Prophylactic efficacy of Boerhavia diffusa L. aqueous extract in toluene induced reproductive and developmental toxicity in Drosophila melanogaster

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Prophylactic efficacy of Boerhavia diffusa L. aqueous extract in toluene induced reproductive and developmental toxicity in Drosophila melanogaster Benil PB a , Sreeja Rani a , Young Ock Kim b , Abdullah Ahmed Al-Ghamdi c , Mohamed S. Elshikh c , Monerah A. Al-Dosary c , Ashraf A. Hatamleh c , Selvaraj Arokiyaraj d,∗ , Hak-Jae Kim e,∗ a

Department of Agadatantra, Vaidyaratnam P.S. Varier Ayurveda College, Kottakkal, Edarikode P.O, Malappuram, Kerala, India Department of Bio-Environmental Chemistry, College of Agriculture and Life Sciences, Chungnam National University, 99 Daehak-Ro, Yuseung-Gu, Daejeon 34134, Republic of Korea c Department of Botany and Microbiology, College of Sciences, King Saud University, P.O. Box 22452, Riyadh 11495, Saudi Arabia d Department of Food Science & Biotechnology, Sejong University, Seoul 05006, Republic of Korea e Department of Clinical Pharmacology, College of Medicine, Soonchunhyang University, Cheonan, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 15 May 2019 Received in revised form 17 July 2019 Accepted 22 July 2019 Keywords: Developmental toxicity Reproductive toxicity Toluene Boerhavia diffusa Antioxidant

a b s t r a c t Background: Environmental exposure to toxicants poses high risk to develop reproductive and developmental chronic toxicity in man. Toluene is one of the commonest industrial agents whose exposure is attributed with potential to induce reproductive and developmental toxicity. Since they contaminate the immediate environment of air and water to which humans are exposed, its containment is of great public health importance. Conventional treatment modalities fail owing to the difficulty to detect these highly volatile agents in environment and human body. The peril of such hazardous exposures is evident only when irreversible structural and functional damages have incurred. In such instances, prevention gains an upper hand when compared to therapeutic interventions. Several natural compounds derived from medicinal herbs possess potential to curb toxicities induced by such xenobiotic agents. Among them Boerhavia diffusa Linn. is a widely distributed and common herb attributed with antitoxic potential and capability for antioxidant defence. A study was performed on the prophylactic efficacy of aqueous extract of B. diffusa in curbing toluene induced developmental toxicity in Drosophila melanogaster. Methods: The study consisted of a preliminary phytochemical screening and HPTLC profiling of B. diffusa aqueous extract (BDAE). LC50 of toluene was assessed and a sublethal dose of 200 ppm was fixed for the study. Four doses of BDAE; 25, 50, 100 and 200 mg/ml designated as Low dose, medium dose 1, medium dose 2 and high dose was used for the study. The parameters used for the study included the determination of larval period, pupal period, percentage of egg hatching, morphometric analysis of egg, larvae, pupae and adults, fertility, fecundity, lifespan and levels of antioxidant enzymes such as catalase, glutathione-S-transferase and superoxide dismutase. Results: The phytochemical and HPTLC characters were as per the pharmacopoeial standards. LC50 of toluene was found to be 430 ppm in this study. BDAE at medium dose 2 and high dose significantly prevented the deterioration of reproductive and developmental toxicity parameters of larval period, pupal period, percentage of egg hatching, morphometric characters of larva, pupa and adult, fertility, fecundity and lifespan in drosophila. Also the drug significantly elevated the levels of antioxidant enzymes. Conclusion: Toluene exposure during lifetime is inevitable. B. diffusa, equipped with its rich active ingredients prevented toluene induced developmental and reproductive toxicity in Drosophila. This medicinal herb provides a ray of hope in preventing environmental toxin induced reproductive and developmental toxicity. © 2019 Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).

