International Journal of Toxicology

http://ijt.sagepub.com/ Reproductive and Fertility Effects of an Extract of Andrographis paniculata in Male Wistar Rats
J. Joshua Allan, Mukul P. Pore, M. Deepak, B. Murali, Anand S. Mayachari and Amit Agarwal International Journal of Toxicology 2009 28: 308 DOI: 10.1177/1091581809339631 The online version of this article can be found at: http://ijt.sagepub.com/content/28/4/308

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Reproductive and Fertility Effects of an Extract of Andrographis paniculata in Male Wistar Rats
J. Joshua Allan, Mukul P. Pore, M. Deepak, B. Murali, Anand S. Mayachari, and Amit Agarwal
The possible effect of extract of Andrographis paniculata Nees (A paniculata) standardized to !10% andrographolide, the main bioactive component, on male fertility in albino Wistar rats was evaluated, by orally administering 0, 20, 200, and 1000 mg/kg of body weight per day, for 65 days prior to mating and 21 days during mating. The treated groups showed no signs of dose-dependent toxicity. The body weight gain and feed consumption were not affected at any of the dose levels. The testosterone levels and fertility indices in treatment groups were found to be comparable with

International Journal of Toxicology Volume 28 Number 4 July/August 2009 308-317 # 2009 The Author(s) 10.1177/1091581809339631 http://ijt.sagepub.com hosted at http://online.sagepub.com

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 and histopathological changes. Based on these findings, it can be concluded that the no-observed adverse effect level of extract of A paniculata ( !10% andrographolide) was found to be more than 1000 mg/kg per day. Keywords: Andrographis paniculata; andrographolide; male fertility; no-observed adverse effect level

erbal remedies, since ancient times, have been used in almost all human races as a source of medicine. Nearly 80% of the worlds population still relies upon medicinal herbs for basic health care needs.1,2 Although they are often considered as natural and relatively harmless, phyto preparations are not always free from toxicity.3 Herbal products have been used frequently without proper evaluation of their actual efficacy in the treatment or the occurrence of undesirable side effects. Currently little information is available on the reproductive effects of popular medicinal plants. There has been growing interest over the safety of natural products in clinical use and evaluation of effects on

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, Bangalore 560 100, India; e-mail: joshua@naturalremedy.com.

reproductive system has been considered as part of the safety studies of widely used medicinal plants.4 Andrographis paniculata, commonly known as Kalmegh or green chiretta and belonging to family Acanthaceae, is an annual herb native to peninsular India and Sri Lanka and is also distributed in different regions of Southeast Asia, China, America, West Indies, and Christmas Island.5 All parts of this herb are extremely bitter in taste and are considered medicinally important in the traditional system of Indian medicine for treatment of various illnesses. The plant extracts are found to have antiviral, antipyretic, antiulcer, hepatoprotective, antiallergic, antiplatelet aggregation, antidiabetic, anticancer, and antimalarial activities.6-17 Two diterpenes isolated from this plant, andrographolide and neoandrographolide, have been shown to possess promising immunomodulatory activity.18 It has recently been reported that andrographolide exerts antiallergic activity by significantly decreasing degranulation of mast cells with consequent reduction in the liberation of histamine.8 A paniculata exerts its

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anti-inflammatory effect by inhibiting reactive oxygen production in neutrophils and lipopolysaccharideinduced nitric oxide production in macrophages.19,20 Several randomized placebo-controlled clinical trials conducted with A paniculata extract standardized to andrographolide and deoxyandrographolide showed its preventive effects against the common cold and the capacity to significantly shorten the duration of the disease.21-25 Despite diverse studies on the therapeutic effectiveness of A paniculata, only a few studies on the toxicological assessment of this medicinal plant have been reported. A preliminary study reported that oral administration of 20 mg of herb powder of A paniculata to male albino rats for 60 days resulted in cessation of spermatogenesis and regression of Leydig cells suggesting the antiandrogenic effect of this plant.26 Andrographolide, administered to male rats at 2 dose levels of 25 and 50 mg/kg of body weight for 48 days, showed testicular toxicity and sperm abnormalities.27 On the other hand, extract of A paniculata containing 6.1% of andrographolide did not produce testicular toxicity in male rats when administered at the doses of 20, 200, and 1000 mg/kg, for 60 days.28 A phase I clinical study of A paniculata fixed combination showed no adverse effects on male semen quality and fertility in healthy human subjects.29 Considering the facts that the plant has been incorporated as an active ingredient in 26 of the 40 polyherbal formulations available for liver disorders,30 and the potential of andrographolide to cause testicular toxicity is still unclear, the present study was undertaken to assess the possible testicular toxicity of extract of A paniculata treated for a prolonged period in albino Wistar rats.

