Antioxidant mediated ameliorative steroidogenesis by Commelina benghalensis L. and Cissus...

Pesticide Biochemistry and Physiology 109 (2014) 18–33

Contents lists available at ScienceDirect

Pesticide Biochemistry and Physiology

journal homepage: www.elsevier .com/locate /pest

Antioxidant mediated ameliorative steroidogenesis by Commelinabenghalensis L. and Cissus quadrangularis L. against quinalphos inducedmale reproductive toxicity

0048-3575/$ - see front matter � 2014 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.pestbp.2014.01.002

⇑ Corresponding author. Fax: +91 431 2407045.E-mail address: [email protected] (S. Achiraman).

Palanivel Kokilavani a, Udhayaraj Suriyakalaa a, Perumal Elumalai b, Bethunaicken Abirami a,Rajamanickam Ramachandran a, Arunachalam Sankarganesh a,c, Shanmugam Achiraman a,⇑a Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli 620 024, Tamilnadu, Indiab Department of Endocrinology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai 600 113, Indiac Department of Bio Sciences and Technology, VIT University, Vellore 632 014, India

a r t i c l e i n f o

Article history:Received 18 December 2013Accepted 14 January 2014Available online 24 January 2014

Keywords:QuinalphosC. benghalensisC. quadrangularisAntioxidantsTestosteroneOxidative stress

a b s t r a c t

Quinalphos (QP) is speculated to cause endocrine disruption through the generation of reactive oxygenspecies (ROS) by oxidative stress (OS). Exposure of QP decreased testosterone level considerably whichresulted in reduced viable sperms in mice. The QP induced toxicity is initiated by the formation of freeradicals as it is evidenced from the increased Lipid peroxidation (LPO) and diminution of antioxidantenzymes in testicular tissue. Increased serum cholesterol and reduced testicular cholesterol indicatedthe inhibition of cholesterol transport and biosynthesis in testicular tissues. Lack of cholesterol in testic-ular tissue impaired the steroidogenesis by down-regulating the expression of StAR protein, CytochromeP450, 3b-HSD and 17b-HSD leading to reduced testosterone level. Treatment of Commelina benganlensis(CBE) and Cissus quadrangularis (CQE) significantly recovered the alterations in antioxidant profiles aswell as increased LPO, thereby recovering the decreased mRNA expression levels of intermediateenzymes. However, CQE effectively protected the OS and prevented the inhibition of steroidogenesisthereby preventing male infertility.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction

A number of recent medical reports have indicated the adversetrends in human health during the last decade. Male reproductivesystem is one of the severely affected organ systems in humanbody. Toxic damage in the testes will lead to reduced sperm count,defective spermatozoa and impaired androgen production. A num-ber of reports have cited the adverse effect of various environmen-tal toxicants on reproductive health in human and experimentalanimals [1–4]. Several reports have indicated that the use of wide-spread chemicals alter the hormonal balance, hence called as endo-crine disruptors (EDCs). Endocrine disruptors are estrogen-likeand/or anti-androgenic chemicals in the environment that havepotentially hazardous effects on male reproductive axis resultingin infertility and on other hormonal dependent reproductive func-tions causing erectile dysfunction (ED). These toxic effects proba-bly occur, because they (i) mimic natural hormones, (ii) inhibitthe action of hormones, and/or (iii) alter the normal regulatoryfunction of the endocrine system. Endocrine function can be dis-rupted through (a) the generation of Reactive Oxygen Species

(ROS) by oxidative stress, (b) impairment of intracellular transportof biomolecules (c) inhibition in the expression of steroidogenicenzymes which are involved in hormone (testosterone) productionwhich finally lead to hormonal imbalance (endocrine disruption)and disruption of germ cell differentiation.

Many pesticides are now suspected of being EDCs that can leadto an increase in birth defects, sexual abnormalities and reproduc-tive failure. Endocrine disrupting pesticides are lipophilic (fat lov-ing), resistant to metabolism, and able to bioconcentrate up thefood chain. This is because these substances become stored in bodyfats and can be transferred to the developing offspring via the egg.Many pesticides have now been found to have estrogenic or anti-androgenic activity. Organophosphate insecticides (OPIs) adverselyaffect the testicular functions by affecting the steroidogenesis; de-crease the weights of testes and its accessory glands thereby lead-ing to male infertility [5–7]. OPIs induce oxidative stress (OS) anddecrease the level of antioxidant enzymes and cause testiculardamages [8]. OS is the primary mechanism of OPIs, which increasesthe level of LPO [9]. Crucially, OPIs compounds block both adreno-corticotropin- and cAMP-stimulated steroidogenesis [10]. More-over, exposures to environmental toxicants particularly OPI havethe potential to reduce the level of testosterone, decrease thesperm count and testicular ascorbic acid which adversely affects

http://crossmark.crossref.org/dialog/?doi=10.1016/j.pestbp.2014.01.002&domain=pdf

http://dx.doi.org/10.1016/j.pestbp.2014.01.002

mailto:[email protected]

http://dx.doi.org/10.1016/j.pestbp.2014.01.002

http://www.sciencedirect.com/science/journal/00483575

http://www.elsevier.com/locate/pest

P. Kokilavani et al. / Pesticide Biochemistry and Physiology 109 (2014) 18–33 19

normal sexual development in humans and spermatogenesis[11,12]. OPIs impair the steroidogenesis via disrupting the StARprotein and increase the number of abnormal sperms [13,14].

Quinalphos, (QP: O, Odiethyl O-2 quinoxalinoxalinyl phosph-arothionate) is an OPI that contaminates our ecosystem widelydue to its application as an insecticide and acaricide in controllingthe pests of a variety of crops in India to replace conventional orga-nochlorine pesticides [15]. Earlier findings indicated that the QPexposure reduced the antioxidant enzymes in testes through thegeneration of ROS and elevated the level of LPO [16]. In rats, QP ad-versely affects the activity of testicular steroidogenic enzymes andcauses massive degeneration of germ cells and reduction in spermcount [17]. QP and its metabolites affect the testicular enzymesand sperm count, cause damage to germ cells and sertoli cells[18] and induce P450 oxidation [19]. In a preliminary study ithas been shown that oral exposure of QP at the higher dose levelincreases the quantity of abnormal sperm [20].

During steroidogenesis the substrate cholesterol is convertedinto testosterone through a series of steroidogenic steps catalyzedby different enzymes [21]. Numerous reports indicate that steroi-dogenic acute regulatory (StAR) protein mediates steroidogenesis[22]. StAR is a 30 kDa protein which increases the movement ofcholesterol from the outer to the inner mitochondrial membrane[23]. Role of ROS in QP induced testicular toxicity mainly on testos-terone level was documented well [19]. However, the direct effectsof QP on StAR protein activity and mRNA expression of steroido-genic enzymes are still unknown.

Recently, considerable emphasis has been made on natural foodsources as preventive and therapeutic agents for the treatment ofreproductive toxicity. Green leafy vegetables have greater antioxi-dant potential, which have been well studied and proved throughfree radical scavenging activities [24]. Plant extracts are rich sourceof phenolic compounds and are commonly found in edible plants.They have been reported to have multiple biological effects, includ-ing antioxidant activity [25]. Several studies have reported the po-tential effect of herbs and fruits in recovering environmentaltoxicants induced rigorous damages in vital organs such as liver,kidney and testes via antioxidants activity [26–28]. Studiesshowed that a number of plant products including phytosteroidsand polyphenolic substances (e.g. flavonoids, alkaloids and tan-nins) exert antioxidant actions [29–32]. In the same way, we havechosen Commelina benghalensis and Cissus quadrangularis which aremedicinally important, prominent edible item and traditionallyused for treating infertility.

C. benghalensis (CBE) is a perennial herb native to tropical Asiaand Africa. In the Indian subcontinent it is widely used as a folkmedicine for the treatment of leprosy, headache, fever, constipa-tion, and jaundice [33]. The whole plant is reported to contain alka-loids, volatile oil, wax [34], vitamin-C and higher levels of bothlutein and b-carotene [35]. The phytochemical screening of CBE re-vealed the presence of carbohydrates, tannins, glycosides, volatileoils, resins, balsams, flavonoids and saponins [36]. Compoundssuch as n-octacosanol, n-triocotanol, stigma-sterol, compesterol,hydrocyanic acid have been reported. A recent study suggestedthat CBE extract improves antioxidant status and protect liveragainst oxidative stress [37]. Few studies reported its use in thetreatment of female infertility in ethnomedicine [38,39]. CBE is re-ported to possess antimicrobial, antioxidant activities, sedative andanxiolytic properties [40,41]. These observations provided a credi-ble scientific justification upon the ethnopharmacological utiliza-tion of CBE [42].

C. quadrangularis Linn. (CQE) is an ancient medicinal plant na-tive to India. It is an edible plant, commonly known as ‘‘bone set-ter’’ found in high temperature regions of India. Phytochemicalscreening of the plant has revealed high contents of ascorbic acid,carotene, anabolic steroidal substances, and calcium. The stem

contains two asymmetric tetracyclic triterpenoids, and two steroi-dal principles. The presence of b-sitosterol, d-amyrin, d-amyrone,and flavanoids (quercetin) having different potential metabolicand physiological effects have also been reported [43–45]. Thisplant plays a role in bone healing and possesses antimicrobial,antiulcer, antioxidative, cholinergic activity and holds beneficialeffect on cardiovascular diseases [46,47]. Stem of CQE is used inthe form of paste to treat infertility in Siddha medicine [48]. How-ever, there is no scientific data to validate the significant role ofCQE on male fertility.

Since humans are exposed to QP in day to day life, it is specu-lated that it might cause endocrine disruption particularly inreproductive hormones. In fact, few reports indicated that theseQP cause hormonal imbalance. However, the mechanism by whichthe pollutants cause damage to the male reproductive physiologyis unclear. Hence, a thorough study is warranted. Few studies indi-cated that CBE and CQE contain higher concentration of antioxi-dants as well as steroid contents which are essential fortesticular function. Hence, it is hypothesized that the toxicity in-duced by QP could be prevented or restored by providing the plantextracts as food supplements. Therefore, the present study was de-signed to (i) to explore the toxic effects of QP on endocrine disrup-tion and following consequences leading to male reproductivetoxicity (ii) to elucidate the possible mechanism by which QP af-fects steroidogenesis, understanding its toxicity on testicular Ley-dig cell function and (iii) to validate the protective and curativepotential of C. benghalensis L. and C. quadrangularis L. on QP in-duced mice.

2. Materials and methods

2.1. Chemicals

Quinalphos (QP) (25% Ekalux) was purchased from Syngenta,Mumbai, India. 5,5-Dithio-bis-2-nitrobenzoic acid (DTNB), thiobar-bituric acid (TBA), xanthine oxidase, ethylenediamine tetra aceticacid (EDTA), hydrogen peroxide (H2O2), trichloroacetic acid, 2,4-dinitrophenyl hydrazine were obtained from Merck, Himedia andSigma aldrich. All other reagents used were of analytical grade.Goat polyclonal anti-b-actin antibody was purchased from Sig-ma–Aldrich Pvt. Ltd., USA. Rabbit polyclonal anti-StAR (FL-285)was procured from Santa Cruz Biotechnology, Inc. Europe. The sec-ondary antibody, horseradish peroxidase (HRP) conjugated goatanti-rabbit IgG was obtained from GENEI, Bangalore, India. Metha-nol and all other chemicals were purchased from Sisco ResearchLaboratory (SRL), Mumbai, India.

2.2. Animals

Healthy adult male Swiss albino mice of Wistar strain Mus mus-culus weighing 30–35 g (60 days old) were purchased from TANU-VAS, Chennai, India. The animals were housed in a controlledenvironment at 23 ± 1 �C with alternating 12 h light–dark cyclesand a relative humidity of 50 ± 5%. The animals were given free ac-cess to pelleted feed (Sai Durga Feeds Ltd., Bangalore) and water.After one week of adaptation period, experiments were started.All experiments conducted were in accordance with the guidelinesfor animal care by the Institutional Animal Ethics Committee(IAEC) (BDU/IAEC no. 2012/36/28.03.2012), Bharathidasan Univer-sity, Tiruchirappalli, India.

2.3. Preparation of plant extracts

2.3.1. Cold water extraction5 g of powdered plant materials were dissolved in distilled

water (100 ml). Then it was kept in mechanical shaker for 48 h.

20 P. Kokilavani et al. / Pesticide Biochemistry and Physiology 109 (2014) 18–33

The extract was filtered through filter paper using Buchner funnel.The filtrate was quickly frozen at –20 �C and dried for 48 h usingvacuum freeze dryer (CHRIST ALPHA, Germany). The resulting ex-tract was reconstituted with distilled water to obtain desired con-centration and used for further analysis.