∗ Corresponding authors. E-mail addresses: [email protected] (S. Arokiyaraj), [email protected], [email protected] (H.-J. Kim). https://doi.org/10.1016/j.jiph.2019.07.020 1876-0341/© 2019 Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Introduction The factors favouring the reproductive and developmental health begins from the life in utero and is determined by the interactions of the social, physical and nutritional environments and other physical and chemical agents [1,2]. The indicators of reproductive adversity are currently attributed to factors like decline in age of onset of puberty, decline in fertility and fecundity, increased rates of poor birth outcomes like prematurely born or small for gestational age or born with birth defects, increased rates of childhood diseases such as autism, certain types of cancer and obesity and declines in life expectancy etc. [3]. In reproductive health, the preconception and prenatal exposure to environmental chemicals is a key area of inquiry because exposure to dangerous chemicals during pregnancy is a norm in almost all countries [4]. Exposure to environmental chemicals during developmental stage is linked to a myriad of health consequences that can manifest across lifetime of an individual and can potentially be transmitted to the next generation [5]. Further, preconception and prenatal exposure to environmental chemicals can be prevented to a greater extend. The important strategies identified for prevention of toxic exposures are manifold. The primary level of prevention is through education aimed at individual or communities. Risk communication, operational control, monitoring, Legislation, product registration and licencing are also considered under primary prevention [6]. These measures however fall short of their aim in many instances. Several researches over the past decades show that the pathology of chronic diseases (referred to as “diseases of civilization”) is regulated by multifactorial elements which includes, apart from environmental agents; nutrition and disease risk [7]. Environmental pollutants bioaccumulate in our body and induce signalling pathways that are associated with the pathogenesis of many chronic diseases [8].The need of the hour is to develop strategies by which the effect of toxicants on the pathogenesis of chronic disease etiology and progression can be minimized and is a matter of public health importance. Data pooled from epidemiologic, basic science and clinical research proves that diet and nutrition along with lifestyle changes can modify pathologies of diseases associated with environmental toxic insults [9]. Environmental exposure biomarkers are desirable to quantify individual level exposure and monitor changes in exposure over time. The major groups of chemicals assigned to have reproductive and developmental toxicity are silica, asbestos, metals, pesticides and persistent organic pollutants, solvents, smoking, hair dyes and cosmetic products etc. [10]. Among these, solvents and hydrocarbons form a major group because their toxicity cannot be ascertained from biomarkers. Toluene forms an indispensable part of natural hydrocarbons and is a raw material and by-product of several industrial processes and is also a solvent in many products of daily human contact [11]. Increased incidences of urogenital, gastrointestinal and cardiac anomalies among offspring of toluene exposed mothers were indicated by a case-referent study of women with occupational exposures to organic solvents [12]. A number of experimental studies investigating the reproductive and developmental toxicity of toluene have been conducted using rats, mice and rabbits [13]. These studies provide convincing evidence that exposure to toluene during gestation causes fetotoxicity. Societies around the world revered medicinal plants by giving them a unique place in culture, medicinal practices and nutrition. Natural products are diverse in having multi-dimensional chemical structures which received considerable attention as biological function modifiers [14]. Countless active principles have been separated from natural products. Use of medicinal plants in human diseases is multipronged, as they provide nutritional as well as therapeutic benefits from the presence of large array of secondary metabolites. For the same reason they have been uti-

lized for chemoprevention and as prophylactics in toxicities. The plethora of herbs and spices mentioned in traditional medical systems like Ayurveda holds an extra advantage due to its time-tested formulations. Boerhavia diffusa, belonging to the family Nyctaginaceae is a wildly growing herb both in tropical and sub-tropical areas ranging from grasslands, wastelands, agroecosystems and forest gaps. It grows vigorously as weeds in countries like India, Pakistan, Saudi Arabia and other countries in Middle East, Africa, South America, Australia etc. Throughout classical Ayurveda texts it is attributed with properties like bitter, astringent, cooling, anthelminthic, diuretic, aphrodisiac, cardiac stimulant, diaphoretic, emetic, expectorant, anti-inflammatory, febrifuge, laxative and a tonic [15,16]. Besides it is widely used in formulations that are antitoxic and meant torejuvenate. B. diffusa is an extensively studied herb and its diuretic, hepatoprotective, anti-inflammatory, anti-fibrinolytic, anti-cancer, anti-diabetic, immune-modulatory, immune-suppressive, anti-lymphoproliferative, analgesic activities has been studied and reported [17]. Owing to the presence of wide range of activities, the present study was designed to test the prophylactic efficacy of B. diffusa aqueous extract (BDAE) in ameliorating toluene induced reproductive and developmental toxicity and oxidative stress in Drosophila melanogaster. Aqueous extract was utilized for the study owing to the fact that general pharmaceutical preparation used in traditional medicines is in the form of decoctions. Drosophila was selected as the model organism owing to its importance in toxicity studies. Further, drosophila having a shorter life span and large number of progenies is an ideal model for studying developmental and reproductive toxicities.

