ghanda 6/1, Chaukambha Sanskrit Sansthan, 1997, Varanasi.

4. Lipsett et. al. 1980; Van Thiel et. al. 1983 5. http://www.nutriline.org/pages/desc.php? nid=99&, and http://www.teslatech.info/ttstore/report/art icles/v2n3art/trib.htm on 16/9/2011 6. http://natural-fertility-info.com/tribulusfertility.html on 16/9/2011 7. Chauhan N. S, Sharma V., Dixit V. K. ; Sexual enhancement, aphrodisiac - Effect of Asteracantha longifolia seeds on the sexual behaviour of male rats â&#x20AC;&#x201C; N. P. R. 2009 Sep: Department of Pharmaceutical Sciences, H.S. Gour Vishwavidyalaya, MP, India 8. Abbas. A, Jamil. S, Qadder. A and Zafar.

Comparative Single-Blind S ; Randomized Placebo-Controlled Clinical Trial of a Polyherbal Formulation Containing Asparagus racemosus and Cheiranthus cheiri in Oligospermia.

9. http://library.ust.edu.ph/journalweb/acta_ abstract6.htm on 28/5/2011 10. http://www.bhj.org/journal/2003_4503_ju ly/semen_416.htm on 28/5/2011 11. Fertil Steril. 2008; Effect of Mucuna pruriens on semen profile and biochemical parameters in seminal plasma of infertile men, 2007, Departments of Biochemistry and Obstetrics and Gynecology, King George's Medical University, Lucknow, India.

12. Shukla K. K, Mahdi A. A, Ahmad M. K, Jaiswar S. P, Shankwar S. N, Tiwari S. C; Mucuna pruriens Reduces Stress and Improves the Quality of Semen in Infertile Men. - Evid Based Complement Alternate Med. 2007. Department of Biochemistry, King George's Medical University, Lucknow, India.

13. http://www.ncbi.nlm.nih.gov/pmc/articles /PMC2816389/ on 20/6/2011

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14. Science, 4 January 2002: Vol. 295 no.

5552 pp. 124-127, DOI: 10.1126/science.

1065570 and http://www.sciencemag.org/content/295/5 552/124.abstract 3/7/2011 15. Human reproduction update, 1999, vol 5,No. 2,pp 108-119, and 16. http://humupd.oxfordjournals.org/content/ 5/2/108.full.pdf on 6/6/2011 17. http://www.ncbi.nlm.nih.gov/pubmed/103 77282 on 8/7/2011 18. African Journal of Pharmacy and Pharmacology Vol. 5(6), pp. 721-725, June 2011, Available online http://www.academicjournals.org/ajpp, and http://www.academicjournals.org/ajpp/PD F/pdf2011/June/Khaki%20et%20al.pdf on 8/5/2011 19. http://en.wikipedia.org/wiki/Tannin 10/6/2011 20. Anne Grette Byskov, Mogens Baltsen, Claus Yding Andersen, J Mol Med, 1998,76/818 â&#x20AC;&#x201C; 823.

21. http://en.wikipedia.org/wiki/sapogenin on 10/6/2011 22. http://en.wikipedia.org/wiki/protease on 10/6/2011 23. http://en.wikipedia.org/wiki//L-Dopa on 10/6/2011 and Domeena C. Renshaw, Sex and Drugs, S. Afr. med. J. o 54, 325 (1978).

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LIST OF TABLES Table I) Ingredients and quantity of the trial drug Gokshuradi Churna Sl. No

Drug Name

Botanical name

Part used

Quantity

1.

Gokshura

Tribulus terrestris Linn.

Fruit

1 Part

2.

Kokilaksha

Asteracantha longifolia Nees

Seed

1 Part

3.

Satavari

Asparagus racemosus Willd.

Root

1 Part

4.

Kapikachu

Mucuna pruriens Baker.

Seed

1 Part

5.

Naagabala

Sida cordata (Sida veronicaefolia)

Root

1 Part

6.

Atibala

Abutilon indicum Linn.

Root

1 Part

Table II) Effect of therapy on Sperm count Mean Sperm Count

%

SD

SE

‘t’ value

‘P’ value

1.7

24.44%

0.36579

0.13878

3.9631

P<0.001

2.25

1.35

40.00%

0.83158

0.20925

4.3011

P<0.001

After 90 days

2.25

0.85

62.22%

0.98947

0.22825

6.1336

P<0.001

AFU

2.25

1.2

46.66%

1.20790

0.25219

4.1635

P<0.001

BT

AT

After 30 days

2.25

After 60 days

Table III) Effect of therapy on Sperm motility Mean Sperm motility

%

SD

SE

‘t’ value

‘P’ value

1.8

23.40%

0.2605

0.11712

4.69596

P<0.001

2.35

1.5

36.17%

0.3447

0.13473

6.30903

P<0.001

After 90 days

2.35

1.05

55.32%

0.5368

0.16813

7.73228

P<0.001

AFU

2.35

1.45

38.29%

0.6210

0.18083

4.97698

P<0.001

BT

AT

After 30 days

2.35

After 60 days

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Table IV) Overall results Result

AT

AFU

Complete cure

2

1

Marked improvement

2

1

Moderate improvement

12

6

Mild Improvement

3

9

Unchanged

1

3

Table V) Overall results on the basis of conception in the partner Result

No. of patients

Percentage

Conception after treatment (CAT)

2

10%

No conception after treatment (NCAT)

18

90%

GRAPHS Graph I) Overall results 14 12 10 8 6 4 2 0

12 9 6 2

1

Complete cure

2

3 1

Marked Moderate Mild improvement improvement Improvement

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3 1

Unchanged

AT AFU