husk. The temperature in the animal room was maintained between 19 C and 25 C with 10 to 15 air changes per hour, a relative humidity of 30% to 70%, and illumination cycle set to 12 h of light and 12 h of dark. Standard certified pelleted diet (Amrut brand; M/s Nav. Maharashtra Chakan Oil Mills Ltd., Pune, India) and filtered water were provided ad libitum.

Preparation of Extract
The raw material (whole plant, the majority of which was leaves of A paniculata; 400 kg) was procured from a local commercial supplier and was authenticated at the National Institute of Science Communication and Information Resources (NISCAIR), New Delhi. It was extracted with ethanol by refluxing at 60 C to 65 C (3 times each with 1200 L of ethanol for 3 h) in the manufacturing facility of M/s Natural Remedies, Bangalore, India. The liquid extract was combined and concentrated by distillation under vacuum to a thick paste (28 kg, extract I). The marc left after ethanol extraction was further refluxed with water at 85 C to 90 C (2 times each with 1200 L water for 3 h). The liquid extract was combined and concentrated by distillation under vacuum to a thick paste (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 paniculata was partitioned between ethyl acetate and water. The ethyl acetate layer was subjected to normal phase silica gel column chromatography to furnish 5 fractions. Fractions 1-5 were purified by reverse phase column chromatography followed by preparative HPLC to obtain andrographolide (1), neoandrographolide (2), andrograpanin (3), and 14-deoxy-11, 12-didehydroandrographolide (4). The isolated compounds were characterized by comparing their 1H and 13C NMR data with literature.33,34 These compounds were used as reference standards for HPLC analysis. Andrographolide from Sigma Aldrich was also used.

Methods
This study was conducted in accordance with the 1993 International Conference on Harmonisation Harmonised Tripartite Guideline31 and in compliance with the Organisation for Economic Cooperation and Development Principles of Good Laboratory Practice.32

Animals
Adult Wistar rats (12-14 weeks old) used in this study were bred and reared at Intox, Pune, India. The animals were housed in polypropylene cages with stainless steel grill tops and bedding of clean paddy

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Table 1.
Validation Parameter Specificity Linearity

Summary of Analytical Method Validation

Result

Very specific for andrographolide with no interference from other compounds. Confirmed by the PDA spectra and the peak purity. 5 concentrations of each, 5 replicates of each 1: correlation coefficient 0.996 2-4: correlation coefficient 1.000 1: 98.8-102.4% 2: 97.9-103.5% 3: 97.8-100.2% 4: 94.0-101.4%

Accuracy by spike recovery method

Precision (of concentration, retention time 5 concentrations of each, 5 replicates of each and reproducibility) 1: Range of quantification 60-500 mg/mL RSD% for retention time <0.05% RSD for peak area: <2.5% 2-4: Range of quantification 25-400 mg/mL RSD% for retention time <0.05% RSD for peak area: <2.5% Robustness Stability of sample and standard solutions 4 different sample concentrations, 2 different HPLC columns, 2 different analysts Actual RSD% 2.3% 2 batches tested after making solution at T 0 and T 12 h.

PDA, photo diode array; HPLC, high performance liquid chromatography; RSD, relative standard deviation.

HPLC Analysis
The extract was analyzed for the content of 4 andrographolides by HPLC. A known volume (20 mL) of the mixed standard and sample solutions were injected to the HPLC system (Shimadzu, Model LC 2010 A, Tokyo, Japan) consisting of quaternary pump with UV detector, auto injector, and column oven with class VP software (Shimadzu, Japan). The stationary phase was an octadecylsilane column (C18, 5 mm, 250 4.6 mm; Hibar RT, Lichrosphere 100; Merck, Darmstadt, Germany). The mobile phase consisted of a mix of phosphate buffer (solvent A; prepared by dissolving 0.136 g of potassium dihydrogen orthophosphate [KH2PO4] in 900 mL of HPLC grade water [obtained from Arium Sartorius water purification system] and by adding 0.5 mL of orthophosphoric acid [H3PO4; AR grade, Rankem, New Delhi, India] and making the volume to 1000 mL) and acetonitrile (solvent B; HPLC grade, Qualigens, Mumbai, India). Both acetonitrile and phosphate buffer were filtered separately through 0.45mm membrane filter and degassed by sonicating for 3 min. The solvents A and B were mixed in such a manner that the concentration of solvent B was increased from 5% to 45% as a linear gradient in the first 18 min. From 18 to 25 min the concentration of solvent B was increased from 45% to 80% as a linear