2.4. Experimental design

The animals were divided into 9 groups randomly and eachgroup contained 6 animals (n = 6). Mode of administration is oraland the period of treatment is 7 days. Group 1 was served as con-trol (Water), group 2 mice were orally treated with QP alone(7.5 mg/kg BW for 7 days), groups 3 and 4 mice received plant ex-tract alone (CBE 400 mg/kg BW, CQE 350 mg/kg BW, respectively).Groups from 5 to 8 mice were treated with QP along with plant ex-tract. Groups 5 and 6 were served as protective (QP and simulta-neous treatment of CBE) and curative groups of CBE (7 days QPtreatment followed by next 7 days CBE treatment). Likewise,groups 7 and 8 mice were used for CQE protective (QP and simul-taneous treatment of CQE) and curative treatment (7 days QPtreatment followed by next 7 days CQE treatment). Group 9 wasused as withdrawal. The dose of CBE, CQE and QP was determinedbased on the previous studies [16,41,49].

2.5. Body weight measurement, blood and organ collection

Body weight was measured at the beginning and at the end ofthe experimental period with the use of electronic balance. Atthe end of the experimental period, animals were overnight fastedand were euthanized by decapitation under deep anesthesia withdiethyl ether inhalation. Blood was collected by cardiac punctureand allowed to clot at room temperature. Serum was collectedfrom the clotted blood by centrifugation (Cooling centrifuge –REMI) at 1500g for 15 min at 4 �C and stored at �80 �C until anal-yses. Serum was used for hormone assay and cholesterol analysis.Organs such as testis, epididymis, seminal vesicle and prostatewere recovered and their weights were recorded. Testes and epi-didymis was processed for further analysis.

2.6. Serum analysis

The level of testosterone and LH was measured by Accu bindELISA (Enzyme linked immuno sorbent assay) microwells standardkit (Monobind Inc., USA).

2.7. Semen analysis

2.7.1. Sperm countSperm counts were made according to Gopalakrishnan et al.

[50]. Semen sample was diluted to 20� by adding diluting medium(50 g of sodium bicarbonate in 10 ml of 40% formalin). High(>100 � 106 ml�1) density semen samples would require furtherdilution while low (<10 � 106 ml�1) density samples would re-quire lesser dilution. Semen sample (10–15 ll) was loaded into aNeubauer counting chamber and the number of sperms in the cen-tral square was counted.

2.7.2. Sperm viabilitySperm viability was done by eosin-nigrosin staining solution.

An intact cell membrane does not take up the red stain eosin while,dead cells (i.e. one with damaged cell membrane) takes it. Nigrosinis used as a background stain to provide contrast for the unstained(White) live cells. A drop (50 ll) of semen was mixed with eosin-nigrosin staining solution and incubated for 30 s and smeared overa microscopic slide. Mounted smears are stored at room tempera-ture. Spermatozoa are assessed at 100� magnification under oil

immersion objective (Kohler illumination). Spermatozoa that arewhite (unstained) are classified as ‘‘live’’ and those that show anypink or red coloration are classified as ‘‘dead.’’ Sperm viabilitywas expressed as percentage.

2.8. Testicular tissue analysis

200 mg of testis was taken and homogenized with 1 ml of lysisbuffer (pH 7.4), centrifuged at 10,000�g for 15 min at 4 �C. Super-natant was used for further analysis. Protein concentration wasestimated by using Bradford reagent (Sigma, Cat # B6916). The col-or intensity was measured at 595 nm.

2.8.1. Estimation of lipid peroxidation (LPO)Level of LPO was determined by measurement of malondialde-

hyde (MDA). MDA was estimated according to the method of Ohk-awa et al. [51]. Briefly, the reaction mixture consisted of 0.3 M TrisHCl buffer (pH 7.4), 2 mM sodium pyrophosphate, 0.2 ml of tissueextract and 10% TBA was incubated at 37 �C with constant shakingfor 30 min. 1 ml of 10% TCA was added to arrest the reaction andmixed well. The mixture was kept in boiling water bath for20 min and centrifuged for 5 min at 2000 rpm. Standard tubes con-taining 10, 20, 30, 40, and 50 nmol/ml were also run simulta-neously. The tubes were centrifuged and the color developed wasmeasured at 532 nm. MDA content of the sample is expressed asnanomoles of MDA formed per milligram protein.

2.8.2. Determination of glutathione peroxidase (GPx)The activity of GPx was determined by the method of Rotruck

et al. [52]. This method depend on the reaction of reduced glutathi-one with dithio-bis-nitrobenzoic acid solution (DTNB), the yellowcolor developed was measured spectrophotometerically at412 nm against blank. GPx was done on the basis of the oxidationof NADPH to NADP+. In brief, the reaction mixture contained 0.4 Msodium phosphate buffer (pH 7), 10 mM sodium azide, 4 mM re-duced glutathione, 2.5 mM H2O2, and 0.1 ml of tissue extract. Thevolume was made up to 2.0 ml with distilled water and incubatedat 37 �C for 10 min and the reaction was terminated by the addi-tion of 10% TCA. The mixture was centrifuged at 3000 rpm for3 min. To the supernatant, 0.3 M of disodium hydrogen phosphateand DTNB was added. The color developed was read at 412 nmagainst a reagent blank containing only phosphate solution andDTNB reagent in a spectrophotometer. The enzyme activity is ex-pressed as units per milligram protein (1 U is the amount of en-zyme that converts 1 lmol reduced glutathione (GSH) to GSSG inthe presence of hydrogen peroxide/min).

2.8.3. Determination of superoxide dismutase (SOD)SOD was estimated by the method of Marklund and Marklund

[53]. The amount of SOD present in the sample was measured onthe principle in which xanthine reacts with xanthine oxidase togenerate SOD. To 0.1 ml of tissue extract, 0.25 ml of absolute alco-hol, 0.15 ml of chloroform and 1 ml of distilled water was added.The mixture was kept in a mechanical shaker for 15 min and thesuspension was centrifuged at 2500 rpm for 15 min. To the super-natant 2 ml of tris EDTA buffer (pH 8.2) and 0.5 ml of pyrogallol(2 mM) was added. The samples were immediately read at470 nm against blank containing all components except the en-zyme and pyrogallol at intervals of 1 min, for 3 min on a spectro-photometer. The enzyme activity is expressed as units permilligram protein.

2.8.4. Determination of catalase (CAT)Estimation of CAT was done according to the method of Sinha

[54]. This assay is based on the determination of the rate constantof hydrogen peroxide decomposition by CAT enzyme. In short, the

Table 1Details of primers employed and expected size of the PCR-amplified cDNA.

Sequence of the primer Productsize (bp)

No. ofcycle

StAR F-50-CCTGGTGGGGCCTCGAGACTT-30

R-30-CTTCGGCAGCCACCCCTTCAG-50229 32

CytochromeP450

F-50CGGGCGGGCTCTTCCTTTGG-30

R-30-GGGGATTGGCTGTGCGGTCC-50230 32

3ß-HSD F-50-GCTGGCAGCCAATGGGAGCA-30

R-30-GGGTTTCGAAGGCCCCTGGC-50231 32

17ß-HSD(III)

F-50-GGGTTATGCAGCCAAACTGT-30

R-30-GCTGACTAGCCCAGTGAAGG-50340 32

RPS16 F-50-CGCGCACGCTGCAGTACAAG -30

R-30-CCCCGAACCTTGAGATGGGC-50331 32


assay mixture contained 0.2 M H2O2, 0.01 M sodium phosphatebuffer (pH 7), and distilled water. The reaction was initiated byadding 0.1 ml tissue extract and dichromate–acetic acid reagentwas added at 15, 30, 45, and 60 s, to arrest the reaction. Tubes werethen heated for 10 min and allowed to cool, the green color devel-oped was read at 590 nm against blank containing all componentsexcept the enzyme in a spectrophotometer. The activity of CAT isexpressed as units per milligram protein (1 U is the amount of en-zyme that utilizes 1 lmol hydrogen peroxide/min).

2.8.5. Estimation of nonenzymatic antioxidantAscorbic acid (vitamin C) was estimated according to the meth-

od of Omaye et al. [55]. Ascorbic acid was oxidized by copper toform dehydro ascorbic and diketoglutaric acid. These productswhen treated with 2,4-dinitrophenyl hydrazine (DNPH) forms thederivative bis-2,4-dinitrophenyl hydrazone. Thiouria provides amild reducing medium, which helps to prevent interference fromacetic acid chromogens. 0.5 ml of tissue homogenate was mixedthoroughly with 6% TCA and centrifuged for 20 min at 3500 rpm.To the supernatant, DNPH reagent was added, mixed well andincubated at 37 �C for 3 h. Placed in ice-cold water and added2.5 ml of 85% sulphuric acid and allowed to stand for 30 min. Aset of standard containing 10–50 lg of ascorbic acid were takenand processed along with a blank containing 0.5 ml of 4% TCA.The color developed was read at 530 nm. Ascorbic acid values areexpressed as lM/mg of tissue.

2.8.6. Estimation of lactate dehydrogenase (LDH)The level of LDH was estimated by the method of Ringoir and

Plum [56]. 1 ml of substrate (lithium lactate – 21.9 ml of glycine,13.1 ml of 0.1 NaOH and 0.7 g of lithium lactate) was added to0.1 ml of sample and then the tubes were incubated at 37 �C for10 min. To this, 0.2 ml of NAD was added, shaken well and againincubated at 37 �C for 15 min followed by adding 0.4 N NaOH.Absorbance was read at 450 nm.

2.8.7. Estimation of cholesterolTotal cholesterol was determined by the method of Zlatkis et al.

[57]. Briefly, to the serum/lipid extract, ferric chloride acetic acidreagent (0.05%) was added and allowed to stand for 15 min andthen centrifuged. To the supernatant, conc. H2SO4 was added andthe color developed was read at 560 nm after 20 min against a re-agent blank. A set of standards were also performed in the similarmanner. Values are expressed as mg/100 g in tissue and mg/dl inserum.

2.8.8. Assay of testicular steroidogenic enzyme activities2.8.8.1. Determination of 3b-HSD enzyme activity (3b-HSD). Theactivity of 3b-HSD was determined by the method described byBergmeyer [58]. In brief, testicular tissue was homogenized inice-cold Tris–HCl buffer (pH 7.2) and centrifuged at 16,000 rpmfor 5 min at 4–8 �C. The supernatant was used as enzyme extractfor the assay of 3b-HSD. The reaction mixture contained 0.6 ml ofpyrophosphate buffer (100 mM), 0.2 ml of NAD (0.5 mM), 2 ml ofdistilled water, and 0.1 ml of dehydroisoandrosterone (0.1 mM).The absorbance at 340 nm was measured immediately after theaddition of enzyme extract at 20-s intervals for 5 min in a spectro-photometer against blank.

2.8.8.2. Determination of 17b-HSD enzyme activity (17b-HSD). Theactivity of 17b-HSD was determined by the method of Bergmeyer[58]. In brief, testicular tissue was homogenized in ice-cold Tris–HCl buffer (pH 7.2) and centrifuged at 10,000 rpm for 5 min at 4–8 �C. The supernatant was used as enzyme extract for the assayof 17b-HSD. The reaction mixture contained 0.6 ml of pyrophos-phate buffer (100 mM), 0.2 ml of NADPH (0.5 mM), 2 ml of distilled

water, and 0.1 ml of 1,4-androstenedine-3,17-dione (0.8 mM). Theabsorbance at 340 nm was measured immediately after the addi-tion of enzyme extract at 30-s intervals for 5 min in a spectropho-tometer against blank.

2.8.9. Reverse transcriptase-polymerase chain reaction (RT-PCR)analysis of testicular gene expressions

Total RNA from the whole testis was isolated using TRIZOL (TRI)reagent single-step kit method (one step RNA reagent product(BS410A), Bio Basic Inc., Canada). The RNA purity and concentra-tion were determined spectrophotometerically at A260/A280 nm.The purity of RNA obtained was 1.7–1.9. 1.5 lg of total RNA wasreverse transcribed by using Genet Bio RT-PCR kit (Suprime ScriptRT premix, SR – 5001) according to the manufactures protocol. ThecDNAs obtained was used as a template for subsequent PCR ampli-fication using ExPrime Taq™ Premix (2�) kit (GENET BIO). The de-tails of the primers used, number of cycles, and size of the PCRamplified products are listed in Table 1. Five microliters of eachPCR product was analyzed by electrophoresis on a 2% agarosegel. The molecular size of the amplified products (StAR, Cyto-chrome P450scc, 3b-HSD, 17b-HSD, and RPS 16) was determinedby comparing with DNA markers (100–1000 bp DNA ladder) runin parallel with RT-PCR products. The gels were then subjected todensitometric scanning (Bio-Rad) to find out the OD units of eachband and then normalized against that of the internal control(RPS 16).