Materials and methods Chemicals and reagents Toluene (Sigma Aldrich, USA), Fehling’s reagent, Molisch’s reagent, Ninhydrin reagent, Mayer’s reagent, Wagner’s reagent, Hager’s reagent, Dragendorff’s reagent, Froehde’s reagent, Marquis reagent, TrisHCl, Catalase Assay Kit (Cayman Chemical, USA), Glutathione-S-Transferase (GST) Assay Kit (Sigma-Aldrich, USA), Superoxide Dismutase Assay Kit (Cayman Chemical, USA).

Collection of plant Fresh plants of B. diffusa was collected from Kottakkal and were authenticated by Centre for Medicinal Plants Research, Arya Vaidya Sala Kottakkal. A voucher specimen of the plant was deposited for future reference (Nyct/09/2018). The plants were cleaned and washed to remove all foreign matter and the roots were collected and made into pieces and dried under shade.

Extraction of the plants The air-dried roots of the plants were powdered and sieved through a mesh of size 40. About 25 gm of the root powder was extracted with 500 ml of distilled water in a rotary shaker for eight hours and filtered through a Whatman filter paper of size 40. The extract was concentrated at reduced temperature and vacuum in a flash evaporator. The dried extract was stored in air tight glass containers in freezer till the beginning of the study.

Phytochemical screening The aqueous extract obtained was subjected to phytochemical screening as per the standard protocols [18].

Please cite this article in press as: PB B, et al. Prophylactic efficacy of Boerhavia diffusa L. aqueous extract in toluene induced reproductive and developmental toxicity in Drosophila melanogaster. J Infect Public Health (2019), https://doi.org/10.1016/j.jiph.2019.07.020

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HPTLC profiling One gram of B. diffusa sample was weighed, powdered and extracted with 10 ml Methanol, and spotted as 10 ␮L. Stationary phase was pre-coated TLC Silica gel 60 F254, 10 × 10 cm Aluminium sheet (Merck). Mobile phase was fixed as Toluene: Ethyl acetate: Formic acid: Methanol in the ratio 7:5:1:0.5. The plates were developed in Camag 10 × 10 cm twin trough chamber. The HPTLC instrumentation used were Camag Linomat 5, Camag TLC Scanner and Camag Reprostar 3. The developed plates were derivatized in 10% sulphuric acid reagent. Experimental organism Wild strains (Oregon) of D. melanogaster was purchased from Drosophila stock centre, university of Mysore, Manasagangotri and was sub-cultured 10 times into new wheat flour agar medium and maintained till the beginning of the study. Dosage of drugs The aqueous extract of B. diffusa was used in four doses for the study. The fixed doses were 25, 50, 100 and 200 mg per ml respectively. They were designated as low dose (LD), medium dose1 (MD1), medium dose 2 (MD2), and high dose (HD) respectively. Preparation of wheat flour agar medium One litre of distilled water was boiled in a glass beaker and hundred grams of jaggery was added and dissolved well. 100gm of wheat flour was added slowly with thorough mixing to avoid formation of lumps. After the mixture was boiled and attained a semi liquid consistency 10 gm of agar was added and stirred well till it melted properly. The mixture was allowed to cool for some time and 7.5 ml of propionic acid was added as an antifungal agent and mixed well. The prepared culture medium was then poured into sterile culture bottle to a height of one inch and allowed to cool to room temperature in a sterile laminar hood. The bottles were plugged with sterile cotton plugs and stored till the transfer of new flies. New media was prepared every third day and the fly culture was transferred regularly to prevent contamination and to promote optimal growth. The culture media containing toluene and drug extracts were prepared separately and layered over the standard medium before the transfer of the flies [19].

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200 mg/ml) separately were prepared and five pairs of three-day old flies were transferred to each bottle. The flies were allowed to exit the bottles after allowing them to lay eggs for a period of eight hours. The bottles were then incubated at 25 ◦ C till the eggs hatched and larvae emerged and waited till they reached the third instar. Larvae from each bottle were collected carefully by washing with tap water through a fine meshed stainless-steel strainer and counted. The larvae were then transferred to fresh media containing exactly the same concentrations and again incubated till they pupated. The number of pupae appearing on the surface and next to the medium was counted. The bottles were again incubated till flies eclosed from the pupae. The number of adult flies emerged were counted by shaking them into flasks containing 70% ethanol. The percentage of larvae, pupae and adult flies emerged were calculated [22]. Duration of larval and pupal period The average time in hours required for the emergence of first larvae, pre-pupae and the adult per each concentration was recorded form the above experiment and the average length of larval period and pupal period were calculated [22]. Percentage of hatched eggs Newly eclosed flies who spent their larval period in respective media containing toluene and BDAE were taken and five pairs of them were transferred to fresh media devoid of toluene and allowed to mate and lay eggs for eight hours. The adult flies were then allowed to exit the bottles and the number of eggs laid was counted under a stereomicroscope. The bottles were then incubated at 25 ◦ C till the eggs hatched and the larvae reached the third instar stage. They were counted and the difference of eggs laid and larvae emerged was calculated and from this the percentage of eggs hatched was calculated from the pooled data of five replicates [22]. Morphometric analysis of eggs Eggs were collected from the flies grown on normal medium(control), toluene alone containing medium, toluene along BDAE at various doses were taken and the length and width of the eggs were measured micrometrically for two generations and the data were combined [22].