gradient. The flow rate of mobile phase was maintained at 1.5 mL per min throughout the analysis and the detector wave length was kept at 223 nm, and the chromatogram was recorded. Quantification of 1-4 was achieved by the external standard method. The peaks in the mixed standards and in the sample were identified by injecting the individual standard solutions.

Standard and Sample Solution Preparation

Solutions of standards 1-4 were prepared by weighing 10 mg of the individual standards and dissolving them separately in 10 mL with methanol (HPLC grade, Qualigens). The stock solution was suitably diluted 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 using the external standard method and was found to be 10.9% (wt/wt). The other compounds identified in

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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 gradient program 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 to 28 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.

the extract (2-4) constituted 2.7% (wt/wt) of the extract. The profiles of the extract of A paniculata analyzed by HPLC and the structural formulae of the components of the extract are shown in Figure 1A and B.

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

Total Sperm Count

Sperm count was performed as described by Seed et al.35 In brief, one of the epididymides was used for sperm count. The cauda epididymis was incised and homogenized in a homogenizer glass vial containing 30 mL of 0.01% (wt/vol) Triton X-100 in 0.9% NaCl for approximately 15 to 20 min. The homogenate probe was rinsed with distilled water and the washing was collected along with the homogenate. The final volume of homogenate was made up to 50 mL using distilled water and then sonicated for 15 to 20 min, and 0.5 mL of 1% eosin Y stain solution was finally added to it. Sperm counts were made in a hemocytometer and recorded.

Experimental Design
Thirty-two adult male albino Wistar rats were divided into 4 groups of 8 animals each. The control group received vehicle (water; 10 mL/kg) and the remaining groups were administered with extract of A paniculata at the dose levels of 20, 200, and 1000 mg/kg of body weight by oral gavage once daily for a period of 65 days prior to mating and 21 days during mating. The doses were prepared freshly every day before administration to animals.

Mortality and Clinical Signs

The treated males were observed daily for mortality and clinical signs throughout the experiment. Any death or abnormal clinical findings were recorded.

Sperm Motility
The sperm from vas deferens were collected and observed under the microscope to determine the percentage of motile sperm. Immediately after sacrifice, one vas deferens was isolated and excised from the testis. The vas deferens was cut into small pieces and placed in a petri dish containing 3 to 5 mL of phosphate-buffered saline maintained at 35 C to 37 C. The sperm suspension was transferred to a test tube placed in a water bath maintained at 35 C to

Body Weight and Feed Intake

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

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Figure 2.

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

37 C and then incubated for 5 to 10 min. Approximately 10 mL of the suspension was transferred to a microscope slide with concave chambers kept on a slide warmer maintained at 35 C to 37 C and roofed with a coverslip for evaluation of sperm motility.35

Necropsy and Histopathology

The males were sacrificed at the end of experiment. Their testes and epididymides were removed, examined macroscopically, preserved in 10% neutral buffered formalin, and processed for histopathological examination. The females were sacrificed on day 15 of gestation and their uteruses were examined for confirmation of pregnancies.

Serum Testosterone
At the time of sacrifice, blood samples were collected, under carbon dioxide anesthesia, from the orbital sinus of all male rats. Serum levels of testosterone were analyzed by an enzyme-linked immunoassay method (Transasia Biomedicals Ltd., Bombay, India).

Statistical Analysis
The body weight and feed consumption data were analyzed using Bartletts test for assessing homogeneity of groups. In case of homogenous data analysis of variance was performed followed by Dunnetts test. In case of heterogeneity, F test was performed for individual comparison followed by t test. A P value < .05 was considered statistically significant.