2.8.10. ImmunoblottingAs many as 45 lg of total protein was mixed with 2� sample

buffer and boiled for 5 min. The sample mixture was run on 12%SDS–PAGE gel in 1� running gel buffer at 100 V for 2.5 h and elec-trotransferred to a PVDF membrane (Millipore, Germany) at 100 Vfor 1 h. The membrane was blocked in blocking buffer containing5% non-fat dry milk powder for overnight. After overnight, theblocked membrane was incubated with rabbit polyclonal anti-StARprotein primary antibody (1:2000). After primary antibody incuba-tion, the membrane was washed thrice with blocking buffer for10 min each. After washing, the blot was incubated with anti-rab-bit antibody conjugated to HRP for 1 h (dilution 1:10,000). The sig-nal detection was visualized using chemiluminescence reagents(ECL) (Enhanced Chemiluminescence, Thermo Scientific, Rockford,IL, USA) by using Chemi Doc Imaging System and the blotted pro-tein was quantified using Quantity one software system (Bio-Rad,USA). Then the membrane was stripped and reprobed to detectgoat polyclonal anti-b-actin (1:1000) and HRP-conjugated anti-rabbit secondary antibody (1:10,000) as internal control.

2.9. Histomorphological analysis

Histology of testis and epididymis was studied by fixing the tis-sues in Bouin’s fluid and adopting the routine paraffin method for


light microscopic studies [59]. 33–5 lm thick transverse or longi-tudinal paraffin sections, as may be applicable of liver, kidney, tes-tis and epididymis were obtained using a rotary microtome (Lieica,jena, Germany) and stained with Harris haematoxylin and eosin,mounted in DPX (Dibutyl phthalate in xylene) mountant and ob-served in a hund wetzlar microscope (Germany). Images were cap-tured through a charge-coupled device (CCD) camera (Canon(PC1356), Canon Inc., made in Japan).

2.10. Statistical analysis

All data were expressed as mean ± SD. The statistical signifi-cance was evaluated by one way analysis of variance (ANOVA)using SPSS (version 17.0, Cary, NC, USA) and the individual com-parisons were obtained by Duncan’s Multiple Range Test. A valueof p < 0.05 was considered to indicate a significant difference be-tween groups.

3. Results

3.1. Effect of C. benghalensis and C. quadrangularis on body andreproductive organs weight of QP intoxicated male mice

3.1.1. Body weightBody weight was significantly decreased in QP exposed mice as

compared to control group. A significant (p < 0.05) increase in bodyweight was seen in both plant extract of protective and curativetreatment. Comparison of protective and curative treatment re-vealed that simultaneous treatment of both plant extracts wasfound to be better than the late treatment (curative). Animals gi-ven plant extracts alone did not show any changes when comparedto the control (Fig. 1).

3.1.2. Weight of reproductive organsThe absolute weight of reproductive organs including testis,

epididymis, seminal vesicle and prostrate were severely decreasedin the QP alone treated mice compared to intact control group,however, the changes in weight was ameliorated to a great extentby the plant extracts treatment (p < 0.05). Concurrent treatment ofplant extracts retrieved more significantly than the late treatment.There was no changes in plant extracts alone supplemented groupscompared to control mice. Among the CBE and CQE treatment, CQEwas found to be more potent than the CBE (Fig. 2).

Fig. 1. Effect of C. benghalensis and C. quadrangularis on body weight of QPintoxicated male mice. All values are mean ± SD. Values with different superscriptsare significantly different among the groups by ANOVA with Duncan’s multiplerange test at p < 0.05. (1) Control, (2) QP treated, (3) CBE alone, (4) CQE alone, (5)QP + CBE (preventive), (6) QP + CBE (curative), (7) QP + CQE (preventive), (8)QP + CQE (curative), (9) withdrawal. All treatments were given orally using oralgavage. [QP: Quinalphos, CBE: C. benghalensis aqueous extract, CQE: C.quadrangularis].

3.2. Effect of C. benghalensis and C. quadrangularis on serumparameters (Testosterone, LH and cholesterol) of QP intoxicated malemice

3.2.1. Level of testosteroneFig. 3a. illustrates the level of testosterone. The level of serum

testosterone decreased to several folds in QP treated mice, com-pared to control. Conversely, the treatment of CBE and CQE notablyretrieved the level of testosterone nearer to normal. However, thesimultaneous treatment of CQE extensively improved (p < 0.05)compared to late treatment as well as CBE treatment, whereas,no difference was observed in mice treated with plant extractsalone when compared with control group. There was no recoveryin group 9.

3.2.2. Level of LHThe Table 2 shows the level of serum LH. There was no signifi-

cant change in LH level after QP exposure.

3.2.3. Level of cholesterolSerum cholesterol level was three fold increased in QP treated

group, compared to control mice. The increased level of cholesterolwas normalized by both plants. Simultaneous treatment of plantextracts maintained the level of cholesterol compared to late treat-ment. Among the two plants, concurrent treatment of CQE sus-tained the cholesterol level compared with post treatment andCBE (Fig. 3b).

3.3. Effect of C. benghalensis and C. quadrangularis on semen analysis(sperm count and viability) of QP intoxicated male mice

Semen quality, in particular, should be considered in a morecomprehensive manner. The assessment of sperm parameters isconsidered an important aspect of identifying and characterizingmale reproductive toxicants. A significant decrease in sperm countwas observed in QP treated mice compared to control mice. In con-trast, the number of sperm was maintained as well as restoredback to the normal by the plant extracts (p < 0.05). There was nomuch difference between the protective and curative treatmentof CBE and CQE. However, comparison of two plants extract, CQEis more effective than the CBE (Fig. 4a). A three-fold reduction inpercentage of viable sperm was observed in QP exposed mice, com-pared to control, whereas, the viability of sperm was significantlyprotected and also restored by the plant extracts (p < 0.05). Amongthe plants, CQE showed significant result than the CBE (Fig. 4b).

3.4. Effect of C. benghalensis and C. quadrangularis on testicularparameters of QP intoxicated male mice

3.4.1. Level of LPO (MDA)Fig. 5 shows the level of lipid peroxidation. A highly significant

increase in the activity of MDA was observed in testis of QP group(group 2), compared to control. In contrast, both protective andcurative treatment of plant extract significantly (p < 0.05) loweredthe level of MDA than the QP group. However, the results demon-strated that simultaneous treatment of CBE and CQE provided sig-nificant protection against the lipid peroxide (LPO) productioninduced by QP, compared to the post treatment. Comparison be-tween CBE and CQE treatment revealed that CQE effectively recov-ered the MDA level to the normalcy.

3.4.2. Antioxidant enzymesFig. 6 depicts the level of antioxidant enzymes. Mice exposed

with QP showed a remarkable reduction in GPx, CAT and SOD levelcompared to control, whereas, simultaneous and post treatment ofplant extracts (CBE and CQE) significantly brought back the level of

Fig. 2. Effect of C. benghalensis and C. quadrangularis on reproductive organs weight of QP intoxicated male mice. (a) Testis weight, (b) epidydimis weight, (c) seminal vesicleweight, (d) prostate weight. All values are mean ± SD. Values with different superscripts are significantly different among the groups by ANOVA with Duncan’s multiple rangetest at p < 0.05. (1) Control, (2) QP treated, (3) CBE alone, (4) CQE alone, (5) QP + CBE (preventive), (6) QP + CBE (curative), (7) QP + CQE (preventive), (8) QP + CQE (curative),(9) withdrawal. All treatments were given orally using oral gavage. [QP: Quinalphos, CBE: C. benghalensis aqueous extract, CQE: C. quadrangularis].

Fig. 3. Effect of C. benghalensis and C. quadrangularis on serum (a) testosterone and(b) cholesterol level of QP exposed male mice. All values are mean ± SD. Values withdifferent superscripts are significantly different among the groups by ANOVA withDuncan’s multiple range test at p < 0.05. (1) Control, (2) QP treated, (3) CBE alone,(4) CQE alone, (5) QP + CBE (preventive), (6) QP + CBE (curative), (7) QP + CQE(preventive), (8) QP + CQE (curative), (9) withdrawal. All treatments were givenorally using oral gavage. [QP: Quinalphos, CBE: C. benghalensis aqueous extract,CQE: C. quadrangularis].

Table 2Effect of C. benghalensis and C. quadrangularis on LH levelof QP administered male mice.

Groups LH(MUI/ML)

Control 0.10 ± 0.03a

QP 0.10 ± 0.01a

CBE alone 0.10 ± 0.05a

CQE alone 0.10 ± 0.02a

QP + CBE (preventive) 0.10 ± 0.04a

QP + CBE (curative) 0.10 ± 0.09a

QP + CQE (preventive) 0.10 ± 0.12a

QP + CQE (curative) 0.10 ± 0.10a

All values are mean ± SD. Values with different super-scripts are significantly different among the groups byANOVA with Duncan’s multiple range test at p < 0.05.

a No significant difference between the groups.


antioxidant enzymes. However, concurrent treatment of CBE sig-nificantly restored when compared to late treatment. In CQE treat-ment, both protective and curative treatment showed similar effect(p < 0.05). Among the CBE and CQE treatment, the level of GPx, CATand SOD was significantly restored by CQE treatment. Among thetwo plants, CQE is known to possess high antioxidant activity thanthe CBE. There is no difference between plant extracts alone trea-ted groups and control.

3.4.3. Level of nonenzymatic antioxidant (vitamin C)Testicular ascorbic acid level was found to be decreased in QP

administered mice, compared to control mice. In contrast, anincreasing trend was noticed in plant extracts given mice(p < 0.05). Both protective and curative treatment of CBE as wellas CQE showed similar results (Fig. 7).

Fig. 4. Effect of C. benghalensis and C. quadrangularis on (a) sperm count and (b)sperm viability of QP exposed male mice. All values are mean ± SD. Values withdifferent superscripts are significantly different among the groups by ANOVA withDuncan’s multiple range test at p < 0.05. (1) Control, (2) QP treated, (3) CBE alone,(4) CQE alone, (5) QP + CBE (preventive), (6) QP + CBE (curative), (7) QP + CQE(preventive), (8) QP + CQE (curative), (9) withdrawal. All treatments were givenorally using oral gavage. [QP: Quinalphos, CBE: C. benghalensis aqueous extract,CQE: C. quadrangularis].

Fig. 5. Effect of C. benghalensis and C. quadrangularis on the level of lipidperoxidation of QP exposed male mice. All values are mean ± SD. Values withdifferent superscripts are significantly different among the groups by ANOVA withDuncan’s multiple range test at p < 0.05. (1) Control, (2) QP treated, (3) CBE alone,(4) CQE alone, (5) QP + CBE (preventive), (6) QP + CBE (curative), (7) QP + CQE(preventive), (8) QP + CQE (curative), (9) withdrawal. All treatments were givenorally using oral gavage. [QP: Quinalphos, CBE: C. benghalensis aqueous extract,CQE: C. quadrangularis].

Fig. 6. Effect of C. benghalensis and C. quadrangularis on the level of testicularenzymatic antioxidants of QP intoxicated male mice. (a) GPx, (b) CAT and (c) SOD.All values are mean ± SD. Values with different superscripts are significantlydifferent among the groups by ANOVA with Duncan’s multiple range test atp < 0.05. (1) Control, (2) QP treated, (3) CBE alone, (4) CQE alone, (5) QP + CBE(preventive), (6) QP + CBE (curative), (7) QP + CQE (preventive), (8) QP + CQE(curative), (9) withdrawal. All treatments were given orally using oral gavage.[QP: Quinalphos, CBE: C. benghalensis aqueous extract, CQE: C. quadrangularis].


3.4.4. Level of LDHThe level of LDH was raised sharply in QP administered mice,

while the level of LDH decreased significantly (p < 0.05) in theplant extract treated animals, when compared with QP exposedmice. Among the protective and curative treatment of plant ex-tract, the simultaneous treatment of CQE showed significant result(Fig. 8).

3.4.5. Level of cholesterolFig. 9 shows the testicular cholesterol level. The level of total

cholesterol of testicular tissue was significantly reduced in QP trea-ted group compared to the control group. However, a significantraise in total cholesterol were recorded in QP combined with plantextracts treated groups comparing to QP group. Protective as wellas curative treatment of CBE and CQE exhibited similar effects. Theactivities of both plants are found to be same.

