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International Journal of Toxicology

http://ijt.sagepub.com/content/28/4/308The online version of this article can be found at:

DOI: 10.1177/1091581809339631

2009 28: 308International Journal of ToxicologyJ. Joshua Allan, Mukul P. Pore, M. Deepak, B. Murali, Anand S. Mayachari and Amit Agarwal

in Male Wistar RatsAndrographis paniculataReproductive and Fertility Effects of an Extract of

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Reproductive and Fertility Effects of anExtract ofAndrographis paniculata in

Male Wistar RatsJ. Joshua Allan, Mukul P. Pore, M. Deepak, B. Murali,Anand S. Mayachari, and Amit Agarwal

The possible effect of extract of Andrographis panicu-lata Nees (A paniculata) standardized to !10% andro-grapholide, the main bioactive component, on malefertility in albino Wistar rats was evaluated, by orally

administering 0, 20, 200, and 1000 mg/kg of bodyweight per day, for 65 days prior to mating and 21 daysduring mating. The treated groups showed no signsof dose-dependent toxicity. The body weight gain and

feed consumption were not affected at any of the doselevels. The testosterone levels and fertility indices in

treatment groups were found to be comparable with

that of the control indicating no effect on fertility. Total

sperm count and sperm motility were not affected.The testes and epididymides did not show any gross andhistopathological changes. Based on these findings, it

can be concluded that the no-observed adverse effectlevel of extract of A paniculata (!10% androgra-pholide) was found to be more than 1000 mg/kgper day.

Keywords: Andrographis paniculata; andrographo-lide; male fertility; no-observed adverse effect level

Herbal remedies, since ancient times, have

been used in almost all human races as asource of medicine. Nearly 80% of the

worlds population still relies upon medicinal herbsfor basic health care needs.1,2 Although they areoften considered as natural and relatively harmless,phyto preparations are not always free from toxicity.3

Herbal products have been used frequently withoutproper evaluation of their actual efficacy in the treat-ment or the occurrence of undesirable side effects.Currently little information is available on the repro-ductive effects of popular medicinal plants. Therehas been growing interest over the safety of naturalproducts in clinical use and evaluation of effects on

reproductive system has been considered as part of

the safety studies of widely used medicinal plants.4

Andrographis paniculata, commonly known asKalmegh or green chiretta and belonging to fam-ily Acanthaceae, is an annual herb native to peninsu-lar India and Sri Lanka and is also distributed indifferent regions of Southeast Asia, China, America,West Indies, and Christmas Island.5 All parts of thisherb are extremely bitter in taste and are consideredmedicinally important in the traditional system ofIndian medicine for treatment of various illnesses.The plant extracts are found to have antiviral, anti-pyretic, antiulcer, hepatoprotective, antiallergic,antiplatelet aggregation, antidiabetic, anticancer,and antimalarial activities.6-17 Two diterpenes iso-lated from this plant, andrographolide and neoandro-grapholide, have been shown to possess promisingimmunomodulatory activity.18 It has recently beenreported that andrographolide exerts antiallergicactivity by significantly decreasing degranulationof mast cells with consequent reduction in theliberation of histamine.8 A paniculata exerts its

From the Natural Remedies Pvt Ltd, Bangalore, India (JJA, MD,BM, ASM, AA); and Intox Pvt Ltd, Pune, India (MP).

Please address correspondence to Dr J. Joshua Allan, MVSc,Department of Pharmacology and Toxicology, R&D Centre,Natural Remedies Pvt Ltd, 5B, Veerasandra Industrial Area,19th KM Stone, Hosur Road, Near Electronic City, Bangalore560 100, India; e-mail: [email protected].