Determination of LC50

Morphometric variation in larvae, pupae and adults

Wild Oregon strains of D. melanogaster flies were grown on standard wheat cream agar medium maintained at 25 ◦ C and 65% humidity. Synchronized egg collection was done as per a standard method [20]. Eggs were transferred to fresh medium and incubated till they hatched and larvae emerged. As the larvae attained 72 h growth, they were washed with tap water carefully several times and collected through a fine meshed stainless-steel strainer. Small vials containing approximately 0.5 gm of wheat cream agar containing graded doses of toluene at 0, 0.625, 1.25, 2.5, 5.0 and 10 mM were taken and ten larvae each were transferred and incubated at 25 ◦ C and the number of flies emerged from each bottle after 10–12 days were counted after shaking them into a flask containing 70% ethanol. Five replications of each concentration were made and the data was pooled to calculate the LC50 by logistic regression [21].

Identical media containing the various doses BDAE were prepared in flat bottomed flasks of 100 ml capacity. Twenty pairs of adult flies were introduced into each bottle and were allowed to mate and lay eggs for eight hours. The flies were allowed to exit the bottles and the bottles were incubated till the larvae emerged. Seven larvae after the first moulting were sampled from each bottle and their length and breadth were estimated micrometrically. Sampling was repeated every 12 h for eight more times after which they pupate. The length and breadth of the pupae are similarly measured and few pupae are allowed to eclose to sample adults to measure their chest width after three days [23].

Determination of pupation and maturity percentage Five replicates of standard wheat cream agar medium containing 200 mM toluene and four doses of BDAE (25, 50,100 and

Determination of fitness Synchronized egg collection was performed as per the method described earlier. Eggs were collected at four-hour intervals and were equally distributed in bottles containing standard wheat cream agar medium containing various doses of BDAE as per the experiment protocol. Three components were measured to assess

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fitness: Dynamics of eclosion, developmental time and egg-toadult survival. Dynamics of eclosion studied the percentage of flies emerged per day and the developmental time was calculated by the formula Dt = (nd × d)/nd , where nd is the number of adults emerged per day, and d is day of hatching. Egg-to-adult survival is expressed as the ratio of adult flies emerged to the number of eggs placed in each bottle [24]. Determination of fertility and fecundity The flies were allowed to grow in their respective medium for five generations. The fifth-generation adults were collected and a pair of male and female flies was transferred to vials containing fresh wheat cream agar medium in five replicates. The male flies after 48 h of mating were allowed to exit and the female flies were transferred to fresh vials in every 24 h and the average number of eggs laid per female per day was calculated to determine the fecundity. The vials were incubated at 25 ◦ C till the larvae pupated and eclosed to emerge adult flies. The number of flies was quantified by capturing them into 70% ethanol. Fertility was determined as the average number of adult flies emerged from each pair of flies [25]. Determination of lifespan For assessing lifespan, thirty flies were taken in a vialcontaining fresh wheat cream agar medium with five replicates for each concentration. Every second day the flies were transferred to new vials containing fresh media. Mortality per day was noted in each vial and survival time for each group was calculated [26]. Determination of antioxidant enzymes Assays of antioxidant enzymes were performed on homogenate of twenty flies in 200ul of Tris buffer (20 mM, pH 7.0) and subsequent centrifugation at 20,000 g for 5 min at 4 ◦ C. This supernatant was used to measure levels of catalase (CAT), Glutathione-Stransferase (GST) and superoxide dismutase (SOD) [27–29].Total protein content was also assessed following the Bradford method [30].