3.4.6. Steroidogenic enzyme activitiesActivities of 3b-HSD and 17b-HSD was drastically reduced to

several fold after QP exposure when compared to control, whereas,plant extracts treatment retrieved the level of 3b-HSD and 17b-HSD compared to QP treated groups. Comparison between simulta-neous and late treatment revealed that simultaneous treatment

Fig. 7. Effect of C. benghalensis and C. quadrangularis on testicular nonenzymaticantioxidant (vitamin C) of QP intoxicated male mice. All values are mean ± SD.Values with different superscripts are significantly different among the groups byANOVA with Duncan’s multiple range test at p < 0.05. (1) Control, (2) QP treated, (3)CBE alone, (4) CQE alone, (5) QP + CBE (preventive), (6) QP + CBE (curative), (7)QP + CQE (preventive), (8) QP + CQE (curative), (9) withdrawal. All treatments weregiven orally using oral gavage. [QP: Quinalphos, CBE: C. benghalensis aqueousextract, CQE: C. quadrangularis].

Fig. 8. Effect of C. benghalensis and C. quadrangularis on the level of LDH in QPinduced male mice. All values are mean ± SD. Values with different superscripts aresignificantly different among the groups by ANOVA with Duncan’s multiple rangetest at p < 0.05. (1) Control, (2) QP treated, (3) CBE alone, (4) CQE alone, (5) QP + CBE(preventive), (6) QP + CBE (curative), (7) QP + CQE (preventive), (8) QP + CQE(curative), (9) withdrawal. All treatments were given orally using oral gavage.[QP: Quinalphos, CBE: C. benghalensis aqueous extract, CQE: C. quadrangularis].

Fig. 9. Effect of C. benghalensis and C. quadrangularis on testicular cholesterol levelof QP administered male mice. All values are mean ± SD. Values with differentsuperscripts are significantly different among the groups by ANOVA with Duncan’smultiple range test at p < 0.05. (1) Control, (2) QP treated, (3) CBE alone, (4) CQEalone, (5) QP + CBE (preventive), (6) QP + CBE (curative), (7) QP + CQE (preventive),(8) QP + CQE (curative), (9) withdrawal. All treatments were given orally using oralgavage. [QP: Quinalphos, CBE: C. benghalensis aqueous extract, CQE: C.quadrangularis].


sustained the activity of 3b-HSD and 17b-HSD. Among the twoplants, CQE treatment significantly protected the level of 3b-HSD(Fig. 10).

3.4.7. mRNA expression of steroidogenic enzymes and steroidogenicacute regulatory protein (StAR)

Level of mRNA expression of StAR protein and steroidogenic en-zymes (Cytochrome P450, 3b-HSD and 17b-HSD) was significantlyreduced to several folds after QP exposure compared to control,whereas, considerable increase was observed after plant extracttreatment. Co-administration of CBE along with QP protected theexpression of StAR protein and steroidogenic enzymes from QPtoxicity, whereas, there was no considerable restoration in CBE latetreatment. In CQE treatment, both simultaneous and late treat-ment significantly protected and rescued the expression of StARas well as steroidogenic enzymes (Cytochrome P450, 3b-HSD and17b-HSD). Comparison of CBE with CQE revealed that CQE wasfound to be more potent than the CBE (Figs. 11 and 12).

3.4.8. Immunoblot of StAR protein expression (30 kDa)Fig. 13 depicts the level of testicular StAR protein. Administra-

tion of QP dramatically altered the level of StAR protein expression(30 kDa) when compared to the corresponding group of controlmice. But, plant extracts treatment significantly recovered the levelof StAR protein expression. Simultaneous as well as late treatmentof plant extract showed similar effect in restoring StAR expression.However, CQE treatment greatly recuperated than the CBE.

Fig. 10. Effect of C. benghalensis and C. quadrangularis on the activities of (a) 3b HSDand (b) 17b HSD enzyme of QP exposed male mice. All values are mean ± SD. Valueswith different superscripts are significantly different among the groups by ANOVAwith Duncan’s multiple range test at p < 0.05. (1) Control, (2) QP treated, (3) CBEalone, (4) CQE alone, (5) QP + CBE (preventive), (6) QP + CBE (curative), (7) QP + CQE(preventive), (8) QP + CQE (curative). All treatments were given orally using oralgavage. [QP: Quinalphos, CBE: C. benghalensis aqueous extract, CQE: C.quadrangularis].

Fig. 11. Effect of C. benghalensis and C. quadrangularis on testicular mRNAexpressions of StAR protein and steroidogenic enzymes following exposure to QP.The gene expressions of steroidogenic enzymes were analyzed by RT-PCR andnormalized to corresponding RPS 16 expressions. The total RNA isolated from testeswas reverse transcribed and cDNA obtained was subjected to PCR. The represen-tative PCR products stained with ethidium bromide. Lane-(1) Control, Lane-(2) QPtreated, Lane-(3) CBE alone, Lane-(4) QP + CBE (preventive), Lane-(5) QP + CBE(curative), Lane-(6) CQE alone, Lane-(7) QP + CQE (preventive), Lane-(8) QP + CQE(curative). All treatments were given orally using oral gavage. [QP: Quinalphos, CBE:C. benghalensis aqueous extract, CQE: C. quadrangularis].


3.5. Effect of C. benghalensis and C. quadrangularis onhistomorphology of testes and epididymis of QP intoxicated male mice

Histological structure of the testis was illustrated in Plate 1.Testis of intact control (Fig. 1) showed normal seminiferous tu-bules lined with germinal epithelial layer. Various types of sper-matogenic cells appeared in their normal shape including:

Fig. 12. The graphs denote the intensity of the signals was quantified by densitometry anlevels of (b) StAR protein, (c) Cytochrome P450scc, (d) 3b-HSD (e) 17b-HSD. All values aregroups by ANOVA with Duncan’s multiple range test at p < 0.05. (1) Control, (2) QP treateQP + CQE (preventive), (8) QP + CQE (curative). All treatments were given orally usiquadrangularis].

spermatogonia, primary spermatocytes, secondary spermatocytes,spermatids and mature spermatozoa. Sertoli and Leydig cells withregular shape were observed. Leydig cells and blood vessels werefound in the interstitial connective tissue between the seminifer-ous tubules, and the tubules appeared to be normal size and shape,whereas, the administration of QP (Fig. 2) caused necrosis, degen-eration, decreasing number of spermatogenic cells in seminiferoustubules, separating of cells from basal region of seminiferous tu-bules and loss of Leydig cells in interstitial tissue.

Administration of CBE and CQE protected and recovered thedamages of testicular tissues due to the QP toxicity greatly as evi-denced by the normal appearance seminiferous tubule of testis,complete spermatogenesis with normal cell association, Leydigcells were found in the interstitial connective tissue between theseminiferous tubules, and the tubules appeared to be uniform insize and shape. They were lined by regularly arranged rows of sper-matogenic cells compared with QP administered group. However,simultaneous treatment of CBE and CQE significantly protectedthe testis from QP induced damages. Comparison between CBEand CQE revealed that CQE treatment provided significant protec-tion against QP induced testicular damages.

Plate 2 demonstrates the histomorphology of epididymis. QPexposure caused loss of sperm from the lumen of epididymis, dis-integration of basal membrane. On contrary, epididymis of controlmice exhibited a well defined peritubular membrane with regularbasal cells and sperm were numerous in the lumens of the (Fig. 1).Simultaneous treatment (Figs. 5 and 7) of both plant extracts re-stored the architecture of epididymis as evidenced by the accumu-lation of sperm in the centre of lumen and regeneration of liningcells and renewal of loss of stereocilia, intertubular connective

d normalized against internal control (RPS 16). The percentage of relative expressionmean ± SD. Values with different superscripts are significantly different among the

d, (3) CBE alone, (4) QP + CBE (preventive), (5) QP + CBE (curative), (6) CQE alone, (7)ng oral gavage. [QP: Quinalphos, CBE: C. benghalensis aqueous extract, CQE: C.

Fig. 13. Effect of C. benghalensis and C. quadrangularis on testicular StAR proteinexpression (30 kDa) of QP intoxicated male mice (top panel). The immunoblot wasprobed with b-actin (42 kDa, bottom panel) to show equal protein loading. Graphrepresenting the densitometrically scanned results (b) corresponding to (a). All valuesare mean ± SD. Values with different superscripts are significantly different among thegroups by ANOVA with Duncan’s multiple range test at p < 0.05. Lane-(1)Control, Lane-(2) QP treated, Lane-(3) CBE alone, Lane-(4) CQE alone, Lane-(5) QP + CBE (preventive),Lane-(6) QP + CBE (curative), Lane-(7) QP + CQE (preventive), Lane-(8) QP + CQE(curative), (9) withdrawal. All treatments were given orally using oral gavage. [QP:Quinalphos, CBE: C. benghalensis aqueous extract, CQE: C. quadrangularis].

Plate 1. Histomorphology of testes observed under light microscope (400X magnification(6) QP + CBE (curative), (7) QP + CQE (preventive), (8) QP + CQE (curative), (9) withdrabenghalensis aqueous extract, CQE: C. quadrangularis].


tissue with stroma was also highly densed than the late treatment(Figs. 6 and 8).

Both protective and curative treatment of plant extracts effec-tively protected and recovered the QP induced epididymal dam-ages. However, simultaneous treatment of CQE showed moreaccumulation of sperm with well defined peripheral membrane.There were no notable changes in plant extracts alone treatedgroups. No considerable recovery in group 9.

4. Discussion

The present study describes the protective and curative effect ofCBE and CQE on QP induced reproductive toxicities in Swiss albinomale mice. To our knowledge, this is the first study which evalu-ates the fertility enhancing properties of plant extracts (CBE andCQE) against testicular damage in experimental animals.

Testosterone has several physiological roles in male reproduc-tive system. One of the major reproductive role of testosterone isthe development of the sperm cell. At the Sertoli cells, testosteroneinduces a nuclear activation process which stimulates and cata-lyzes the maturation and development of sperm during the processof spermatogenesis. In the present study, QP exposure leads to asignificant reduction in serum testosterone concentration, whichhas caused a drastic decrease in epididymal sperm count and via-bility. Sperm parameters such as sperm count and viability are thekey indices of male fertility which are the prime markers in testic-ular spermatogenesis. Our results have shown that oral adminis-tration of QP caused focal degeneration of seminiferousepithelium, deterioration of Sertoli cells and germ cells, shrinkageof seminiferous tubules and drastic damages at the laminar mem-brane lead to loss of testicular mass and ultimately a decrease intestis weight which was also confirmed by the histological analysis

). (1) Control, (2) QP treated, (3) CBE alone, (4) CQE alone, (5) QP + CBE (preventive),wal. All treatments were given orally using oral gavage. [QP: Quinalphos, CBE: C.

Plate 2. Histomorphology of epididymis observed under light microscope (400� magnification). (1) Control, (2) QP treated, (3) CBE alone, (4) CQE alone, (5) QP + CBE(preventive), (6) QP + CBE (curative), (7) QP + CQE (preventive), (8) QP + CQE (curative), (9) withdrawal. All treatments were given orally using oral gavage. [QP: Quinalphos,CBE: C. benghalensis aqueous extract, CQE: C. quadrangularis].


of testes indicating testicular atrophy due to QP toxicity. In addi-tion, histological analysis of epididymis also clearly shows the lossof sperms from the lumen of epididymis by QP, which lead to a de-crease in sperm count. Exposure of QP caused a decrease in bodyweight associated with a reduction in reproductive organ (testes,epididymis, seminal vesicle and prostate gland) weights. Reductionin weight of reproductive organs is a measurement of testiculardamages [16]. High level of male intra-testicular testosterone iscritically required for normal spermatogenesis, development andmaintenance of morphology and normal physiology of seminifer-ous tubules [60]. Correlating the above findings it could be inter-preted that reduced sperm counts observed could be because oflow levels of testosterone.

The exact mechanism for this lower testosterone effect is uncer-tain, but may be due to interference of QP on endocrine function.However, it has been reported that OPIs induce oxidative stress(OS) and decrease the level of antioxidant enzymes and cause tes-ticular damages [8]. An oxidant mechanism might be involved inthe reproductive toxicity because OS has been identified as oneof the factor that affect male fertility status and various environ-mental toxicants are reported to produce ROS, thereby causing oxi-dative changes in testis [61]. OS is the primary mechanism of OPIs,which increases the level of LPO [9]. Lipids are considered to be themost susceptible macromolecules and are present in sperm plasmamembrane in the form of polyunsaturated fatty acids (PUFA) [62].ROS attacks PUFA leading to production of malondialdehyde(MDA) one of the by-products of lipid peroxidation [63]. Further,increased testicular lipid peroxidation causes a decrease in the pro-duction of testicular androgens [64]. The present results clearlypointed that QP initiated its reproductive toxicity by the formationof free radicals, as is evident from the increased LPO and diminu-

tion of antioxidant enzymes in testicular tissue. The increasedLPO is due to the depletion of lipids leading to alterations in mem-brane associated functions, changes in the membrane fluidity andultimately tissue damages. In fact, increased LPO (MDA) levelcaused severe tissue damage and hence the level of viable spermwas lost [18]. These results evidently demonstrate correlation be-tween the increased LPO level and reduction in sperm count.