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anti-inflammatory effect by inhibiting reactive oxygenproduction in neutrophils and lipopolysaccharide-induced nitric oxide production in macrophages.19,20

Several randomized placebo-controlled clinical trialsconducted with A paniculata extract standardized toandrographolide and deoxyandrographolide showed

its preventive effects against the common cold andthe capacity to significantly shorten the duration ofthe disease.21-25

Despite diverse studies on the therapeutic effec-tiveness of A paniculata, only a few studies on thetoxicological assessment of this medicinal plant havebeen reported. A preliminary study reported that oraladministration of 20 mg of herb powder of A panicu-lata to male albino rats for 60 days resulted incessation of spermatogenesis and regression ofLeydig cells suggesting the antiandrogenic effect ofthis plant.26 Andrographolide, administered to malerats at 2 dose levels of 25 and 50 mg/kg of bodyweight for 48 days, showed testicular toxicity andsperm abnormalities.27 On the other hand, extractof A paniculata containing 6.1% of andrographolidedid not produce testicular toxicity in male rats whenadministered at the doses of 20, 200, and 1000 mg/kg,for 60 days.28A phase I clinical study ofA paniculatafixed combination showed no adverse effects onmale semen quality and fertility in healthy humansubjects.29 Considering the facts that the plant hasbeen incorporated as an active ingredient in 26 of the

40 polyherbal formulations available for liver disor-ders,30 and the potential of andrographolide to causetesticular toxicity is still unclear, the present studywas undertaken to assess the possible testicular toxi-city of extract ofA paniculata treated for a prolongedperiod in albino Wistar rats.

Methods

This study was conducted in accordance with the 1993

International Conference on Harmonisation Harmo-nised Tripartite Guideline31 and in compliance withthe Organisation for Economic Cooperation andDevelopment Principles of Good Laboratory Practice.32

Animals

Adult Wistar rats (12-14 weeks old) used in thisstudy were bred and reared at Intox, Pune, India. Theanimals were housed in polypropylene cages withstainless steel grill tops and bedding of clean paddy

husk. The temperature in the animal room was main-tained between 19C and 25C with 10 to 15 airchanges per hour, a relative humidity of 30% to70%, and illumination cycle set to 12 h of light and12 h of dark. Standard certified pelleted diet (Amrutbrand; M/s Nav. Maharashtra Chakan Oil Mills Ltd.,

Pune, India) and filtered water were provided adlibitum.

Preparation of Extract

The raw material (whole plant, the majority of whichwas leaves of A paniculata; 400 kg) was procuredfrom a local commercial supplier and was authenti-cated at the National Institute of Science Communi-cation and Information Resources (NISCAIR), NewDelhi. It was extracted with ethanol by refluxing at

60C to 65C (3 times each with 1200 L of ethanolfor 3 h) in the manufacturing facility of M/s NaturalRemedies, Bangalore, India. The liquid extract wascombined and concentrated by distillation undervacuum to a thick paste (28 kg, extract I). The marcleft after ethanol extraction was further refluxed withwater at 85C to 90C (2 times each with 1200 Lwater for 3 h). The liquid extract was combined andconcentrated by distillation under vacuum to a thickpaste (16 kg, extract II). The concentrated extracts(I and II) were mixed and dried in a vacuum tray

dryer at less than 70

C to get the final extract.

Reference Standards for High

Performance Liquid Chromatography

(HPLC) Analysis

The methanolic extract from the leaves of A panicu-lata was partitioned between ethyl acetate and water.The ethyl acetate layer was subjected to normalphase silica gel column chromatography to furnish5 fractions. Fractions 1-5 were purified by reversephase column chromatography followed by prepara-tive HPLC to obtain andrographolide (1), neoandro-grapholide (2), andrograpanin (3), and 14-deoxy-11,12-didehydroandrographolide (4). The isolated com-pounds were characterized by comparing their 1Hand 13C NMR data with literature.33,34 These com-pounds were used as reference standards for HPLCanalysis. Andrographolide from Sigma Aldrich wasalso used.

