International Journal of Applied Research in Natural Products Vol. 1(2), pp. 26-31, June/July 2008 Available online http://www.healthy-synergies.com ©2008 Healthy Synergies Publications

26

Original Article Spermatogenic activity of rhizomes of Curculigo orchioides Gaertn in male rats Chauhan NS and Dixit VK * Department of Pharmaceutical Sciences, Dr. H. S. Gour University Sagar (M.P.) 470003 India Summary: The rhizomes of Curculigo orchioides Gaertn have been traditionally acclaimed as aphrodisiac. In the present study ethanolic extract of rhizomes was evaluated for its effect on orientation behavior and spermatogenesis in albino rats. A change in orientation behavior was assessed by orientation towards female, towards environment, towards self and type of mobility. Administration of 100 mg/Kg b. w. of ethanolic extract had pronounced effect on orientation of male towards the female rats. Males treated with the extract displayed more frequent and vigorous anogenital sniffing and mounting as compared to untreated animals. The increased spermatogenesis in treated group was confirmed by change in histoarchitecture as evidenced by increase in number of spermatocyte and spermatids. These findings support the folk use of this plant as aphrodisiac.

Industrial relevance: Study proposes to elucidate and demarcate the potential of Rasayan herbs in the treatment as well as uprooting the causes of disease; which is the underlying principle of Rasayana therapy. This leads to discovery of newer phytoconstituents with better activities and provide source of new biomolecules for biotechnologists to work on. The study explore the utilization of Rasyana herbs for effective management of sexual dysfunction. It brings out a competent literature on rasayana, validating their utilization. It gives a larger platform for the herbal cultivators by providing scientific support and data to the traditional unvalidated herbal drugs as rejuvenative tonics.

Keywords: Aphrodisiac, Curculigo orchioides, Rasayana drugs, Spermatogenesis

Introduction Rasayana is a unique concept of Ayurveda which means vital nourishment (Rasa + Ayana) representing a holistic

approach, responsible for preventive aspects against ageing as well as curative aspect against diseases (Mehrotra & Ojha 2006). In Ayurveda Vajikarana constitute the medicines which confer upon a man sexual power similar to that of a stallion are called “Vaajkarana” (literally, horse-making) (Sengupta & Nath 1994). There are number of plant derived compounds which are heavily promoted in commercial products for their procedural effects (Rowland & Tai 2003). The rhizomes of Curculigo orchioides Gaertn (Amaryllidaceae) are described in Ayurveda as a Vajikarana rasayana (Chunekar & Yadav 2005). Rhizomes of the plant are used as tonic, demulcent, diuretic and restorative (Chopra et al., 1956). The plant is reported to possess estrogenic (Vijayanarayana et al., 2007), pendiculatiory (Thakur & Dixit 2007), hepatoprotective (Rao & Mishra 1996a, b), immunostimulant (Lakshmi et al., 2003: Bafna & Mishra 2006) and antioxidant (Venukumar & Latha 2002) activities. We showed in a preliminary pharmacological study that ethanolic extract increases sexual behavior and mating performance male rats (Chauhan et al., 2007) and investigated antihyperglycemic activity in alloxan induced diabetic rats (Chauhan & Dixit, 2007). Present investigations were undertaken with a view to explain the effect of extract on spermatogenesis and sexual behavior.

Material and Methods Animal Stock: The protocol for experimentation was approved by Institutional Animal Ethics Committee (Ref. no:

NOEC/DB/365/5) of Dr H. S. Gour University, Sagar, India. Albino rats of either sex weighing 120-150 g were fed on standard diet and water ad libitum. The animals were housed at room temperature (24± 2 0 C) on a reversed day-night cycle (06:00 hrs to 18:00 hrs.).