Table 1 Phytochemical screening of Boerhavia diffusa aqueous extract. Test Carbohydrates Fehling’s test Molish’s test Flavonoids Lead acetate test Shinoda’s test Alkaline Reagent test Protein Xanthoproteic test Biuret test Amino acids Ninhydrin test Phenols Ferric chloride test Phytosterols Salkowski’s test Liberman-Burchard’s test Saponins Froth test Foam test Fixed oils & fats Stain test Resins Acetone water test Tannins Braemer’s test Glycosides Modified Borntrager’s test Legal’s test Keller-Killiani test Alkaloids Mayer’s test Wagner’s test Dragendroff’s test Hager’s test Froehde’s test Marquis test

Reaction + + + + + + + + + + + + + + + + + + + + + + + + +

Statistical analysis All the observations were tabulated as mean + standard deviation. The statistical evaluation of data was done by one-way ANOVA followed by Dunnett’s multiple comparison test and two-way ANOVA with Tukey’s multiple comparison test. The significance of longevity was tested between groups using Kruskal-Wallis test. Five percent was taken as the level of significance for all tests. LC50 was determined using logistic regression. Results Phytochemical screening and HPTLC profiling The results of the qualitative estimation of BDAE are tabulated in Table 1. The HPTLC fingerprint of B. diffusa showed 12 peaks at 254 nm while in the longer wavelength of 366 nm it produced 10 peaks. Determination of LC50 of toluene in D. melanogaster and determination of pupation and maturity percentage The LC50 of toluene in D. melanogaster was found to be 430 ppm. The influence of environmental toxicants prolongs the developmental milestones in the lifecycle of Drosophila. The study assessed the success rate of the larvae to become pupae and pupae to adults

Fig. 1. Length of Larval period and pupal period in Drosophila melanogaster treated with toluene and BDAE.

due to the effect of toluene and the influence of BDAE to mitigate such changes. The observations from the experiments were compared with the control group, toluene alone group and the groups containing the various doses of BDAE along with toluene. In hatching rate, the fly eggs which spent their embryonic life and larval period in medium containing toluene were significantly reduced. The length of larval period and pupal period were increased in the presence of toluene (Fig.1). The study thus reiteratesthe potential of toluene to delay the developmental stages of Drosophila. The study also demonstrated the concomitant reduction in the pupal percentage, the maturity percentage and the percentage of egg hatching in

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Fig. 2. Larval length in Drosophila melanogaster treated with toluene and BDAE.

Fig. 3. Pupal length and width in Drosophila melanogaster treated with toluene and BDAE.

the presence of toluene. The administration of BDAE however significantly elevated the percentage of egg hatching, larval period, pupal period, pupal percentage and maturity percentage in flies treated with toluene. The lower dose (25 mg/ml) and medium dose 1 (50 mg/ml) failed to invoke any positive response in the flies. While the medium dose 2 (100 mg/ml) and High dose (200 mg/ml) significantly reduced the prolonged larval and pupal periods and enhanced the percentage of egg hatching, pupation and maturation.

Drosophila pupae mean length and width measurements Treatment of toluene has significantly reduced the mean length and width of the pupae (p < 0.001). On supplementing BDAE in the growing medium of the flies, the toxic effects due to toluene has been reduced and the pupae with normal proportions has been maintained (p < 0.001) (Fig. 3). Measuring the chest width in adult Drosophila

Effect of BDAE on larval length and width Mean larval length and width were taken from each group by taking ten larvae at random from each bottle collected at 12 h intervals. The data are tabulated in Fig. 2.The length of the larvae exposed to toluene was reduced significantlywith subsequent treatment with BDAE has significantly elevated equivalent to that of the control flies (p < 0.001 and F (5, 47) = 22.23) and similarly the width of the flies were also decreased in the presence of toluene embedded medium and the BDAE has increased the width of the larvae in a dose dependent manner (p < 0.05, F(5, 49) = 3.05).

Following treatment with toluene, the average width of the chest of adult flies was significantly reduced (p < 0.001). BDAE treatment athigh dose has maintained the normal chest width (Table 2). Measuring the length and width of eggs On comparing the length and width of the insect eggs grown in medium laden with toluene, the study failed to invoke any statistically significant change in the said parameters. It shows that the spending the embryonic stage and maturation in toluene containing media does not affect the morphometry of the eggs (Fig.4).

Please cite this article in press as: PB B, et al. Prophylactic efficacy of Boerhavia diffusa L. aqueous extract in toluene induced reproductive and developmental toxicity in Drosophila melanogaster. J Infect Public Health (2019), https://doi.org/10.1016/j.jiph.2019.07.020

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6 Table 2 Results of quantifying developmental studies.