Effects of Extract of Andrographis paniculata / Allan et al 309



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HPLC Analysis

The extract was analyzed for the content of 4 andro-grapholides by HPLC. A known volume (20 mL) ofthe mixed standard and sample solutions wereinjected to the HPLC system (Shimadzu, Model

LC 2010 A, Tokyo, Japan) consisting of quaternarypump with UV detector, auto injector, and columnoven with class VP software (Shimadzu, Japan). Thestationary phase was an octadecylsilane column(C18, 5 mm, 250 4.6 mm; Hibar RT, Lichrosphere100; Merck, Darmstadt, Germany). The mobilephase consisted of a mix of phosphate buffer (solventA; prepared by dissolving 0.136 g of potassium dihy-drogen orthophosphate [KH2PO4] in 900 mL ofHPLC grade water [obtained from Arium Sartoriuswater purification system] and by adding 0.5 mLof orthophosphoric acid [H3PO4;AR grade, Rankem,New Delhi, India] and making the volume to 1000mL) and acetonitrile (solvent B; HPLC grade, Quali-gens, Mumbai, India). Both acetonitrile and phos-phate buffer were filtered separately through 0.45-mm membrane filter and degassed by sonicating for3 min. The solvents A and B were mixed in such amanner that the concentration of solvent B wasincreased from 5% to 45% as a linear gradient in thefirst 18 min. From 18 to 25 min the concentration ofsolvent B was increased from 45% to 80% as a linear

gradient. The flow rate of mobile phase was main-tained at 1.5 mL per min throughout the analysisand the detector wave length was kept at 223 nm,and the chromatogram was recorded. Quantificationof 1-4 was achieved by the external standard method.The peaks in the mixed standards and in the samplewere identified by injecting the individual standardsolutions.

Standard and Sample Solution

Preparation

Solutions of standards 1-4 were prepared by weigh-ing 10 mg of the individual standards and dissolvingthem separately in 10 mL with methanol (HPLCgrade, Qualigens). The stock solution was suitablydiluted to obtain 0.5 mg/mL of (1) and 0.1 mg/mL

of (2-4) as a mix and also in the individual solutions.

Validation of Methods

The method was validated for specificity, linearity,precision, accuracy, and range of quantification(Table 1).

Content of (1) in the extract was measured usingthe external standard method and was found to be10.9% (wt/wt). The other compounds identified in

Table 1. Summary of Analytical Method Validation

Validation Parameter Result

Specificity Very specific for andrographolide with no interference from other compounds.Confirmed by the PDA spectra and the peak purity.

Linearity 5 concentrations of each, 5 replicates of each

1: correlation coefficient

0.9962-4: correlation coefficient 1.000

Accuracy by spike recovery method 1: 98.8-102.4%2: 97.9-103.5%3: 97.8-100.2%4: 94.0-101.4%

Precision (of concentration, retention timeand reproducibility)

5 concentrations of each, 5 replicates of each1: Range of quantification 60-500 mg/mL

RSD% for retention time


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the extract (2-4) constituted 2.7% (wt/wt) of theextract. The profiles of the extract of A paniculataanalyzed by HPLC and the structural formulae ofthe components of the extract are shown inFigure 1A and B.

Experimental Design

Thirty-two adult male albino Wistar rats were dividedinto 4 groups of 8 animals each. The control groupreceived vehicle (water; 10 mL/kg) and the remain-ing groups were administered with extract of A pani-culata at the dose levels of 20, 200, and 1000 mg/kgof body weight by oral gavage once daily for a periodof 65 days prior to mating and 21 days during mating.The doses were prepared freshly every day beforeadministration to animals.

Mortality and Clinical SignsThe treated males were observed daily for mortalityand clinical signs throughout the experiment. Anydeath or abnormal clinical findings were recorded.

Body Weight and Feed Intake

The body weight of males was recorded at beginningof the treatment and thereafter twice a week till theywere sacrificed. The feed consumption was recorded

1 day before initiation of treatment and thereafterweekly during the premating period of 65 days.

Total Sperm Count

Sperm count was performed as described by Seedet al.35 In brief, one of the epididymides was used forsperm count. The cauda epididymis was incised and

homogenized in a homogenizer glass vial containing30 mL of 0.01% (wt/vol) Triton X-100 in 0.9% NaClfor approximately 15 to 20 min. The homogenateprobe was rinsed with distilled water and the washingwas collected along with the homogenate. The finalvolume of homogenate was made up to 50 mL usingdistilled water and then sonicated for 15 to 20 min,and 0.5 mL of 1% eosin Y stain solution was finallyadded to it. Sperm counts were made in a hemocyt-ometer and recorded.