Plant material: Rhizomes of Curculigo orchioides Gaertn were collected at Sagar M.P. (India) and taxonomically identified at the Department of Botany, Dr H. S. Gour University, Sagar. A voucher specimen of the same has been deposited (No. NSC-CO-2005) at departmental herbarium centre. The dried powdered rhizomes were defatted by extraction with petroleum ether (60-80 0 C). The defatted plant material was then extracted with ethanol (95%), and dried under vacuum (4.08 % w/w). The presence of alkaloids (Dragendroff, Mayer, Wagner and Hagers test), phenolic (Ferric

*Corresponding Author: Tel: +1-91-7582264582 Fax: +1-91-7582264236 E-mail: vkdixit2011@rediffmail.com Accepted 08 April 2008

Aphrodisiac activity of Curculigo orchioides

27

Chloride Test), tannins (Gelatin Solution Test), saponins (Foam test and Haemolysis test) and steroids (Lieberman Burchards Test) were confirmed by qualitative tests( Kokate 2003). The TLC of ethanolic extract in solvent system n-butanol saturated with water and chloroform: acetic acid: methanol: water (16:8:3:2) give 10 and 6 spots respectively (Chauhan 2006).

Preparation of test samples: Ethanolic extract (1%) and sildenafil citrate (0.05%) were suspended in 0.2% of gum acacia and administered orally (p.o) using metal canula. Testosterone propionate was suspended in arachis oil and was administered sub cutaneously (s. c.).

Treatment: The animals were divided in groups of 6 male rats each. Group I animals served as control and received only vehicle i.e. 0.2 % gum acacia suspension. Group II were administered with ethanolic extract of C. orchioides (100mg/kg) daily. Group III animals were given subcutaneously 0.5 mg/Kg dose of Testosterone propionate suspension (Sun Pharma) twice weekly and served as positive control for anabolic and histopathalogical studies. Animals of Group IV received dose of Sildenafil citrate (Hetro Drugs) 5mg/Kg orally daily and served as positive control for behavior studies.

Effect on spermatogenesis: T The method reported by Saksena & Dixit 1987 as modified by Thakur & Dixit. 2006 was used. In brief, after 30 days of treatment the body weights of animals were taken after which the animals of control as well as treated groups were killed by rapid decapitation. Testis were removed and cut into small pieces, fixed in Bovine’s fixative, dehydrated with varying percentage of ethanol for histological studies. Sections were cut (6µ), stained with eosin and analyzed microscopically. Histometric measurements such as diameter of testes, seminiferous tubules and Leydigs cell nucleus were made by random selections of 30 circulars sections by using ocular and stage micrometers. The numbers of different spermatogenic elements were also determined.

Orientation activity: The orientation of male rats towards female (licking, anogenital sniffing), towards self (non-genital grooming, genital grooming), type of immobility (restricted and non restricted) and towards environment (exploration, climbing, raring). The orientation behaviour was observed at 15, 30, 45 and 60 min after treatment to all groups. The severity of response can be scored as 1=presence, 2=moderately severe and 3= intense and continuous action. The cumulative score for each activity in the one-hour was calculated.

Statistical analysis: Results are expressed as mean ± SD. The significance of the data was evaluated using student t-test. The statistical analysis was carried out using Instat 2.1 software.

Results Histopathological studies: The testis section of control group animals showed normal histological texture. The

diameter of semniferous tubules varied within a range. The tubules having maximum diameter, were not abundant and well within range. The cuboidal germinal epithelium exhibited normal shape and size. Sertoli cells had many cytoplasmic processes which were normal in size. Spermatozoa were embedded in the sertoli cells and showed normal cytoplasmic granulation. Leydigs cells had normal nuclear size. Luminal part of the tubule were normal in number with bundles of spermatozoa. Spermatozoa with long tail with small distinct head were more visible (Figure 1 and 4).