Duration of larval period Duration of pupal period Percentage of egg hatch Larval percent Pupal percent Adult percent Egg length Egg width Larval length Larval width Adult chest width

Control

Toluene

Low dose

Medium dose 1

Medium dose 2

High dose

105.4 + 5.46 87.2 + 8.75 73.6 + 12.8 79.2 + 14.8 88.9 + 41.9 86.7 + 17.1 0.35 + 0.05 0.13 + 0.02 4.94 + 0.5 1.75 + 0.6 1.08 + 0.17

194.2 + 17.9c 115 + 13.5c 46.2 + 20.5c 15.6 + 5.5c 27.3 + 19.3c 29.0 + 19.7c 0.348 + 0.04ns 0.134 + 0.02ns 2.66 + 0.6c 0.78 + 0.09b 0.74 + 0.09c

189.8 + 13.9c 111.6 + 6.8c 53.4 + 13.4c 56.3 + 24.9c 44.0 + 17.2c 39.4 + 13.4c 0.35 + 0.03ns 0.14 + 0.02ns 3.3 + 0.3a 1.02 + 0.09b 0.78 + 0.04C

165.6 + 14.4b 99.8 + 11.8a 59.8 + 46.5c 66.0 + 16.3a 73.8 + 8.4a 54.6 + 11.9b 0.35 + 0.04ns 0.13 + 0.03ns 3.5 + 0.2a 1.77 + 0.4ns 0.82 + 0.1b

133.2 + 9.23ns 97.8 + 15.06ns 63.8 + 21.8ns 75.2 + 68.9ns 82.1 + 11.0ns 68.9 + 10.2a 0.35 + 0.02ns 0.14 + 0.03ns 3.72 + 0.7ns 1.82 + 0.47ns 1.03 + 0.12ns

115.0 + 17.6ns 95.6 + 6.84ns 71.1 + 11.8ns 76.9 + 5.7ns 82.1 + 8.4ns 84.2 + 13.2ns 0.34 + 0.02ns 0.14 + 0.02ns 4.72 + 0.7ns 1.86 + 0.26ns 0.87 + 0.17ns

All comparisons are with the control group. a Significance at 5% (p < 0.05) level. b Significant at 1% (p < 0.01) level. c Significant at 0.1% (p < 0.001) level and ns not significant (p > 0.05).

Fig. 4. Egg length and width in Drosophila melanogaster treated with toluene and BDAE.

Determination of fitness Dynamics of eclosion and developmental time Dynamics of eclosion and mean developmental time of flies grown on different groups were measured. The flies in control group emerged from day 10 to day 21 with the largest number emerging on day 17. Their mean developmental time was 16.6 + 4.9 days. In toluene alone treated group, the flies emerged from day 15 to 22 with the largest number emerging on day 19 and a mean developmental time of 19.5 + 2.7 days. Treatment with BDAE has normalised the mean developmental time significantly (in low dose 18.5 + 3.6, in medium dose 1 18.9 + 4.7, medium dose 2 16.0 + 3.3 and in higher dose 15.5 + 4.6 days respectively) in a dose dependent manner (Fig.5).

(Table 3). Similarly, the number of progenies evolved from a single pair of flies is expressed as the fertility parameter. In this study fertility was decreased on exposure to toluene and this was regained significantly on treating with BDAE (p < 0.01, F(3, 30) = 3.49). Determination of lifespan The mean lifespan of the flies treated with toluene was significantly reduced to 17.43 + 14.6 (p < 0.05). Treatment with BDAE significantly elevated the mean survival of the flies by both the medium doses 1 and 2 and high doses to 20.43 + 2.6, 22.0 + 14.8 and 24.14 + 19.5 respectively (p > 0.05). The lower dose of the drug had a mean survival of 19.5 + 9.9 (p < 0.05), while the control group had a mean survival of 25.5 + 11.8 h. Determination of antioxidant enzyme levels

Egg-to-adult survival The mean egg-to-adult survival in control group was 82.5 + 9.8. Treatment with toluene decreased the mean egg-to-adult survival to 23.1 + 12.5 days. Supplementation of BDAE in the growth medium significantly elevated the survival time in dose dependent manner (p < 0.001, F (530) = 9.44). Post hoc comparison with Dunnett’s test against the control flies showed equivalent means in the control and high dose flies. Determination of fertility and fecundity The number of progenies produced from a single mated female is computed as the fecundity parameter. Egg production was considerably reduced on treatment with toluene and the egg producing capability was regained to a great extent by treating with BDAE at various doses. The mean number of eggs produced were elevated significantly on treating with BDAE (p < 0.001, F(5, 30) = 11.02)

Toluene has considerably reduced the antioxidant enzyme levels in D. melanogaster. Treatment with BDAE has increased the levels of catalase, glutathione-S-transferase and superoxide dismutase significantly by the higher doses of the drug (Table 4). Discussion Toluene is a common pollutant of environment and it reaches the atmosphere principally from the volatilization of petroleum fuels, solvents and thinners and motor vehicle exhaust [31]. Presence of toluene has been detected in surface water, ground water, drinking water and soil samples [12]. Several studies prove its association with reproductive and developmental effects in humans as well as other lower animals [32]. Pregnancy loss, congenital malformations are highly related with a greater odd from the exposure to

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Fig. 5. Dynamics of eclosion in Drosophila melanogaster treated with toluene and BDAE.