Sperm MotilityThe sperm from vas deferens were collected andobserved under the microscope to determine the per-centage of motile sperm. Immediately after sacrifice,one vas deferens was isolated and excised from thetestis. The vas deferens was cut into small pieces andplaced in a petri dish containing 3 to 5 mL ofphosphate-buffered saline maintained at 35C to37C. The sperm suspension was transferred to a testtube placed in a water bath maintained at 35C to

Figure 1. High performance liquid chromatogram of standard mix of andrographolides (A) and of the extract (B). Compounds 1-4

eluted using a C18 column and mix of phosphate buffer (pH 2.8; solvent A) and acetonitrile (solvent B) as mobile phase. The gradientprogram used 0 to 18 min, linear change from A-B (95:5) to A-B (55:45) from 18 to 25 min, A-B (55:45) to A-B (20:80) from 25 to28 min, A-B (20:80) to A-B (20:80) and from 28 to 45 min, (20:80) to A:B (95:5). Detection length 223 nm.




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37C and then incubated for 5 to 10 min. Approxi-mately 10 mL of the suspension was transferred to amicroscope slide with concave chambers kept on aslide warmer maintained at 35C to 37C and roofed

with a coverslip for evaluation of sperm motility.

35

Serum Testosterone

At the time of sacrifice, blood samples werecollected, under carbon dioxide anesthesia, from theorbital sinus of all male rats. Serum levels of testos-terone were analyzed by an enzyme-linked immu-noassay method (Transasia Biomedicals Ltd.,Bombay, India).

Male Fertility Index

After 65 days of treatment period, each male rat inthe control and treated groups was mated (1:1) withan untreated female. Females were cohabitated withthe males till the vaginal smear examination con-firmed the presence of spermatozoa. The femalesthat showed positive vaginal smears were consideredsuccessfully mated and that day was designated day 0of gestation. Females were sacrificied on day 15 ofgestation and their uteruses were examined for con-firmation of pregnancies.

Necropsy and Histopathology

The males were sacrificed at the end of experiment.Their testes and epididymides were removed, exam-ined macroscopically, preserved in 10% neutral buf-

fered formalin, and processed for histopathologicalexamination. The females were sacrificed on day 15of gestation and their uteruses were examined forconfirmation of pregnancies.

Statistical Analysis

The body weight and feed consumption data wereanalyzed using Bartletts test for assessing homoge-neity of groups. In case of homogenous data analysisof variance was performed followed by Dunnettstest. In case of heterogeneity, F test was performedfor individual comparison followed by t test. AP value < .05 was considered statistically significant.

Results

Mortality and Clinical Signs

All treated animals survived throughout the experi-mental period (before mating and during mating)except for 2 males from the 200-mg/kg dose group,

Figure 2. Group mean body weights of male rats treated with A paniculata extract.

312 International Journal of Toxicology / Vol. 28, No. 4, July/August 2009



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which died on day 63 and day 80, and 1 male from1000 mg/kg dose group, which died on day 62 duringthe study period. The treated animals appeared nor-mal and did not show any signs of toxicity during thetreatment period.

Body Weight and Feed Intake

Body weights of the male rats treated at differentdose levels were not affected due to treatment asvalues were found to be comparable with the control

group (Figure 2). Nonsignificant variation in bodyweight gain of the male rats was observed throughoutthe experimental period. Feed consumption was notaffected due to treatment up to or including the doseof 1000 mg/kg (Table 2 and Figure 2).

Semen Analysis

Sperm characteristics such as total sperm count andsperm motility did not show any treatment-related

alterations. Total number of epididymal sperm andpercentage of motile sperm were comparable indifferent treatment groups and the control group(Table 3).

Serum Testosterone

Serum levels of testosterone in treated rats atselected dose levels were found to be unaffected andcomparable with that of control animals (Table 3).