Both enzymatic and non-enzymatic antioxidants system pro-tect the cells from oxidative injury. The enzymatic antioxidantssystem comprises CAT, GPx, SOD and non-enzymatic system isascorbic acid. Ascorbic acid (vitamin C) is an important antioxidantcontributing up to 65% of the total antioxidant capacity of seminalplasma found intracellular and extracellular. It neutralizes hydro-xyl, superoxide, hydrogen peroxide radicals and prevents spermagglutination. It prevents lipid peroxidation and protects againstDNA damage induced by the H2O2 radical. Studies have also shownthat antioxidants have a widespread effect in andrology. Antioxi-dants protect spermatozoa from ROS producing abnormal sperma-tozoa, scavenge ROS and prevent DNA fragmentation [65]. Thestatus of cellular antioxidants determines the impact of OS in cel-lular physiology [66]. The results of the present study revealed thatthe activities of CAT, GPx, SOD and ascorbic acid were greatlyinhibited by QP. The delayed accumulation of ascorbic acid in thetestis could have also left the oxidative damage abandoned thusinducing the effect on spermatogenesis. The decreased level ofascorbic acid is well correlated with the diminished sperm countas a result of QP exposure [11]. Further, increased MDA in testisindicates enhanced lipid peroxidation owing to tissue injury andmalfunctions of antioxidant defence mechanism caused by QPexposure. In addition, QP might have induced over production ofsuperoxide radicals which inhibited the activity of SOD, CAT and


GPx. A drastic decrease in body and reproductive organs weightafter QP exposure clearly indicates the effect of oxidative stressin overall physiology of the animal.

Studies have reported the effects of OS on steroidogenesis. Insteroidogenic cells, ROS are produced by the electron transportchain. In Leydig cells, ROS have been shown to have harmful effectson essential components of the steroidogenic pathway [67]. Inaddition, the depletion of Leydig cellular antioxidant enzymes,and increase in the levels of ROS and lipid peroxidation obviouslyreduce the steroidogenic and antioxidant enzymes [68]. Cumula-tive oxidative damage from prolonged exposure to ROS is thoughtto be one of the major causes of cellular aging [69]. Particularly,Leydig cells, which reside in the testicular interstitium, are moresusceptible to extracellular sources of ROS because of their closeproximity to testicular interstitial macrophages [70]. The releaseof ROS by activated macrophages not only affects invading micro-organisms but also exposes adjacent tissues and cells, such as Ley-dig cells, to oxidative stress. Testicular macrophages are known toproduce ROS during inflammation or infection [71].

Most enzymes involved in steroid biosynthesis are either Cyto-chrome P450s (CYPs) or HSDs. Steroidogenesis begins by Cyto-chrome P450, is a cholesterol side-chain cleavage enzyme, whichcatalyzes the first step in steroidogenesis. Conversion of choles-terol to pregnenolone in mitochondria is the first, rate-limiting,and hormonally regulated step in the synthesis of all steroid hor-mones [72,73]. 3b-HSD converts pregnenolone to progesteronei.e., 17-hydroxypregnenolone to 17-hydroxyprogesterone (17-OHP), dehydroepiandrosterone (DHEA) to androstenedione, andandrostenediol to testosterone. 3b-HSD catalyzes both conversionof the hydroxyl group to a keto group on carbon 3 and the isomer-ization of the double bond from the B ring (D5 steroids) to the Aring(D4 steroids) [74,75]. 17b-HSD reduces DHEA, 5-androstanedi-one, andandrosterone, the C19 17-ketosteroids that serve as precur-sors of testosterone and dihydrotestosterone (DHT) [76]. Theconversion of DHEA to androsta-5-ene-3b, 17b-diol by17b-HSDmay contribute significantly to testicular testosterone synthesis[77]. Thus, synthesis of testosterone in Leydig cells is dependentupon the expression of highly regulated genes 3b-HSD and 17b-HSD.

Since, 3b-HSD and 17b-HSD plays an important role in testos-terone conversion, we first checked the activities of 3b-HSD and17b-HSD. The results revealed a significant reduction in the activ-ities of the steroidogenic enzymes, 3b-HSD and 17b-HSD after QPexposure. Further, the expression of 3b-HSD and 17b-HSD waschecked by RT-PCR analysis. The level of mRNA expression of 3b-HSD and 17b-HSD were severely decreased upon QP treatment.Studies on regulation of mRNA expression of steroidogenicenzymes suggested that expression of 3b-HSD and 17b-HSD areregulated by several factors including growth factors, macro-phage-derived factors, steroidogenic-inducing protein (SIP), chlo-ride ions, and calcium (Ca2+) messenger systems. Particularly,cAMP (Cyclic adenosine monophosphate) is an important secondmessenger for trophic hormone-stimulated steroid biosynthesis,regulate the expression of steroidogenic enzymes. Steroidogenesisin Leydig cells is predominantly mediated by luteinizing hormone(LH)/human chorionic gonadotropin (hCG), via interactions with itsspecific receptor, which results in activation of cAMP-dependentprotein kinase A (PKA) and phosphorylation of proteins [78,79].Trophic hormones (LH or hCG) activate G proteins that stimulateadenylate cyclase activity and produce increased intracellular lev-els of cAMP and PKA [80,81]. PKA activation results in the phos-phorylation of proteins such as cholesteryl ester hydrolase aswell as the phosphorylation of transcription factors including ste-roidogenic factor 1(SF1), GATA-4, and cAMP response-elementbinding protein (CREB)/cAMP response element modulator thatfunction to activate genes involved in steroidogenesis. Increased

cAMP level activates the synthesis of Cytochrome P450 proteinthereby regulating the steady levels of their mRNA expression aswell as 3b-HSD mRNA expression [82]. Furthermore, Anakwe andPayne [83] suggested that the most critical role for cAMP is main-taining the enzymes necessary for testosterone production in Ley-dig cells. From the above discussions it is clear that the expressionof 3b-HSD is regulated by the Cytochrome P450 through the activa-tion cAMP. So, we checked the expression of Cytochrome P450after QP exposure. RT-PCR analysis of Cytochrome P450 revealeda significant reduction in mRNA expression after QP exposure.

Increase in ROS level cause damages to Cytochrome P450 en-zymes [67]. Normally, ROS generation occur during steroid hydrox-ylation by the Cytochrome P450scc enzymes in Leydig cells, as aby-product of their catalytic reaction mechanisms [84]. Unfortu-nately, environmental toxicants or their metabolites act as pseudo-substrate for CYP enzymes resulting in increased generation of ROSvia the formation of CYP pseudosubstrate–O2 complex [85].Expression of Cytochrome P450 was found to be decreased afterQP exposure. Down-regulation of Cytochrome P450 in QP exposedmice might be due to elevations in LPO and H2O2 levels, becausefree radicals react with lipids and cause peroxidative changes thatresults enhanced lipid peroxidation. Antioxidants CAT and GPxhave been shown to be responsible for the detoxification of H2O2

[86]. Decreased activities of CAT and GPx in QP exposed testiculartissue may be endorsed to be ineffective in scavenging of H2O2,thus leading to increased MDA. From the results, it is suspectedthat QP and it metabolites might have acted as pseudosubstratethereby inhibiting the expression of CYP enzymes. Decrease inmRNA expression of steroidogenic enzymes and activities of anti-oxidant enzymes confirm the deleterious effects of QP throughROS on testicular steroidogenic enzymes.

Environmental toxicants inhibit the expression of StAR proteinvia ROS generation suggesting that StAR protein is particularly sen-sitive to OS [87]. Previous reports indicated that ROS blocks thehormone-sensitive cholesterol transfer step [68]. ROS have alsobeen shown to inhibit steroidogenesis in MA-10 tumor Leydigcells, at the level of cholesterol transfer [88]. Analysis of mRNAexpression of StAR reveals that exposure of QP drastically down-regulated the transcription of StAR gene which is ultimatelyreflected in StAR protein expression confirmed by western blotanalysis. Transcriptional or translational inhibition of StAR expres-sion leads to dramatic decrease in steroid biosynthesis, whereas10–15%, of steroid synthesis appears to be mediated throughStAR-independent mechanisms [23,89–91].

ROS can also be produced in Leydig cells through the mitochon-drial respiration [92], because mitochondrial respiration consumes85–90% of the oxygen used by cells and represents the greatest po-tential source of ROS within the cell and is very susceptible to oxi-dative damage. ROS mediated perturbation of Leydig cellmitochondria decrease testosterone production by inhibiting StARprotein expression and function. So, cholesterol transfer activity ofStAR absolutely required integral mitochondria [93]. Presently, de-crease in StAR expression and increased level of MDA suggestingthat QP treatment might have caused non-specific tissue injurythereby it would have depolarized the mitochondrial membranepotentiality.

From these results we also suspected that QP might have inter-rupted with LH through which it would have down-regulated theCytochrome P450. LH binds to specific, high-affinity receptors onthe surface of Leydig cells thereby increasing the production ofintracellular cAMP and consequently regulating the expression ofsteroidogenic enzymes [94]. Thus, the biosynthesis of testosteroneis dependent on stimulation of Leydig cells by the pituitary hor-mone LH. Some studies have reported that the exposure to envi-ronmental toxicants have adverse effects on testicular functionby decreasing pituitary LH secretion and reducing Leydig cell


steroidogenesis by diminishing the number of cell surface LHreceptors through the increased generation ROS and LPO [95,96].But, the results of the present study revealed that QP did not affectthe LH level. Earlier reports have shown that OPIs can directlyblock the steroidogenic pathway between the formation of cAMPand the production of pregnenolone in MA-10 cells [97].

Further, LH mediated PKA pathway maintaining steady statelevels of StAR protein, and post-translational modification of pro-teins by PKA thereby regulating the steroidogenic function [98].This indicates the significant role of LH in regulating StAR expres-sion. In addition, the biosynthesis of steroid hormones in the go-nads is dependent on the action of the StAR, which transferscholesterol across the inner mitochondrial space from the outermitochondrial membrane [82,99]. Several observations imply thatenvironmental toxicants, such as lindane, manganese and dimeth-oate block steroidogenesis via the disruption of StAR proteinexpression [100–102]. In addition, several in vitro studies havedemonstrated the effects of various toxicants on the levels of StARproteins [103,104,96]. StAR protein is more susceptible to modula-tion by xenobiotic when compared with the steroidogenic en-zymes [13].

The acute regulation of steroidogenesis is determined by the ac-tion of StAR and P450scc. StAR promotes steroidogenesis byincreasing the movement of cholesterol into mitochondria. Since,StAR functions upstream of the steroidogenic enzymes, a reductionin StAR protein levels alone could account for the observed level ofsteroidogenic inhibition [101]. At the same time, availability of suf-ficient amount of cholesterol is important for steroidogenesis, be-cause, cholesterol is the main precursor for testosteroneproduction. Eventhough Leydig cells synthesis cholesterol by denovo path way, it requires extracellular cholesterol for testosteronebiosynthesis. Much of the precursor cholesterol for steroid hor-mone synthesis is derived from plasma lipoproteins in the formof cholesteryl esters (CE) [105,106]. Rodent steroidogenic cellspreferentially obtain most of their cholesterol via the selective up-take pathway [107]. Particularly, Leydig cells take up the choles-terol from its environment to synthesize testosterone [108] viaSR-B1 receptor (scavenger receptor class B type 1). Moreover, highlevel of steroid biosynthesis depends on the constitutive expres-sion of the components involved in the uptake of cholesterol esters(SR-B1), their conversion to free cholesterol (HSL), and its mobili-zation to the inner mitochondrial membrane (StAR) [109].