The extract treated group animals showed pronounced effects in terms of testis weight and histological alterations. Since the weight and size of the testis was greater in extract treated groups almost all semniferous tubules showed greater diameter. The germinal epithelium cells appeared to be hyperactive. Large numbers of different cells at different stages of spermatogenesis were evident. Lumen of every semniferous tubules had enormous number of spermatozoa. Sertoli cells were enlarged highly processed and rich in nutrients as evidenced by highly granulated cytoplasm. This was the normal response of the sertoli cells when they were in readiness for providing nutritional supplementation to large number of spermatozoa (Majumdar 1995). Almost all leydig cells showed hypertrophy with enlarged nucleus and darkly stained cytoplasm. Increment in the volume of cells and nucleus was strongly suggestive of steroid synthesis under the direct or indirect influence of the drug. Almost all tubules were overcrowded with sperm bundles. In some tubules, spermatids were found scattered amidst spermatozoa. The blood vessels of testis were slightly dilated. Histoarchitecture of testosterone treated group also exhibited similar profile. Increased spermatogenesis was evident by high number of spermatozoa in semniferous tubules and which is evident by increase in spermatogenic elements as compared to control (Figure 2, 3, 5 and 6 and Table-2).

Orientation activities: Orientation activities (Table - 1) studies show that with treatment of the extract there is increase in attraction of male towards female. Increasing mounting frequency and anogenital sniffing was noticed as compared to control .The attraction towards environment is more in control than drugs treated group. There is also increase in attraction towards female and genital grooming in treated rats which is comparable with standard treatment.

Discussion The present investigations bring forth the spermatogenic activity of ethanolic extract of rhizomes of C. orchioides in

albino rats. Spermatogenesis involves a complex interplay between the structural elements of testis and the endocrine system. Hypothalamic gonadotrophic releasing hormone induces pituitary gonadotrophin (McLachlan 2000).

Abundance of spermatozoa in seminiferous tubule clearly indicates spermatogenesis which is regulated by hormone (Hadziselimovic & Herzog 1997. Hypertrophy of Leydigs cells is also suggestive of steroids synthesis. Earlier phytochemical investigations have shown the presence of glycoside, saponins and sterols (Rao et al., 1978, Xu et al., 1992) in rhizomes. It is likely that these steroidal constituent increase the steroidogenesis and elevate androgen levels which results in observed effect. Involvement of hypothalmo-pitutary axis by way of FSH stimulation can not be ruled out. Rhizome has been demonstrated to have antioxidant properties in-vitro and in-vivo (Bafna & Mishra 2005,

Chauhan and Dixit

28

Table 1. Effect of Curculigo orchioides on Orientation Activities

Orientation Towards Mean activity Score Control group Extract group Standard group

Female

Mounting 21 74 90 Licking 20 48 49 Anogential smelling 27 36 48 Environment Exploration 44 23 27 Rarring 58 28 11 Climbing 52 26 25

Self

Nongential grooming 26 25 12 Gential grooming 24 40 58 Mobility Restricted 16 31 60 Not restricted 24 21 16

Scored Point 1=presence, 2=moderately severe and 3= intense and continuous action.

Table-2. Effect of ethanolic extract of C. orchioides on spermatogenic element in male rats

S. No Size of Seminiferous tubules (µm) Number of spermatogenic elements

Length Breadth Spermatogonia Spermatocyte Control Group

240.2±48.9

110.4±29.4

17.56±2.67

72.26±4.77

Extract Group (100 mg/kg) 308.8±41.5** 108.0±29.9** 12.56±1.99** 107.46±6.07** Testosterone Group (0.5 mg/kg) 336.8±76.5** 116.0±23.0** 12.66±1.66** 118.16±5.31**

Values are Mean ± S.D; p < .001; All treated groups are compared with control group

Figure 1: Histoarchitecture of testis section of control group X 100. LC = Leydig’s cell; SGM = Spermatogonia; SB = Sperm bundles; ST= Seminiferous tubules S= Space

Aphrodisiac activity of Curculigo orchioides

29

Figure 2: Histoarchitecture of testis section of Ethanolic extract treated group at X 100. LC = Leydig’s cell; GE = Germinal epithelium; SGM = Spermatogonia; SB = Sperm bundles; ST= Seminiferous tubules

Figure 3: Histoarchitecture of testis section of Testosterone treated group at X 100. LC = Leydig’s cell; GE = Germinal epithelium; SGM = Spermatogonia; SB = Sperm bundles; ST= Seminiferous tubules