Male Fertility Index

During the period of mating 8 of 8 males were foundfertile in the control group, whereas 7 of 8, 6 of 7,and 7 of 7 males were found fertile in the 20, 200,and 1000 mg/kg groups, respectively. The percentmale fertility index was thus 100 in the controlgroup and 87.50, 85.71, and 100 in the 20, 200, and1000 mg/kg groups, respectively.

Table 2. Group Mean Feed Consumption of Male Rats Given Extract ofA paniculata

Control 20 (mg/kg) 200 (mg/kg) 1000 (mg/kg)

Weeks Average Average Percentage of Control Average Percentage of Control Average Percentage of Control

0 27.51 23.66 86 22.57 82 22.25 811 24.95 24.50 98 26.00 104 24.26 97

2 22.16 22.11 100 25.19 114 21.52 973 22.51 24.03 107 23.16 0 22.59 1004 23.20 20.30 88 21.14 0 20.38 885 20.22 19.56 97 15.70 78 19.80 986 20.93 20.64 99 15.66 75 21.61 1037 20.13 20.15 100 20.51 102 21.34 1068 17.03 18.22 107 18.87 111 17.16 1019 23.30 20.46 88 20.75 89 18.67 8010 20.29 19.55 96 17.24 85 18.08 89Study average 22.02 21.20 97 20.64 94 20.70 95

Values indicate average feed intake per rat per day (g).

Table 3. Group Mean Sperm Count, Motility Percentage, and Serum Testosterone Levels of Male Rats GivenExtract ofA paniculata

Dose Group (mg/kg) Sperm Count per Cauda Epididymis ( 107) Motility (%) Testosterone (ng/mL)

Control(n 8) 7.58 + 0.62 82.25 + 8.48 4.23 + 1.9220(n 8) 6.95 + 1.38 84.25 + 5.91 5.11 + 2.28200(n 6) 8.34 + 0.52 82.00 + 8.76 4.28 + 1.631000(n 7) 9.01 + 1.79 84.00 + 6.89 5.03 + 2.09

Values are expressed as mean + SD.




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Necropsy and Histopathology

No gross pathological changes were observed inmales that died during the study or during the termi-nal sacrifice. Histopathological examination of testesand epididymides of male rats from the controlgroup and those treated with extract at and up to thedose level of 1000 mg/kg did not reveal any signifi-cant and treatment-related alterations (data notshown).

Discussion

A paniculata and its extracts have been studied exten-sively for a wide variety of beneficial effects andincluded in herbal preparations to treat various ail-ments, especially for prevention and treatment of thecommon cold21,22,24,36,37 and liver disorders.7,15,18

The toxic nature of A paniculata is not well-known28

and only little information is available with regard tothe reproductive toxicity of this plant.26-29 To ascertainthe safety of the plant extract in clinical use, a testicu-lar toxicity study was carried out in male Wistar rats.

Absence of treatment-related mortality oradverse clinical signs up to the dose level of1000 mg/kg indicated the relatively harmless natureof the test compound. The toxic effects of test sub-stances are normally analyzed by monitoring altera-tions in the body weight and feed intake.3,4,38,39

When a substance reduces the body weight of ani-mals subjected to treatment by more than 10%, itis considered an adverse effect.4,40 Such a fact, how-ever, has not been observed in the treated groups ofthis study. This finding was in accordance with theprevious study reported by Akbarsha et al.26

In the male reproductive system, reduced spermcount, sperm motility, and reduced testosterone levelare considered standard criteria for the characteriza-tion of toxic agents that may cause fertility problemsin the treated subjects.3,28,41-44 Moreover, satisfac-tory assessment of the toxic potential of an agenton sperm counts requires prolonged treatment peri-ods, and to show such effects on spermatogonialstem cells to be expressed in all evaluations of caudaepididymal or in ejaculated sperm, treatment of adultmales should be continued for a minimum of 6 cyclesof the germinal epithelium, which corresponds toapproximately 80 days in the rat.3 No alterations inthe sperm count, motility, and serum testosteronelevels in this study suggested the normal functioningof Leydig cells. These findings were in accordance

with the previous study wherein ethanolic extractof A paniculata did not show any toxic effect onserum testosterone levels and Leydig cells.28