Recently, studies have reported that functions of SR-B1 can bemodulated by OS, either exogenous or endogenous [110]. Environ-mental toxicants significantly reduce the level of SR-B1, therebyreducing the level of testosterone [111,112]. Further, low levelsof SR-B1 mRNA decrease the synthesis of progesterone in MA-10cells caused by U0126, which could be a result of a decreased up-take of cholesterol into cells due to the repression of SR-B1. In thepresent study, surprisingly, the cholesterol is noted to be at a high-er concentration in serum after QP exposure. By contrast, signifi-cant reduction in cholesterol level was evidenced in the testis.This clearly indicates that the QP might have affected the SR-B1receptor by OS and it could be realized that there might be an im-paired intercellular transport of cholesterol into the Leydig cellsthereby affecting the steroidogenic pathway. This study clearlyindicates StAR expression and steroid synthesis are tightly corre-lated with the expression of scavenger receptor class B type 1(SR-B1) [113].

Eventhough, multiple and complex intracellular events appearto be involved in regulating the steroidogenic response in Leydigcells, the reduction in testicular cholesterol concentration clearlyindicated that QP exposure cruelly disrupted the Leydig cell steroi-dogenesis in male mice by altering the expression of SR-B1 recep-tor via OS. From the results, we are reporting that QP might havealtered the expression SR-B1 by OS, because most of EDCs disrupts

the endocrine system via ROS generation. So, it is speculate thatthe alterations in SR-B1 due to QP might have significantly dimin-ished the mRNA expression of StAR, which lead to reduction inCytochrome P450 scc, 3b-HSD and 17b-HSD mRNA expression.Reduction in the rate of transcription of StAR gene and steroido-genic enzymes gene may account for the reduction in steroidogen-esis which confirmed by significant inhibition of testicularsteroidogenesis i.e., reduction in testosterone synthesis after theexposure to QP. Also, decrease in expression of StAR protein afterQP exposure might be due to alterations at the level of transcrip-tion or translation. So, we strongly believe that QP triggered itsanti-androgenic effect via disrupting the SR-B1 receptor.

Dietary antioxidants particularly green leafy vegetables rein-force the cellular antioxidants defence system. Treatment withCBE and CQE significantly retrieved the testosterone level, spermcount and viability against QP induced adverse effects on spermparameters as well as hormone level which might be due to theirandrogenic activity [45], because phytochemical analysis revealedthat among the various phytochemical constituents steroid wasfound to be higher in both plants and also by their antioxidant effi-ciency [114]. In addition, the phytochemical screening of plant ex-tracts revealed high contents of ascorbic acid, which couldprobably play a role in spermatogenesis. Dietary antioxidants areusually present in the form of vitamin C, vitamin E, beta-carotenes,carotenoids, and flavonoids. SOD involving in disputation of H2O2

and prevents the further generation of free radicals, whereas,CAT helps to remove the H2O2 formed during the reaction cata-lyzed by the SOD. This could possibly be a reason for the plant ex-tracts treatment significantly (p < 0.05) restoring testicularantioxidant activities thereby rescuing the reproductive toxicityof toxicants. To support the above mentioned concept, previousstudies have shown that administration of 200 mg of vitamin C or-ally along with vitamin E and glutathione for two months signifi-cantly reduced 8-OH-dG levels in spermatozoa and also lead toan increase in sperm count [115]. Changes in antioxidant activitieswere restored back to normal by CBE and CQE treatment. CBE andCQE treatment seems to confer a protective antioxidant defencecapacity on the toxicants as evidenced by a significant reductionin the level of malondialdehyde and an increase in antioxidantsactivities along with increased in ascorbic acid. The anti-oxidativeconstituents present in plant extracts might be responsible for thefree radical scavenging activity leading to anti-lipidperoxidativeand anti-superoxide formation.

Supplementation of CBE and CQE significantly brought back theexpression of StAR protein to normal. Further, treatment of CBEand CQE restored the expression of Cytochrome P450 and exten-sively enhanced the testicular 3b-HSD and 17b-HSD expressionsand their activities. We believe that this might be due to the pro-tection of SR-B1 from ROS induced LPO by the plant extracts viaantioxidant activities. The antioxidant compounds present in theplant extracts might have scavenged the ROS generated by QP,thereby it would have enhanced the expression SR-B1 which facil-itated the Leydig cell to take up extracellular cholesterol. Resultsclearly indicate that plant extracts effectively scavenged the ROSgenerated by QP through elevating testicular antioxidants (GPx,CAT, SOD and Ascorbic acid) thereby preventing the oxidativedamage to testicular tissue particularly Leydig cells. Further, the le-vel of testicular cholesterol was also found to be increased in plantextract treated groups compared to QP group. The cholesterolmobilization by the plant extract would have activated the cho-lesteryl ester hydrolase which lead to subsequent activation ofPKA mediated StAR expression and steroidogenic enzymes. Also,the up regulation of StAR protein by CBE as well as CQE confirmingthat simultaneous treatment of plant extracts significantly pro-tected the mitochondrial structural integrity from the depolariza-tion due to QP induced OS. So, the upregulated of StAR protein


facilitated the transport of cholesterol from outer to inner mito-chondrial membrane, and subsequently activation of downstreamprocess i.e., activation of steroidogenic enzymes (CytochromeP450, 3b-HSD and 17b-HSD).

Overall, our results imply that QP induced oxidative damageplays a very crucial role in affecting the male reproductive systemand confirmed the reduction in testosterone synthesis in Leydigcells. Cholesterol transport into the Leydig cells could have beeninhibited through QP’s anti-androgenic effect via disrupting SR-B1 receptor. Lack of cholesterol in Leydig cells impaired the steroi-dogenesis by down-regulating the expression of StAR protein,Cytochrome P450, b-HSD and 17b-HSD. CBE and CQE effectivelyprevented and restored the oxidative damage by elevating antiox-idant enzymes thereby, restoring steroidogenesis. Flavanoids andsaponins are well known for their antioxidant activities[116,117]. Hence, we believe that the treatment of CBE and CQEcould have healed testicular toxicity through the antioxidantsmechanisms.

Among the two plants, simultaneous treatment of CQE effi-ciently recovered the changes caused by QP. This study evaluatedthe potentiality of C. benghalensis and C. quadrangularis on repro-ductive toxicity. However, among the two plants C. quadrangularisis found to be more potent than C. benghalensis. These results sug-gest that both plants effectively protected as well as cured QP in-duced adverse effects on male reproductive system and alsoconfirmed that both plants are having fertility enhancing proper-ties. CBE and CQE could be a potent natural antioxidant in prevent-ing testicular toxicity induced by environmental toxicants.

5. Conclusion

This study concluded that QP exhibiting a central role in affect-ing the male reproductive system through oxidative stress andconfirmed the reduction in testosterone synthesis thereby, affect-ing sperm count and viability. Lack of cholesterol in testicular tis-sue impaired the steroidogenesis by down-regulating theexpression StAR protein, Cytochrome P450, 3b-HSD and 17b-HSD.CBE and CQE prevented and restored the oxidative damage byameliorating the antioxidant enzymes and thereby leading to nor-mal steroidogenesis. The CQE effectively maintained the normaltransport of cholesterol and prevented the inhibition of steroido-genesis due to QP exposure. This is the first study suggesting thatthe transport of cholesterol from serum to Leydig cells has beeninhibited by the exposure of QP. We are speculating that the SR-B1 receptor might have been inhibited by QP exposure.

Conflict of interest statement

The authors declared no conflict of interest. The authors aloneare responsible for the content and writing of the paper.

Acknowledgments

We gratefully acknowledge to UGC-RGNF, UGC, DST-FASTTRACK, CSIR, Government of India, New Delhi for their financialsupport. The authors wish to thank Bharathidasan University,Tiruchirappalli, Tamilnadu, for the University Research Fellowship.

References

[1] S.C. Thakur, S.S. Thakur, S.K. Chaube, S.P. Singh, Subchronic supplementationof lithium carbonate induces reproductive system toxicity in male rat,Reprod. Toxicol. 17 (2003) 683–690.

[2] M. Ohi, P.R. Dalsenter, A.J.M. Andrade, A.J. Nascimento, Reproductive adverseeffects of fipronil in Wistar rats, Toxicol. Lett. 146 (2004) 121–127.

[3] N. Prashanthi, K. Narayana, A. Nayanatara, H.H. Chandra Kumar, K.L. BairyD’Souza, The reproductive toxicity of the organophosphate pesticide O,O-

dimethyl O-4-nitrophenyl phosphorothioate (methyl parathion) in the malerat, Folia Morphol. 65 (2006) 309–321.

[4] A.M. Al-Attar, Antioxidant effect of vitamin E treatment on some heavymetals-induced renal and testicular injuries in male mice, Saudi J. Biol. Sci. 18(2010) 63–72.

[5] S.C. Joshi, R. Mathur, N. Gulati, Testicular toxicity of chlorpyrifos (anorganophosphate pesticide) in albino rat, Toxicol. Ind. Health 23 (2007)439–444.

[6] G.G. Moustafa, Z.S. Ibrahim, Y. Hashimoto, A.M. Alkelch, K.Q. Sakamoto, M.Ishizuka, S. Fujita, Testicular toxicity of profenofos in matured male rats, Arch.Toxicol. 81 (2007) 875–881.

[7] F. Sayim, Histopathological effects of dimethoate on testes of rats, Bull.Environ. Contam. Toxicol. 78 (2007) 479–484.

[8] Y. Kalender, S. Kaya, D. Durak, F.G. Uzun, F. Demir, Protective effects ofcatechin and quercetin on antioxidant status, lipid peroxidation and testis-histoarchitecture induced by chlorpyrifos in male rats, Environ. Toxicol.Pharmacol. 33 (2012) 141–148.

[9] I.S. Taib, S.B. Budin, A.R. Ghazali, P.A. Jayusman, S.R. Louis, J. Mohamed,Fenitrothion induced oxidative stress and morphological alterations of spermand testes in male sprague-dawley rats, Clinics (Sao Paulo) 68 (2013) 93–100.

[10] M. Civen, C.B. Brown, The effect of organophosphate insecticides on adrenalcorticosterone formation, Pestic. Biochem. Physiol. 4 (1974) 254–259.

[11] K. Narayana, N. Prashanthi, A. Nayanatara, H.H. Kumar, K. Abhilash, K.L. Bairy,Effects of methyl parathion (O,O-dimethyl O-4-nitrophenyl phosphorothioate)on rat sperm morphology and sperm count, but not fertility, are associated withdecreased ascorbic acid level in the testis, Mutat. Res. 588 (2005) 28–34.

[12] E. Fattahi, K. Parivar, S.G.A. Jorsaraei, A.A. Moghadamnia, The effects ofdiazinon on testosterone, FSH and LH levels and testicular tissue in mice, Iran.J. Rep. Med. 7 (2009) 59–64.

[13] L.P. Walsh, C. McCormick, D.M. Stocco, Roundup inhibits steroidogenesis bydistrupting StAR expression, Environ. Health Persp. 108 (2000) 769–776.

[14] F.G. Uzun, S. Kalender, D. Durak, F. Demir, Y. Kalender, Malathion-inducedtesticular toxicity in male rats and the protective effect of vitamins C and E,Food Chem. Toxicol. 47 (2009) 1903–1908.

[15] N. Adityachaudhury, H. Banerjee, R.K. Kole, An appraisal of pesticide use inIndian Agriculture with Special reference to their consumption in WestBengal, Sci. Cult. 63 (1997) 223–228.

[16] D. Debnath, T.K. Mandal, Study of quinalphos (an environmental estrogenicinsecticide) system formulation (eka IUX 25 EC) induced damage of thetesticular tissue and antioxidant defense in Sprague–Dawley rats, J. Appl.Toxicol. 20 (2000) 197–204.

[17] R. Sarkar, K.P. Mohanakumar, M. Chowdhury, Effects of an organophosphatepesticide, quinalphos, on the hypothalamo–pituitary–gonadal axis in adultmale rats, J. Reprod. Fertil. 118 (2000) 29–38.

[18] N. Pant, S.P. Srivastava, Testicular and spermatotoxic effects of quinalphos inrats, J. Appl. Toxicol. 23 (2003) 271–274.

[19] P.D. Dwivedi, M. Das, S.K. Khanna, Role of cytochrome P-450 in quinalphostoxicity: effect on hepatic and brain antioxidant enzymes in rats, Food Chem.Toxicol. 36 (1998) 437–444.

[20] D.S. Rupa, P.P. Reddy, O.S. Reddy, Cytogenetic effects of quinalphos in mice,Food Chem. Toxicol. 29 (1991) 115–117.

[21] A.H. Payne, D.B. Hales, Overview of steroidogenic enzymes in the pathwayfrom cholesterol to active steroid hormones, Endocr. Rev. 25 (2004) 947–970.

[22] B.J. Clark, J. Wells, S.R. King, D.M. Stocco, The purification, cloning, andexpression of a novel luteinizing hormone-induced mitochondrial protein inMA-10 mouse Leydig tumor cells. Characterization of the steroidogenic acuteregulatory protein (StAR), J. Biol. Chem. 269 (1994) 28314–28322.

