GJRMI - Volume 3, Issue 8, August 2014

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INDEX – GJRMI - Volume 3, Issue 8, August 2014 MEDICINAL PLANTS RESEARCH Life & Earth Sciences EVALUATION OF INSECTICIDAL EFFECT OF VEGETABLE OILS EXTRACTED FROM THE RIPE SEEDS OF AZADIRACHTA INDICA AND KHAYA SENEGALENSIS IN ADULT ANOPHELES GAMBIAE S.L. IN MAROUA, FAR NORTH REGION CAMEROON Saotoing P, Tchuenguem Fohouo F.N, Febal H.A, Amba A.H.B, Amadou H, Nawe T.U.S

312–322

INDIGENOUS MEDICINE Ayurveda – Review Article EVALUATION OF PUMSAVANA VIDHI IN THE CONTEXT OF PRE-CONCEPTION AND PRE NATAL DIAGNOSTIC TECHNIQUE ACT (1994) Vaikos C D

323–330

COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R, PLANT ID – TENDER LEAVES OF PRIYANGU – CALLICARPA MACROPHYLLA VAHL., OF THE FAMILY LAMIACEAE PLACE – KOPPA, CHIKKAMAGALUR DISTRICT, KARNATAKA, INDIA


Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 8 | August 2014 | 312–322 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Research Article EVALUATION OF INSECTICIDAL EFFECT OF VEGETABLE OILS EXTRACTED FROM THE RIPE SEEDS OF AZADIRACHTA INDICA AND KHAYA SENEGALENSIS IN ADULT ANOPHELES GAMBIAE S.L. IN MAROUA, FAR NORTH REGION CAMEROON Saotoing P1*, Tchuenguem Fohouo F N2, Febal H A3, Amba A H B4, Amadou H5, Nawe T U S6 University of Maroua, Higher Teachers’ Training College, Department of Life and Earth Sciences Ngaoundéré University, Faculty of Science, Department of Biological Sciences *Corresponding author: psaotoing@yahoo.fr; psaotoing@gmail.com 1,3,4,5,6 2

Received: 23/06/2014; Revised: 25/07/2014; Accepted: 01/08/2014

ABSTRACT The insecticidal effect of vegetable Oils of two plants in the Far North Region Cameroon was evaluated on the adult population of Anopheles gambiae s.l. in Maroua through of net bed in the laboratory. The plant oils of Azadirachta indica and Khaya senegalensis were extracted from mature seeds, then diluted with petroleum ether. Plots of net bed were impregnated with these solutions. After 24 hours of exposure, the plant oil extracts showed a repellent effect on insecticide in adult A. gambiae. A. indica provided an LC50 of 2.77g/m², and K. senegalensis an LC50 of 2.79g/m². For lethal 50 hours (HL50), A. indica provided 11 hours 56 mins 42 seconds of HL50 and K. senegalensis gave 14 h51mn19s. As cons, the Mosquito Impregnated Net has an LC50 of 2.30 × 10-4g/m2 and an HL50 of 2 hours 52 min 28seconds. Vegetable oil of A. indica presented quite high toxicity compared to that of K. senegalensis in A. gambiae. However, these two vegetable oils have a lower toxicity than the deltamethrin usually used for to the concentration of 5.5 × 10-4g/m2 insecticide effect. KEYWORDS: Evaluation, insecticidal effect, Vegetable Oils, bed net, Anopheles gambiae, Cameroon.

Cite this article: Saotoing P, Tchuenguem Fohouo F.N, Febal H.A, Amba A.H.B, Amadou H, Nawe T.U.S (2014), EVALUATION OF INSECTICIDAL EFFECT OF VEGETABLE OILS EXTRACTED FROM THE RIPE SEEDS OF AZADIRACHTA INDICA AND KHAYA SENEGALENSIS IN ADULT ANOPHELES GAMBIAE S.L. IN MAROUA, FAR NORTH REGION CAMEROON, Global J Res. Med. Plants & Indigen. Med., Volume 3(8): 312–322 Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||


Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 8 | August 2014 | 312–322

INTRODUCTION In Africa, Malaria is the main cause of medical consultation.Nearly 50% of mortality and morbidity among children under 6 years are caused by malaria (PNLP, 2006). According to the World Malaria Report" of 2005, the World Health Organization (WHO) reported that 107 countries and territories have areas which are concerned by the transmission of malaria and some 3.2 billion people live in endemic areas. The fight against the malaria vector must be adapted to target vectors. Preventive control is based on long lasting impregnated nets, on the indoor residual spraying of insecticides and, to a lesser extent, on the destruction of breeding area. Active materials of insecticides which are mostly used, belong to organophosphates, pyrethroids and carbamates synthesis (Djogbénou, 2009). Plants sampling based on ethnobotanical survey are known to show greater percentage yield of bioactive useful compounds than other methods (Usman et al., 2014). Vector resistance to insecticides synthesis constitutes the main limits to their usage. Resistance to organophosphates and carbamates has been recently discovered in West Africa in the Anopheles gambiae the main vector’s malaria (WHO, 2006). The World Health Organization recognizes that the most practical to control resistance, in alternate effect spraying consists in using alternatively insecticide of different types following a pre-established program or according to the results of resistance. Very few studies have been conducted on the present effect interest of the insecticides extracts of local plants that could be prevented. Azadirachta indica and Khaya senegalensis are very used in Far North Region Cameroon and they have insect repellents and insecticides effects (Dansou et al., 2007, Fawrou et al., 2006). Some farmers function on the basis of solution reward leaves of these plants as insecticide so as to destroy and drive away pests (Mamadou, 1997). Unlikely, the information provided by the population reveals that extracts have a repellent effect on mosquitoes, once they ambolmed themselves with. On the social plan, a new efficient

method to fight against some malaria vector agents must significantly reduce the mortality rate of this disease. On the economic plan, oils extracted from the ripe seeds of local plants are accessible to all at low cost. This could enormously reduce budgets, funding for the prevention and fight against malaria and all the diseases transmitted by mosquitoes. The overall objective of this work is done to evaluate the effect of insecticide vegetable oils extracted from the ripe seeds of Azadirachta indica and Khaya senegalensis in adult Anopheles gambiae sl in the city of Maroua. MATERIALS AND METHODS Presentation and study of the site Maroua, the Capital of Diamaré Division and the Far North Region of Cameroon, is located between 10° 31 ' - 10° 39' North latitude and 14° 13 ' - 14° 24' East longitude. It has an in altitude of 400 m in the Savannah (Casalprime, 2008). Maroua town is divided into three sub-divisions. Maroua town stretches along the shores of the Mayo - Kaliao which passes longitudinally from West to East. On a global view, the relief of this Region is made up of all the plains combined with the Mandara Mountains and mountains on the border with Nigeria. With an altitude of 1000 m of mountains and 300 m of plain, Maroua is a transitional zone between the desert areas of North and the wet climate of the South Sudanese (Yann, 2000). The Region is of the Sudano - Sahelian climate with an average temperature of 35°C, with the highest in March and April which goes up to 45°C and with a minimum of 25°C (Yann, 2000). A very short rainy season alternates with a dry season relatively long, depending on the years. The annual interest rainfall of about 800-900 mm is distributed over 50 to 60 days of precipitation (Mechia, 2005). The harmattan is the most dominant wind of the Region and blows almost all the year (Méchia 2005). The vegetation consists of thorny steppes and Sahelian interest dominated by some species such as Azadirachta indica, Khaya senegalensis, Balanites aegyptiaca, Anogeissus leiocarpus

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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 8 | August 2014 | 312–322

and Boswellia dalzieli which grow on the rocky soils (Boutrais, 1984). The population of the Maroua town has gone from 201, 372 inhabitants (GPC, 1987) to about 400, 000 inhabitants in 2013 according to the Maroua I council. More than forty different ethnic groups live in the Region (Yengue et Yann, 2002). METHODOLOGY Sampling larvae The harvesting of the Anopheles larvae was conducted during the months of March, April, May and June 2013. Samples were made using ladles enamel handle. The larvae were collected by using a pipette bulb and placed in a metal tray of 50 cm of diameter in the laboratory of the Institute of Agricultural Research for Development (IRAD) of Maroua. Rearing larvae of Anopheles gambiae The larvae were maintained in breeding conditions with different water deposits. The identification and recognition of Anopheles larvae were made from their parallel position with respect to the water surface (Fig. 1). The study took into consideration the only Anopheles genus. Containers for mosquito