Figure 4: Histoarchitecture of testis section of control group X 450. GE = Germinal epithelium; SGM = Spermatogonia; SB = Sperm bundles; 1PS = Primary spermatocyte; 2PS = Secondary spermatocyte ; ST= Seminiferous tubules

Chauhan and Dixit

30

Figure 5: Histoarchitecture of testis section of C. orchioides ethanolic extract treated group at X 450. LC = Leydig’s cell; BV = Blood vessels; GE = Germinal epithelium; SGM = Spermatogonia; SC = Serotoli cells; SB = Sperm bundles; 1PS = Primary spermatocyte; 2PS = Secondary spermatocyte SS = Spermitids

Figure 6: Histoarchitecture of testis section of Testosterone treated group at X 450. LC = Leydig’s cell; SGM = Spermatogonia; SB = Sperm bundles; 1PS = Primary spermatocyte

Venukumar & Latha 2002, Tang et al., 2004). The phenol and phenolic glycoside shows antioxidative property (Wu et

al., 2005). These antioxidant defence systems are of major importance because peroxidative damage is currently regarded as the single most important cause of impaired testicular function underpinning the pathological consequences of a wide range of conditions from testicular torsion to diabetes and xenobiotic exposure (Aitken & Roman 2007). In a normal situation, the antioxidant mechanisms present in the reproductive tissues and their secretions are likely to quench these reactive oxygen species (ROS) and protect against oxidative damage to gonadal cells and mature spermatozoa (Sikka SC 2001). Antioxidant compounds also alter androgen level (Islam et al., 1991) and change in androgen level like testosterone may be responsible for spermatogenesis.

Reduced number of spermatozoa, mal formed spermatozoa or their reduced or insufficient motility are the leading causes of disturbed fertility or infertility in patient. The drug may thus provide an alternative for management of infertility due to reduced spermatogenesis. Further studies are necessary to elucidate the compounds of the ethanolic extract is responsible for enhances spermatogenesis in rats.

Acknowledgments Authors gratefully acknowledge Hetro Drugs for providing Sildenafil citrate as a gift sample. One of the authors

Nagendra S.Chauhan is also thankful to UGC Major Research Project for providing financial support. References

Aitken RJ and Roman Shaun D. 2007. Antioxidant Systems and Oxidative Stress in the Testes. In: Cheng CY, editor. Molecular Mechanisms in Spermatogenesis. Landes Bioscience p 1.

Aphrodisiac activity of Curculigo orchioides

31

Bafna AR, Mishra SH. 2006. Immunostimulatory effect of methanol extract of Curculigo orchioides on immunosuppressed mice. J Ethnopharmacol 104:1–4.

Bafna AR, Mishra SH. 2005. In vitro antioxidant activity of methanol extract of rhizomes of Curculigo orchioides Gaertn. Ars Pharm 46 (2):125-138.

Chauhan NS. 2006. Phytochemical and Pharmacological Studies on Curculigo orchioides Gaertn [Dissertion]. Sagar: Dr. H. S. Gour University. p 15-25.

Chauhan NS, Rao CV, Dixit VK. 2007. Effect of Curculigo orchioides rhizomes on sexual behaviour of male rats. Fitoterapia 78:530-534.

Chauhan NS, Dixit VK. 2007. Antihyperglycemic activity of the ethanolic extract of Curculigo orchioides Gaertn. Phcog Mag. 3(12):237-240.

Chopra R.N.,. Nayar S.L, Chopra I.C. 1956. Glossary of Indian Medicinal Plants., New Delhi, India CSIR p. 84 Chunekar KC, Yadav CL. 2005. Medicinal plants of Sushruta Samhita. Vol.1. New Delhi : Vedam ebooks pvt Ltd. P.

201. Hadziselimovic F, Herzog B. 1997. Treatment with a Luteinizing Hormone-Releasing Hormone Analogue after

Successful Orchiopexy Markedly Improves the Change of Fertility Later in Life The Journal of Urology 158(3):1193-1195.