[23] D.M. Stocco, B.J. Clark, Regulation of the acute production of steroids insteroidogenic cells, Endocr. Rev. 17 (1996) 221–244.

[24] R.P. Singh, K.N.C. Murthy, G.K. Jayaprakasha, Studies on the antioxidantactivity of pomegranate (Punica granatum) peel and seed extracts usingin vitro models, J. Agric. Food Chem. 50 (2002) 81–86.

[25] P.K.J.P.D. Wanasundara, F. Shahidi, Optimigation of hexametaphosphate-assisted extraction of flaxseed proteins using response surface methodology,J. Food Sci. 61 (1996) 604–607.

[26] J.A. Olagunju, A.A. Adeneye, B.S. Fagbohunka, N.A. Bisuga, A.O. Ketiku, A.S.Benebo, O.M. Olufowobi, A.G. Adeoye, M.A. Alimi, A.G. Adeleke,Nephroprotective activities of the aqueous seed extract of Carica papayaLinn. in carbon tetrachloride induced renal injured Wistar rats: a dose- andtime-dependent study, Biol. Med. 1 (2009) 11–19.

[27] E.B. Saafi, M. Louedi, A. Elfeki, A. Zakhama, M.F. Najjar, M. Hammami, L.Achour, Protective effect of date palm fruit extract (Phoenix dactylifera L.) ondimethoate induced-oxidative stress in rat liver, Exp. Toxicol. Pathol. 63(2011) 433–441.

[28] S.M.S. Jalali-e-Emam, B. Alizadeh, M. Zaefizadeh, R.A. Zakarya, M.Khayatnezhad, Superoxide dismutase (SOD) activity in NaCl stress in salt-sensitive and salt-tolerance genotypes of colza (Brassica napus L.), Middle-East J. Sci. Res. 7 (2011) 7–11.

[29] T. Yokozawa, C.P. Chen, E. Dong, T. Tanaka, G.I. Nonaka, I. Nishioka, Study onthe inhibitory effect of tannins and flavonoids against the 1, 1-diphenyl-2-picrylhydrazyl radical, Biochem. Pharmacol. 56 (1998) 213–222.

[30] P.K. Marja, I.H. Anu, J.V. Heikki, R. Jussi-Pekka, P. Kalevi, S.K. Tytti, M.Heinonen, Antioxidant activity of plant extracts containing phenoliccompounds, J. Agric. Food Chem. 47 (1999) 3954–3962.

[31] F. Liu, T.B. Ng, Antioxidative and free radical scavenging activities of selectedmedicinal herbs, Life Sci. 66 (2000) 725–735.

http://refhub.elsevier.com/S0048-3575(14)00009-1/h0005






























































































[32] T.A. Woyengo, V.R. Ramprasath, P.J.H. Jones, Anticancer effects ofphytosterols, Eur. J. Clin. Nutr. 63 (2009) 813–820.

[33] S.M.R. Hasan, M.M. Hossain, A. Faruque, M.E.H. Mazumder, M.S. Rana, R.Akter, M.A. Alam, Comparison of antioxidant potential of different fractions ofCommelina benghalensis Linn, Bangladesh J. Life Sci. 20 (2008) 9–16.

[34] J. Paresh, S.V. Chanda, Antibacterial activity of aqueous and alcoholic extractof 34 Indian medicinal plants against some Staphylococcus species, Turk. J.Biol. 32 (2008) 63–71.

[35] R. Lakshminarayana, M. Raju, T.P. Krishnakantha, V. Baskaran, Lutein andZeaxanthin in leafy greens and their bioavailability: olive oil influences theabsorption of dietary lutein and its accumulation in adult rats, J. Agric. FoodChem. 101 (2007) 1598–1605.

[36] J. Ibrahim, V.C. Ajaegbu, H.O. Egharevba, Pharmacognostic and phytochemicalanalysis of Commelina benghalensis L, Ethno. Leaflets 14 (2010) 610–615.

[37] N. Sambrekar-Sudhir, P.A. Patil, A. Patil Suhas, Protective effect of Commelinabenghalensis – root extracts on isoniazid induced hepatotoxicity in wistarrats, Int. J. Adv. Pharm. Biol. Sci. 4 (2012) 265–271.

[38] W.J. Vander-Burg, Commelina benghalensis L., in: G.J.H. Grubben, O.A.Denton (Eds.), PROTA 2: Vegetables/Légumes. [CD-Rom], PROTA,Wageningen, Netherlands, 2004.

[39] M.A. Akbarsha, S. Kamalakkannan, K.V. Krishnamurthy, Ethnomedical Usesand Phytochemical Basis of Selected Indian Medicinal Plants, BharathidasanPublication, 2009. p. 55.

[40] M.C. Sharma, S. Sharma, Preliminary phytochemical and antimicrobialinvestigations of the aqueous extract of Ixora coccinea Linn and Commelinabenghalensis L. on gram positive and gram negative microorganisms, Middle-East J. Sci. Res. 6 (2010) 436–439.

[41] S.M.R. Hasan, M.M. Hossain, R. Akter, M. Jamila, M.E.H. Mazumder, M.A. Alam,A. Faruque, S. Rana, S. Rahman, Analgesic activity of the different fractions ofthe aerial parts of Commelina benghalensis Linn, Int. J. Pharmacol. 6 (2010) 63–67.

[42] V.G. Mbazima, M.P. Mokgotho, F. February, D.J.G. Rees, L.J. Mampuru,Alteration of Bax-to-Bcl-2 ratio modulates the anticancer activity ofmethanolic extract of Commelina benghalensis (Commelinaceae) in Jurkat Tcells, Afr. J. Biotechnol. 20 (2008) 3569–3576.

[43] S. Jakikasem, P. Limsiriwong, T. Kajsongkarm, T. Sontorntanasart,Phytochemical study of Cissus quadrangularis, Thai. J. Pharm. Sci. 24 (2000)25.

[44] M. Jainu, C.S. Devi, Effect of Cissus quadrangularis on gastric mucosal defensivefactors in experimentally induced gastric ulcer – a comparative study withSucralfate, J. Med. Food 7 (2004) 372–376.

[45] G. Mishra, S. Srivastava, B.P. Nagori, Pharmacological and therapeutic activityof Cissus quadrangularis: an overview, Int. J. Pharm. Tech. Res. 2 (2010) 298–310.

[46] A. Anoop, M. Jagdeesan, Gastric and duodenal antiulcer and cytoprotectiveeffect Cissus quadrangularis Linn. Variant II in rats, Nigeria J. Nat. Prod. Med. 6(2002) 1–7.

[47] K.N.C. Murthy, A. Vanitha, M.M. Swamy, G.A. Ravishankar, Antioxidant andantimicrobial activity of Cissus quadrangularis L, J. Med. Food 6 (2003) 99–105.

[48] http://www.blazelead.com/Cissus-Quadrangularis-L/Ms-Sheikh-International-/773371-1061143/.

[49] G.S. Mate, N.S. Naikwade, C.S. Magdum, A.A. Chowki, S.B. Patil, Evaluation ofanti-nociceptive activity of Cissus quadrangularis on albino mice, Int. J. GreenPharm. 2 (2008) 118–121.

[50] K. Gopalakrishnan, A.H. Bandivdekar, A.R. Sheth, Agglutination of humanspermatozoa with antibodies to inhibin, Ind. J. Exp. Biol. 25 (1987) 433–435.

[51] H. Ohkawa, N. Ohishi, K. Yagi, Assay for lipid peroxides in animal tissues bythiobarbituric acid reaction, Anal. Biochem. 95 (1979) 351–358.

[52] J.T. Rotruck, A.L. Pope, H.E. Ganther, A.B. Swanson, D.G. Hafeman, W.G.Hoekstra, Selenium: biochemical role as a component of glutathioneperoxidase, Science 179 (1973) 588–590.

[53] S. Marklund, G. Marklund, Involvement of the superoxide anion radical in theautoxidation of pyrogallol and a convenient assay for superoxide dismutase,Eur. J. Biochem. 47 (1974) 469–474.

[54] A.K. Sinha, Colorimetric assay of catalase, Anal. Biochem. 47 (1972) 389–394.[55] S.T. Omaye, J.D. Turnbull, H.E. Sauberlich, Selected methods for the

determination of ascorbic acid in animal cells, tissues, and fluids, MethodsEnzymol. 62 (1979) 3–11.

[56] S. Ringoir, J. Plum, LDH isozymes of human T and B lymphocytes, Clin. Chim.Acta 60 (1975) 379–383.

[57] A. Zlatkis, B. Zak, A.J. Boyle, A new method for the direct determination ofserum cholesterol, J. Lab. Clin. Med. 41 (1953) 486–492.

[58] H.U. Bergmeyer, b-Hydroxysteroid dehydrogenase, in: H.U. Bergmeyer (Ed.),Methods of Enzymatic Analysis, Academic Press, New York, 1974, pp. 447–489.

[59] G.L. Humason, Animal Tissue Techniques, fourth ed., W.H. Freeman and Co.,San Francisco, 1979. p. 661.

[60] R.M. Sharpe, S. Maddocks, M. Millar, J.B. Kerr, P.T. Saunders, C. McKinnell,Testosterone and spermatogenesis. Identification of stage-specific, androgen-regulated proteins secreted by adult rat seminiferous tubules, J. Androl. 13(1992) 172–184.

[61] I.D. Sharlip, J.P. Jarow, A.M. Belker, L.I. Lipshultz, M. Sigman, A.J. Thomas, P.N.Schlegel, S.S. Howards, A. Nehra, M.D. Damewood, J.W. Overstreet, R.Sadovsky, Best practice policies for male infertility, Fertil. Steril. 77 (2002)873–882.

[62] R.J. Aitken, Free radicals, lipid peroxidation and sperm function, Reprod.Fertil. Dev. 7 (1995) 659–668.

[63] J.S. Armstrong, M. Rajasekaran, W. Chamulitrat, P. Gatti, W.J. Hellstrom, S.C.Sikka, Characterization of reactive oxygen species induced effects on humanspermatozoa movement and energy metabolism, Free Radic. Biol. Med. 26(1999) 869–880.

[64] M.F. Kashko, A.M. Khokha, S.N. Antsulevich, N.A. Doroshkevich, P.P. Voronov,Influence of ethanol and ethanol-induced lipid peroxidation on thesteroidogenic activity of testicles, Ukr Biokhim Zh 65 (1993) 89–94.

[65] A. Agarwal, K.P. Nallella, S.S. Allamaneni, T.M. Said, Role of antioxidants intreatment of male infertility: an overview of the literature, Reprod. Biomed.Online 8 (2004) 616–627.

[66] V. Seth, B.D. Banerjee, A. Bhattacharya, A.K. Chakravorty, Lipid peroxidation,antioxidant enzymes, and glutathione redox system in blood of humanpoisoning with propoxur, Clin. Biochem. 33 (2000) 683–685.

[67] M. Georgiou, L.M. Prekins, A.H. Payne, Steroid synthesis dependent oxygen-mediated damage of mitochondrial and microsomal Cytochrome P-450enzymes in rat Leydig cell cultures, Endocrinology 121 (1987) 1390–1399.

[68] H.R. Behrman, R.F. Aten, Evidence that hydrogen peroxide blocks hormone-sensitive cholesterol transport into mitochondria of rat luteal cells,Endocrinology 128 (1991) 2566–2958.

[69] K. Hensley, R.A. Floyd, Reactive oxygen species and protein oxidation inaging: a look back, a look ahead, Arch. Biochem. Biophys. 397 (2002) 377–383.

[70] D.B. Hales, T. Diemer, K.H. Hales, Role of cytokines in testicular function,Endocrine 10 (1999) 201–217.

[71] R.Q. Wei, J.B. Yee, D.C. Straus, J.C. Hutson, Bactericidal activity of testicularmacrophages, Biol. Reprod. 38 (1988) 830–835.

[72] I.D. Halkerston, J. Eichhorn, O. Hechter, Arequirement for reducedtriphosphopyridine nucleotide for cholesterol side-chain cleavage bymitochondrial fractions of bovine adrenal cortex, J. Biol. Chem. 236 (1961)374–380.

[73] S.B. Koritz, A.M. Kumar, On the mechanism of action of theadrenocorticotrophic hormone. The stimulation of the activity of enzymesinvolved in pregnenolone synthesis, J. Biol. Chem. 10 (1970) 152–159.