Table 3 Results of quantifying Reproductive studies.

Fitness – mean developmental time Fitness – egg-to-adult survival Fecundity Fertility Mean Life span

Control

Toluene

Low dose

Medium dose 1

Medium dose 2

High dose

16.6 + 4.9 82.5 + 9.8 449.6 + 153.7 224.0 + 59.9 25.5 + 11.8

19.5 + 2.7a 23.1 + 12.5c 112.3 + 45.4c 99.0 + 19.4ns 17.43 + 14.6a

18.5 + 3.6a 22.0 + 8.7c 110.8 + 16.9c 103.5 + 9.2ns 19.5 + 9.9a

18.9 + 4.7a 53.7 + 18.5c 203.0 + 104.8b 189.5 + 104.7b 20.43 + 2.6ns

16.0 + 3.3ns 68.4 + 12.8ns 427.8 + 229.7ns 213.0 + 187.0ns 22.0 + 14.8ns

15.5 + 4.6ns 78.8 + 14.6ns 400.8 + 70.5ns 218.5 + 102.2ns 24.14 + 19.5ns

All comparisons are with the control group. a Significance at 5% (p < 0.05) level. b Significant at 1% (p < 0.01) level. c Significant at 0.1% (p < 0.001) level and ns not significant (p > 0.05). Table 4 Antioxidant enzyme levels.

Catalase Glutathione-S-transferase Superoxide dismutase

Control

Toluene

Low dose

Medium dose 1

Medium dose 2

High dose

282.7 + 12.5 830.5 + 34.0 88.67 + 19.9

76.5 + 9.6c 67.5 + 21.9c 23.59 + 26.8c

80.18 + 24.5c 73.0 + 12.8c 28.33 + 41.1c

104.8 + 19.4b 327.3 + 11.6b 37.7 + 39.3b

115.3 + 10.6ns 557.3 + 16.9a 78.67 + 18.4ns

251.5 + 31.5ns 888.2 + 22.8ns 84.2 + 22.7ns

All comparisons are with the control group. a Significance at 5% (p < 0.05) level. b Significant at 1% (p < 0.01) level. c Significant at 0.1% (p < 0.001) level and ns not significant (p > 0.05).

toluene [33]. Mechanism of such maladies is vested on the chromosomal aberrations induced from toluene exposure [34]. In this light of events a solid remedy is sort and for this the wealth of knowledge inscribed in the traditional medical practices are revoked. Ayurvedic system of medicine, being practiced from time immemorial has a wealth of drugs in the form of flora and fauna having a wider distribution and thus ensures ease in availability and cost effectiveness. Pertaining to toxic conditions, a drug that is mentioned often is B. diffusa, a common weed widely distributed all over India and in many other parts of the world. In this study the potential of B. diffusa root aqueous extract is utilized to reverse the developmental toxicity and hampered fertility, fecundity, lifespan and antioxidant defence mechanisms induced by toluene in the well-established toxicity model of D. melanogaster. A preliminary phytochemical screening and HPTLC profiling of the drug was performed and the results almost coincide with the reported works done elsewhere [35]. The LC50 of toluene in Drosophila was tested initially and the observed value of 430 ppm. The aqueous extract of the roots of the plant B. diffusa showed the presence of major groups of secondary metabolites like carbohydrates, alkaloids, amino acids, glycosides, tannins, proteins, flavonoids, steroids and sugars. The rich pharmacological use of Boerhavia in traditional medicines is due to this vast array of phyto-constituents present in them. HPTLC profiling yielded 12 peaks in 254 nm and 10 peaks