Islam MW, Tariq M, Ageel, AM, Al-said MS, Al-Yhya AM. 1991. Effects of Saliva haematodes on sexual behaviour of male rats. J Ethanopharmacol 33:67-72.

Kokate CK. 2003. Practical Pharmacognosy. IV ed. New Delhi: Vallabh Prakashan. 107-111p. Lakshmi V, Pandey K, Puri A, Saxena R.P, Saxena KC. 2003. Immunostimulant principles from Curculigo orchioides. J

Ethnopharmacol 89:181–184. McLachlan RI. 2000. The endocrine control of spermatogenesis. BaillieÁ re's Clinical Endocrinology and Metabolism

14(3):345-362. Majumdar SS, Tsuruta J, Griswold MD, Bartke A. 1995.Isolation and Culture of Sertoli Cells from the Testes of Adult

Siberian Hamsters: Analysis of Proteins Synthesized and Secreted by Sertoli Cells Cultured from Hamsters Raised in a Long or a Short Photoperiod. Biol Reprod 52: 658-666.

Mehrotra NN, Ojha SK. 2006. Ayurvedic rasayana therapy and rejuvenation(Kaya Kalp). Current R&D Highlight Jan-Mar:6-10.

Rao KS, Mishra SH. 1996a. Studies of Curculigo orchioides Gaertn for anti-inflammatory and hepatoprotective activities. Indian Drugs 33(11):20-25.

Rao KS, Mishra SH. 1996b. Effect of rhizomes of Curculigo orchioides Gaertn. on drug induced hepatoxicity. Indian Drugs 33(9):458-461.

Rao RVK, Ali N and Reddy MN. 1978. Occurrence of both Sapogenin and alkaloid Lycorine in Curculigo orchioides, Indian J Pharma Sci 40:104-105.

Rowland D, Wendi T. 2003. A review of plant-derived and herbal approaches to the treatment of sexual dysfunctions. J Sex Mari Thera 29(3):185–205.

Saksena S, Dixit VK. 1987. Role of total alkaloids of Mucuna pruriens Baker in Spermatogenesis in Albino Rats. Indian J Nat Prod 2:3-7.

Sengupta K, Nath N.1994. The Ayurvedic system of medicine,vol-II. 2nd Indian reprint, New Delhi: Logos press. 773p. Sikka SC. 2001. Relative Impact of Oxidative Stress on Male Reproductive Function. Curr Medici Chem 8: 851-862. Tang SY, Whiteman M, Peng ZF, Jenner A, Yong EL, Halliwell B. 2004. Characterization of antioxidant and

antiglycation properties and isolation of active ingredients from traditional chinese medicines. Free Radical Biology and Medicine 36: 1575–1587.

Thakur M, Dixit VK. 2006. Effect of Chlorophytum borivilianum on androgenic and sexual behavior in male rats. Indian drugs 43(4): 300-306.

Thakur M, Dixit VK. 2007. Effect of some vajikaran herbs on pendiculation activities and in vitro sperm count in male. Sexu Disabi 25(4): 203-207.

Venukumar MR and Latha MS. 2002. Antioxidant activity of Curculigo orchioides in carbon tetrachloride induced hepatopathy in rats. Indian J Clini Biochem17(2): 80-87.

Vijayanarayana K, Rodrigues RS, Chandrashekhar KS, Subrahmanyam EVS. 2007. Evaluation of estrogenic activity of alcoholic extract of rhizomes of Curculigo orchioides. J Ethnopharmacol 114(2): 241-245.

Wu Q, Fu DX, Hou AJ, Lei GQ, Liu ZJ, Chen JK, Zhou TS. 2005. Antioxidative phenols and phenolic glycosides from Curculigo orchioides. Chem Pharm Bull 53(8): 1065-7.

Xu JP, Xu RS, Li XY. 1992. Glycosides of Cycloartane sapogenin from Curculigo orchioides. Phytochemistry 31: 233–236.