Nonexistence of any gross or histopathologicalchanges in testes and epididymis of this study werefound to be in agreement with the observations of

Burgos et al.28

However, Akbarsha et al26

reportedantifertility effects of A paniculata based on theregression and disintegration of Leydig cells in malerats after administration of 20 mg of dry powder for60 consecutive days. Contrarily, no pathologicalchanges in Leydig cells were observed in the subse-quent study by the same author, after oral adminis-tration of andrographolide in rats at 25 and 50 mg/kg of body weight for 48 days.27 It is currentlyaccepted that evaluation of testicular toxicity bymicroscopic abnormalities is more subjective thanquantitative,28 and it is very difficult to appreciatemorphological changes in Leydig cells by the usualhistological procedures alone.45

To further authenticate the safety ofA paniculatain the male reproductive system, the treated malerats were allowed to mate with the untreated virginfemales. Fertility of the treated males was unaffectedexcept for 1 male from each of the 20 and 200 mg/kgdose groups, which was not able to impregnate themated females. However, this effect was observedin the absence of significant effects on sperm moti-lity, sperm count, microscopic features of the testes,

and serum testosterone level. Besides, all males fromthe high-dose group (1000 mg/kg) revealed a 100%fertility index. This clearly indicated that infertilityin the 2 males was neither dose dependent nor treat-ment related. Based on the pregnancy rates in thefemales mated with treated males, it is evident that

A paniculata at the treated doses did not inducefunctional infertility in male rats.

As outlined briefly elsewhere in the article, con-flicting results have been reported that are pertinentto the effects of A paniculata on the male reproduc-tive system. The earlier investigation by Akbarshaand coworkers in rats was based on the oral adminis-tration of crude dry leaf powder of A paniculata andattempted to show that A paniculata could poten-tially affect spermatogenesis and produce degenera-tive changes in normal histological characteristicsof reproductive tissues in rats.26 Lack of proper phy-tochemical and analytical specification details of thetest material and also the nonavailability of data oncritical androgenic parameters like sperm count,sperm motility, and serum testosterone levels seem




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to be the major drawbacks of this study comparedwith other similar works. In contrast, detailed ultra-structural evaluations of Leydig cells using electronmicroscopy by Burgos et al28 revealed that driedextract of A paniculata (standardized to 6.11% [wt/wt] andrographolide) at the dose levels of 20, 200,

and 1000 mg/kg for 60 days did not produce anyalterations in number of mitochondria, smoothendoplasmic reticulum, or nucleus in Leydig cellsand thereby did not induce subchronic testiculartoxicity. The HPLC quantification method for andro-grapholide content in A paniculata dried extract usedfor treatment is also described. In addition, the studyconfirmed that there were no significant changes intestosterone levels in treated rats and in concordancewith the results of the present study. Thus, to com-pare the varied results obtained during these safetyassessments, it is noteworthy to refer to the experi-mental conditions such as the nature of investiga-tional substance, experimental conditions, studymethodologies, analysis, and interpretation ofresults.29,46

Considering differences in reports of the toxiceffects of A paniculata in animal models, clinicaltrials can provide more reliable information. A phaseI clinical study revealed that the daily administrationof different doses of fixed combination extract of

A paniculata for 10 days was found to be safe withrespect to effects on human fertility parameters. All

study subjects showed good tolerance to treatment,none of the semen samples revealed critical altera-tions, and no pathological implications werereported in any study subjects.29

Conclusions

Our study on the possible reproductive toxicity ofA paniculata administered to male Wistar rats showedno interference with gamete production and androgensecretion, and fertility was confirmed even after

the treatment period. The no-observed adverse effectlevel of extract ofA paniculata was found to be morethan 1000 mg/kg/day in male albino Wistar rats.

Acknowledgments

The authors declare that there are no conflicts ofinterest. The authors thank Sri. R. K. Agarwal, Chair-man, M/s Natural Remedies Pvt. Ltd., Bangalore,

India, for his encouragement and support in complet-ing this work successfully. Also, the authors thankM/s Shiratori Pharmaceutical Co. Ltd, Chiba, Japan,for partly funding the project.

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