[74] Y. Lachance, V. Luu-The, C. Labrie, J. Simard, M. Dumont, Y. de Launoit, S.Guerin, G. Leblanc, F. Labrie, Characterization of human 3-hydroxysteroiddehydrogenase/D 5 to D 4 -isomerase gene and its expression in mammaliancells, J. Biol. Chem. 265 (1990) 20469–20475.

[75] M.C. Lorence, B.A. Murry, J.M. Trant, J.I. Mason, Human 3-hydroxysteroiddehydrogenase/D5 to D 4 isomerase from placenta: expression innonsteroidogenic cells of a protein that catalyzes the dehydrogenation/isomerisation of C21 and C19 steroids, Endocrinology 126 (1990) 2493–2498.

[76] S. Andersson, W.M. Geissler, L. Wu, D.L. Davis, M.M. Grumbach, M.I. New, H.P.Schwarz, S.L. Blethen, B.B. Mendonca, W. Bloise, S.F. Witchel, G.B. Cutler Jr.,J.E. Griffin, J.D. Wilson, D.W. Russel, Molecular genetics and pathophysiologyof 17-hydroxysteroid dehydrogenase 3 deficiency, J. Clin. Endocrinol. Metab.81 (1996) 130–136.

[77] C.E. Fluck, W.L. Miller, R.J. Auchus, The 17, 20 lyase activity of cytochromeP450c17 from human fetal testis favors the delta5 steroidogenic pathway, J.Clin. Endocrinol. Metab. 88 (2003) 3762–3766.

[78] M. Ascoli, F. Fanelli, D.L. Segaloff, The lutropin/choriogonadotropin receptor, aperspective, Endocr. Rev. 23 (2002) 141–174.

[79] D.M. Stocco, X. Wang, Y. Jo, P.R. Manna, Multiple signaling pathwaysregulating steroidogenesis and steroidogenic acute regulatory proteinexpression: more complicated than we thought, Mol. Endocrinol. 19 (2005)2647–2659.

[80] G. Selstam, S. Rosberg, J. Liljekvist, L. Gronquist, T. Perklev, K. Ahren,Differences in action of LH and FSH on the formation of cyclic AMP in theprepubertal rat ovary, Acta Endocrinol. (Copenh.) 81 (1976) 150–164.

[81] X. Zhu, L. Birnbaumer, G protein subunits and the stimulation ofphospholipase C by Gs-and Gi-coupled receptors: lack of receptorselectivity of Galpha(16) and evidence for a synergic interaction between Gbeta gamma and the alpha subunit of a receptor activated G protein, Proc.Natl. Acad. Sci. U.S.A. 93 (1996) 2827–2831.

[82] D. Stocco, Star protein and the regulation of steroid hormone biosynthesis,Annu. Rev. Physiol. 63 (2001) 193–213.

[83] O.O. Anakwe, A.H. Payne, Noncoordinate regulation of de novo synthesis ofcytochrome P-450 cholesterol side-chain cleavage and cytochrome P-450 17alpha-hydroxylase/C17-20 lyase in mouse Leydig cell cultures: relation tosteroid production, Mol. Endocrinol. 1 (1987) 595–603.

[84] P.J. Hornsby, Steroid and xenobiotic effects on the adrenal cortex: mediationby oxidative and other mechanisms, Free Radic. Biol. Med. 6 (1989) 103–115.

[85] P.G. Quinn, A.H. Payne, Steroid product-induced, oxygen-mediated damage ofmicrosomal cytochrome P-450 enzymes in Leydig cell cultures. Relationshipto desensitization, J. Biol. Chem. 260 (1985) 2092–2099.

[86] L. Cheng, E.W. Kellogg III, Packer, photoinactivation of catalase, Photochem.Photobiol. 34 (1981) 125–129.

[87] T. Diemer, J.A. Allen, K.H. Hales, D.B. Hales, Reactive oxygen disruptsmitochondria in MA-10 tumor Leydig cells and inhibits steroidogenic acuteregulatory (StAR) protein and steroidogenesis, Endocrinology 144 (2003)2882–2891.

[88] D.M. Stocco, J. Wells, B.J. Clark, The effects of hydrogen peroxide onsteroidogenesis in mouse Leydig tumor cells, Endocrinology 133 (1993)2827–2832.



















































































































































































[89] D. Lin, T. Sugawara, J.F. Strauss 3rd, B.J. Clark, D.M. Stocco, P. Saenger, A. Rogol,W.L. Miller, Role of steroidogenic acute regulatory protein in adrenal andgonadal steroidogenesis, Science 267 (1995) 1828–1831.

[90] B.J. Clark, R. Combs, K.H. Hales, D.B. Hales, D.M. Stocco, Inhibition oftranscription affects synthesis of steroidogenic acute regulatory protein andsteroidogenesis in MA-10 mouse Leydig tumor cells, Endocrinology 138(1997) 4893–4901.

[91] P.R. Manna, J. Kero, M. Tena-Sempere, P. Pakarinen, D.M. Stocco, I.T.Huhtaniemi, Assessment of mechanisms of thyroid hormone action inmouse Leydig cells: regulation of the steroidogenic acute regulatoryprotein, steroidogenesis, and luteinizing hormone receptor function,Endocrinology 142 (2001) 319–331.

[92] H. Chen, D. Cangello, S. Benson, J. Folmer, H. Zhu, M.A. Trush, B.R. Zirkin, Age-related increase in mitochondrial superoxide generation in the testosterone-producing cells of brown Norway rat testes: relationship to reducedsteroidogenic function?, Exp Gerontol. 36 (2001) 1361–1373.

[93] I.P. Artemenko, D. Zhao, D.B. Hales, K.H. Hales, C.R. Jefcoate, Mitochondrialprocessing of newly synthesized steroidogenic acute regulatory protein(StAR), but not total StAR, mediates cholesterol transfer to cytochrome P450side chain cleavage enzyme in adrenal cells, J. Biol. Chem. 276 (2001) 46583–46596.

[94] M.L. Dufau, Endocrine regulation and communicating functions of the Leydigcell, Annu. Rev. Physiol. 50 (1988) 483–508.

[95] B.T. Akingbemi, C.M. Sottas, A.I. Koulova, G.R. Klinefelter, M.P. Hardy,Inhibition of testicular steroidogenesis by the xenoestrogen bisphenol A isassociated with reduced pituitary luteinizing hormone secretion anddecreased steroidogenic enzyme gene expression in rat Leydig cells,Endocrinology 145 (2004) 592–603.

[96] P. Murugesan, T. Muthusamy, K. Balasubramanian, J. Arunakaran, Effects ofvitamin C and E on steroidogenic enzymes mRNA expression inpolychlorinated biphenyl (Aroclor 1254) exposed adult rat Leydig cells,Toxicology 232 (2007) 170–182.

[97] Y.S. Choi, D.M. Stocco, D.A. Freeman, Diethylumbelliferyl phosphate inhibitssteroidogenesis by interfering with long-lived factor acting between proteinkinase A activation and induction of the steroidogenic acute regulatoryprotein (StAR), Eur. J. Biochem. 234 (1995) 680–685.

[98] B.J. Clark, S.C. Soo, K.M. Caron, Y. Ikeda, K.L. Parker, D.M. Stocco, Hormonaland developmental regulation of the steroidogenic acute regulatory protein,Mol. Endocrinol. 9 (1995) 1346–1355.

[99] L.K. Christenson, I. Strauss, F. Jerome, Steroidogenic acute regulatory protein;an update on its regulation and mechanism of action, Arch. Med. Res. 32(2001) 576–586.

[100] L.P. Walsh, D.M. Stocco, The role of the steroidogenic acute regulatory (StAR)protein in environment endocrine disruptor inhibited steroidogenesis, in:Proc. 52nd Universal, Tayoko, Japan, Acadamy press Inc., 2000, pp. 253–266.

[101] J. Cheng, J.L. Fu, Z.C. Zhou, The inhibitory effects of manganese onsteroidogenesis in rat primary Leydig cells by disrupting steroidogenicacute regulatory (StAR) protein expression, Toxicology 187 (2003) 139–148.

[102] P. Elumalai, G. Krishnamoorthy, K. Selvakumar, R. Arunkumar, P.Venkataraman, J. Arunakaran, Studies on the protective role of lycopeneagainst polychlorinated biphenyls (Aroclor 1254)-induced changes in StAR

protein and cytochrome P450 scc enzyme expression on Leydig cells of adultrats, Reprod. Toxicol. 27 (2009) 41–45.

[103] E.P. Murono, R.C. Derk, The effects of the reported active metabolite ofmethoxychlor, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane, ontestosterone formation by cultured Leydig cells from young adult rats,Reprod. Toxicol. 19 (2004) 135–146.

[104] R. Zachow, M. Uzumcu, The methoxychlor metabolite, 2,2-bis-(phydroxyphenyl)-1,1,1-trichloroethane, inhibits steroidogenesis in ratovarian granulosa cells in vitro, Reprod. Toxicol. 22 (2006) 659–665.

[105] R.C. Pedersen, Cholesterol biosynthesis, storage, and mobilization insteroidogenic organs, in: P.L. Yeagle (Ed.), Biology of Cholesterol, CRC PressInc., Boca Raton, FL, 1988, pp. 39–69.

[106] E. Reaven, L. Tsai, S. Azhar, Cholesterol uptake by the ‘‘selective’’ pathway ofovarian granulose cells: early intracellular events, J. Lipid Res. 36 (1995)1602–1617.

[107] C. Glass, R.C. Pittman, D.B. Weinstein, D. Steinberg, Dissociation of tissueuptake of cholesteryl ester from that of apoprotein A-1 of rat plasma highdensity lipoprotein: selective delivery of cholesteryl ester to liver, adrenal,and gonad, Proc. Natl. Acad. Sci. U.S.A. 80 (1983) 5435–5439.

[108] A.H. Payne, M.P. Hardy, L.D. Russell (Eds.), The Leydig Cell, Cache River Press,Vienna, IL, 1996.

[109] R.M. Rao, Y. Jo, S. Leers-Sucheta, H.S. Bose, W.L. Miller, S. Azhar, D.M. Stocco,Differential regulation of steroid hormone biosynthesis in R2C and MA-10Leydig tumor cells: role of SR-B1-mediated selective cholesteryl estertransport, Biol. Reprod. 68 (2003) 114–121.

[110] G. Valacchi, P.A. Davis, E.M. Khan, R. Lanir, E. Maioli, A. Pecorelli, C.E. Cross, T.Goldkorn, Cigarette smoke exposure causes changes in scavenger receptor B1level and distribution in lung cells, Int. J. Biochem. Cell Biol. 43 (2011) 1065–1070.

[111] N.J. Barlow, S.L. Phillips, D.G. Wallace, M. Sar, K.W. Gaido, P.M. Foster,Quantitative changes in gene expression in fetal rat testes following exposureto di(n-butyl) phthalate, Toxicol. Sci. 73 (2003) 431–441.

[112] K.P. Lehmann, S. Phillips, M. Sar, P.M. Foster, K.W. Gaido, Dose-dependentalterations in gene expression and testosterone synthesis in the fetal testes ofmale rats exposed to di (n-butyl) phthalate, Toxicol. Sci. 81 (2004) 60–68.

[113] P.R. Manna, Y. Jo, D.M. Stocco, Regulation of Leydig cell steroidogenesis byextracellular signal-regulated kinase 1/2: role of protein kinase A and proteinkinase C signaling, J. Endocrinol. 193 (2007) 53–63.

[114] M. Jainu, C.S. Devi, Gastroprotective action of Cissus quadrangularis extractagainst NSAID induced gastric ulcer: role of proinflammatory cytokines andoxidative damage, Biol. Interact. 161 (2006) 262–270.

[115] H. Kodama, R. Yamaguchi, J. Fukuda, H. Kasai, T. Tanaka, Increased oxidativedeoxyribonucleic acid damage in the spermatozoa of infertile male patients,Fertil. Steril. 68 (1997) 519–524.

[116] N.L. Baek, Y.S. Kim, J.S. Kyung, K.H. Park, Isolation of antihepatotoxic agentfrom the roots of Astragalus membranaceous, Korean J. Phamacog. 27 (1996)111–117.

[117] M. Yoshikawa, T. Morikawa, Y. Kashima, K. Ninomiya, H. Matsuda, Structureof new dammarane-type triterpene Saponins from the flower buds of Panaxnotoginseng and hepatoprotective effect of principal ginseng Saponins, J. Nat.Product. 66 (2003) 922–927.









































































































Date post:	23-Dec-2016
Category:	Documents
Upload:	shanmugam
View:	227 times
Download:	5 times

Antioxidant mediated ameliorative steroidogenesis by Commelina benghalensis L. and Cissus...

Documents