in 366 nm photo densitograms. The increased number of peaks in the present study may be due to the selection of the ideal solvent system (Toluene: ethyl acetate: formic acid: methanol) that gave a very good separation of the compounds. The developmental toxicity exerted by toluene was well evident in the study by the elongation of the larval period and the period of maturation. Percentage of pupation and adult eclosion also indicates the effect of toxin on fly development. These assays measured the effect of toxin exposure over a substantial development period (larvae to pupae). This assay is considered a high throughput method to measure the genetic and nutritional influences on a toxic insult [22]. Reduction in foetal body weight and retardation of skeletal development is the most consistent fetotoxic effect demonstrated in animal studies [13]. The statistically significant results obtained for the above parameters highlights the role of B. diffusa as a potent antitoxic agent capable for reversing the toxic insults produced by toluene. However, the study failed to demonstrate any significance in the morphometry of eggs grown in medium containing toluene. The fitness parameters utilized for the present study was the dynamics of eclosion, developmental time and egg-to-adult survival. Rather than a drug entity traditional medicines also have a nutraceutical aspect. Supplementing BDAE in the culture medium enhances the nutritional quality of the medium along with its

Please cite this article in press as: PB B, et al. Prophylactic efficacy of Boerhavia diffusa L. aqueous extract in toluene induced reproductive and developmental toxicity in Drosophila melanogaster. J Infect Public Health (2019), https://doi.org/10.1016/j.jiph.2019.07.020

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antitoxic potential against toluene. Changes in the fitness components may be due the transcriptional dynamics. It is reported that the nutritional regimes provided to the flies induces developmental plasticity in flies and is considered an adaptive process resultant of changes manifested at physiological, morphological and behavioural levels [24,25]. Changes in fitness components after transferring eggs could be the result of plastic responses to the drug present in the medium. The developmental time as well as the egg-to-adult survival was significantly increased on treating with toluene. BDAE significantly reduced the same to the level as that of the control flies. Fecundity and fertility are two fitness parameters used to assess the reproductive value of female flies. Fecundity is influenced by the genotype of the flies, their body size, age, their mate as well as the environmental factors. In toxicology, fecundity and fertility are important factors as they are directly linked to the effect of the toxicant to the physiological factors and is expressed in terms of egg and offspring production [36]. In the present study, there is a significant increase in egg laying in different groups of flies compared to toluene treated group. Enhancement of fecundity in males makes them fast maters thereby performing large number of copulations and produces more offspring. In female flies, fast mating provides an ample opportunity to receive largest number of sperms and thereby largest number of eggs and offspring. Thus, fecundity and fertility go hand in hand and this study proved the potential of BDAE in enhancing both fecundity and fertility in toluene treated drosophila flies. Aging is the sum total of deteriorating structural and functional changes occurring in an organism culminating in its death which is dependent on time. Factors that play a role in the cascade of events leading to the aging process affect the longevity of the organism proportionately. Several molecular mechanisms have been studied with respect to the lifespan of drosophila. In drosophila, histone deacetylation of Sirtuin (SIR2) and RPD3 has been shown related to shortening of lifespan [36,37]. Toxic substances present in the environment could initiate such molecular mechanisms shortening the lifespan of the organism. In the present study BDAE significantly prolonged the lifespan of flies treated with toluene. The underlying molecular mechanisms may be established. The chief toxic manifestations of toluene are targeted on nervous tissues, liver, heart and kidneys [38]. Toluene toxicity cannot be attributed to any metabolite that is biologically active. Severe oxidative stress is given prime importance in the pathogenesis of the toxicity. Toluene generates free oxygen radical through an NADPH dependent reduction reaction culminating in generating superoxide anion [39]. Consequent to toluene administration, the levels of antioxidant enzyme levels of Catalase, Glutathione-Stransferase and superoxide dismutase levels significantly reduced. Administering BDAE at various doses significantly elevated the levels of these antioxidant enzymes.

Conclusion This study concludes the capability of B. diffusa aqueous extract in reversing the developmental toxicity induced by toluene and it is proved to be a significant agent that enhances toxicity induced fitness and fertility in drosophila flies. The major mechanism of toxicity induced by toluene is the generation of free radicals. B. diffusa has high potential to curb the free radical induced damages in the flies. Thus, BDAE proves to be a cheap yet powerful tool in addressing the toxicity induced by solvents like toluene. Though many of the assays involved in this study only utilized biological parameters, extensive studies to unlock the molecular mechanisms should be rigorously taken. The role of natural herbs in addressing the emerging health challenges is yet to be tapped to its fullest.

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Please cite this article in press as: PB B, et al. Prophylactic efficacy of Boerhavia diffusa L. aqueous extract in toluene induced reproductive and developmental toxicity in Drosophila melanogaster. J Infect Public Health (2019), https://doi.org/10.1016/j.jiph.2019.07.020