Male Fertility Index

After 65 days of treatment period, each male rat in the control and treated groups was mated (1:1) with an untreated female. Females were cohabitated with the males till the vaginal smear examination confirmed the presence of spermatozoa. The females that showed positive vaginal smears were considered successfully mated and that day was designated day 0 of gestation. Females were sacrificied on day 15 of gestation and their uteruses were examined for confirmation of pregnancies.

Results
Mortality and Clinical Signs
All treated animals survived throughout the experimental period (before mating and during mating) except for 2 males from the 200-mg/kg dose group,

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Table 2. Group Mean Feed Consumption of Male Rats Given Extract of A paniculata
Control Weeks 0 1 2 3 4 5 6 7 8 9 10 Study average Average 27.51 24.95 22.16 22.51 23.20 20.22 20.93 20.13 17.03 23.30 20.29 22.02 Average 23.66 24.50 22.11 24.03 20.30 19.56 20.64 20.15 18.22 20.46 19.55 21.20 20 (mg/kg) Percentage of Control 86 98 100 107 88 97 99 100 107 88 96 97 Average 22.57 26.00 25.19 23.16 21.14 15.70 15.66 20.51 18.87 20.75 17.24 20.64 200 (mg/kg) Percentage of Control 82 104 114 0 0 78 75 102 111 89 85 94 Average 22.25 24.26 21.52 22.59 20.38 19.80 21.61 21.34 17.16 18.67 18.08 20.70 1000 (mg/kg) Percentage of Control 81 97 97 100 88 98 103 106 101 80 89 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 Given Extract of A paniculata
Dose Group (mg/kg) Control(n 8) 20(n 8) 200(n 6) 1000(n 7) Values are expressed as mean + SD. Sperm Count per Cauda Epididymis ( 107) 7.58 6.95 8.34 9.01 + + + + 0.62 1.38 0.52 1.79 Motility (%) 82.25 + 8.48 84.25 + 5.91 82.00 + 8.76 84.00 + 6.89 Testosterone (ng/mL) 4.23 5.11 4.28 5.03 + 1.92 + 2.28 + 1.63 + 2.09

which died on day 63 and day 80, and 1 male from 1000 mg/kg dose group, which died on day 62 during the study period. The treated animals appeared normal and did not show any signs of toxicity during the treatment period.

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

Body Weight and Feed Intake

Body weights of the male rats treated at different dose levels were not affected due to treatment as values were found to be comparable with the control group (Figure 2). Nonsignificant variation in body weight gain of the male rats was observed throughout the experimental period. Feed consumption was not affected due to treatment up to or including the dose of 1000 mg/kg (Table 2 and Figure 2).

Serum Testosterone
Serum levels of testosterone in treated rats at selected dose levels were found to be unaffected and comparable with that of control animals (Table 3).

Male Fertility Index

During the period of mating 8 of 8 males were found fertile 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 percent male fertility index was thus 100 in the control group and 87.50, 85.71, and 100 in the 20, 200, and 1000 mg/kg groups, respectively.

Semen Analysis
Sperm characteristics such as total sperm count and sperm motility did not show any treatment-related

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Necropsy and Histopathology

No gross pathological changes were observed in males that died during the study or during the terminal sacrifice. Histopathological examination of testes and epididymides of male rats from the control group and those treated with extract at and up to the dose level of 1000 mg/kg did not reveal any significant and treatment-related alterations (data not shown).

Discussion
A paniculata and its extracts have been studied extensively for a wide variety of beneficial effects and included in herbal preparations to treat various ailments, especially for prevention and treatment of the common 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 to the reproductive toxicity of this plant.26-29 To ascertain the safety of the plant extract in clinical use, a testicular toxicity study was carried out in male Wistar rats. Absence of treatment-related mortality or adverse clinical signs up to the dose level of 1000 mg/kg indicated the relatively harmless nature of the test compound. The toxic effects of test substances are normally analyzed by monitoring alterations in the body weight and feed intake.3,4,38,39 When a substance reduces the body weight of animals subjected to treatment by more than 10%, it is considered an adverse effect.4,40 Such a fact, however, has not been observed in the treated groups of this study. This finding was in accordance with the previous study reported by Akbarsha et al.26 In the male reproductive system, reduced sperm count, sperm motility, and reduced testosterone level are considered standard criteria for the characterization of toxic agents that may cause fertility problems in the treated subjects.3,28,41-44 Moreover, satisfactory assessment of the toxic potential of an agent on sperm counts requires prolonged treatment periods, and to show such effects on spermatogonial stem cells to be expressed in all evaluations of cauda epididymal or in ejaculated sperm, treatment of adult males should be continued for a minimum of 6 cycles of the germinal epithelium, which corresponds to approximately 80 days in the rat.3 No alterations in the sperm count, motility, and serum testosterone levels in this study suggested the normal functioning of Leydig cells. These findings were in accordance

with the previous study wherein ethanolic extract of A paniculata did not show any toxic effect on serum testosterone levels and Leydig cells.28 Nonexistence of any gross or histopathological changes in testes and epididymis of this study were found to be in agreement with the observations of Burgos et al.28 However, Akbarsha et al26 reported antifertility effects of A paniculata based on the regression and disintegration of Leydig cells in male rats after administration of 20 mg of dry powder for 60 consecutive days. Contrarily, no pathological changes in Leydig cells were observed in the subsequent study by the same author, after oral administration of andrographolide in rats at 25 and 50 mg/ kg of body weight for 48 days.27 It is currently accepted that evaluation of testicular toxicity by microscopic abnormalities is more subjective than quantitative,28 and it is very difficult to appreciate morphological changes in Leydig cells by the usual histological procedures alone.45 To further authenticate the safety of A paniculata in the male reproductive system, the treated male rats were allowed to mate with the untreated virgin females. Fertility of the treated males was unaffected except for 1 male from each of the 20 and 200 mg/kg dose groups, which was not able to impregnate the mated females. However, this effect was observed in the absence of significant effects on sperm motility, sperm count, microscopic features of the testes, and serum testosterone level. Besides, all males from the high-dose group (1000 mg/kg) revealed a 100% fertility index. This clearly indicated that infertility in the 2 males was neither dose dependent nor treatment related. Based on the pregnancy rates in the females mated with treated males, it is evident that A paniculata at the treated doses did not induce functional infertility in male rats. As outlined briefly elsewhere in the article, conflicting results have been reported that are pertinent to the effects of A paniculata on the male reproductive system. The earlier investigation by Akbarsha and coworkers in rats was based on the oral administration of crude dry leaf powder of A paniculata and attempted to show that A paniculata could potentially affect spermatogenesis and produce degenerative changes in normal histological characteristics of reproductive tissues in rats.26 Lack of proper phytochemical and analytical specification details of the test material and also the nonavailability of data on critical androgenic parameters like sperm count, sperm motility, and serum testosterone levels seem

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to be the major drawbacks of this study compared with other similar works. In contrast, detailed ultrastructural evaluations of Leydig cells using electron microscopy by Burgos et al28 revealed that dried extract 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 any alterations in number of mitochondria, smooth endoplasmic reticulum, or nucleus in Leydig cells and thereby did not induce subchronic testicular toxicity. The HPLC quantification method for andrographolide content in A paniculata dried extract used for treatment is also described. In addition, the study confirmed that there were no significant changes in testosterone levels in treated rats and in concordance with the results of the present study. Thus, to compare the varied results obtained during these safety assessments, it is noteworthy to refer to the experimental conditions such as the nature of investigational substance, experimental conditions, study methodologies, analysis, and interpretation of results.29,46 Considering differences in reports of the toxic effects of A paniculata in animal models, clinical trials can provide more reliable information. A phase I clinical study revealed that the daily administration of different doses of fixed combination extract of A paniculata for 10 days was found to be safe with respect to effects on human fertility parameters. All study subjects showed good tolerance to treatment, none of the semen samples revealed critical alterations, and no pathological implications were reported in any study subjects.29

India, for his encouragement and support in completing this work successfully. Also, the authors thank M/s Shiratori Pharmaceutical Co. Ltd, Chiba, Japan, for partly funding the project.

References
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Conclusions
Our study on the possible reproductive toxicity of A paniculata administered to male Wistar rats showed no interference with gamete production and androgen secretion, and fertility was confirmed even after the treatment period. The no-observed adverse effect level of extract of A paniculata was found to be more than 1000 mg/kg/day in male albino Wistar rats.

Acknowledgments
The authors declare that there are no conflicts of interest. The authors thank Sri. R. K. Agarwal, Chairman, M/s Natural Remedies Pvt. Ltd., Bangalore,

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