Figure 1. Morphology of larvae Anophelinae (a) and Culicinae (b)

larvae bore the characteristics of the sampling sites. In each tray, a maximum of 100 larvae were introduced. Nutritional powder made from prowns and biscuits were served as food to the larvae. The water from the trays were from time to time renewed to avoid water pollution resulting from the presence of the nutritional powder. Two to three days after the introduction of the larvae in large trays, the nymphs appeared. The nymphs were in turn removed from the plates and transferred to transparent glasses. The glasses containing water house were placed inside cubic cages made of tulle net with a volume of 8000 cm3 (Fig.2). In each cage, a petridish containing a sugar juice based on 10 % sucrose was placed, to serve as an emerging food to adult mosquitoes. With their emergence in cages, adult mosquitoes were fed with blood of Rabbits before being submitted to the various toxicity tests to avoid biased results that would be linked to poor diet to the Anopheles. The determination of adult Anopheles species was made using the identification keys of Gillies & De Meillon (1968) and Gillies & Coetzee (1987). Meanwhile, seed collection Azadirachta indica and Khaya senegalensis continued.

Figure 2: Adult of Anopheles gambiae in cage

Fig 1: Image Courtesy: Google Images

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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 8 | August 2014 | 312–322

Figure 3: Azadirachta indica tree

Figure 4: Khaya senegalensis tree

Fruits

Collection of fruits and seeds

Sedimentation and filtration

The seeds were collected from ripe fruits. In fact, these fruits were harvested in Pitioaré quarter (Camp IRAD) and at the Camp SIC neighborhood. Azadirachta indica (Fig.3) produces berries that fall when ripe. Seeds are collected within a shell in fruit lying below a thin layer of chair. Khaya senegalensis (Fig.4) produces fruit capsular, dehiscent, 4 or 5 valves, 5 to 10 cm of diameter, and light gray at maturity. Dried fruits open and release seeds.

It consists of letting in rest the extract obtained by pressing. The heaviest substances settle at the bottom of the container can be recovered with floating oil above. The operation was repeated several times until the obtention of clear oil. The oil obtained was then filtered to remove the impurities.

Treatment of fruits and seeds The ripe fruits of Azadirachta indica were having very high water content. They were dried before removing the seeds. This allows the seed to separate itself easily from the shell. Dried fruits of Khaya senegalensis do not need to be dried first. For cones, the fruit should be picked because they fall after having released their seeds. After drying the fruits, the seeds were then subjected to roasting and grinding, and then pulped between the millstones. The molding thus obtained is steamed until oil oozes. The following steps were followed to obtain vegetable oils. Pressing by traditional method Pressing is optimized with 5 % to 8 % with an impurity content of 2 % of humidity. A manual pressing was used, which is actually home made equipment but which provides good extraction rates.

Impregnating of bed nets with extracts Nets industrially impregnated with delthamétrin, pyrethroids, DDT were done with the concentrations of 55 mg/m²; 0.2 g/m² and 8 mg/m² respectively. The obtention of effective concentration was made using several ranges of concentration tests: 0.1 g/m²; 0.2 g/m²; 0.3 g/m²; 0.4 g/m²; 0.5 g/m²; 1.5 g/m²; 2.5 g/m²; 3.5 g/m²; 5 g/m², with the surfaces of the cones being very low compared to the areas (m²) used industrially. A similarity was used to determine the masses of extracts on the surface of a cone. The following formula was obtained: X = 0.0169 × M (M is the mass per square meter, X is the mass value brought to the surface of 0.0169 m²). Knowing the different masses needed for testing, oil volumes corresponding to each mass were determined. It was enough indeed to find the density of oils and deduct the volumes. To determine the density, a known volume of oil was weighed on an electronic balance and the density of vegetable oils has been obtained by applying the formula d = m/v (where m is the mass and v the volume), and each volume was obtained using the formula m = v/d. After calculation, we obtained: (d1 =

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density of the Azadirachta indica oil, d1 = 0.82) and (d2 = oil Khaya senegalensis density, d2 = 0.80). Cones used for the tests were made thanks to the model cones of WHO for this purpose. The end caps serving as bottle packaging of mineral water were used. These nozzles, like WHO-cones have the characteristics of being made of polyethylene, a material on which the mosquito scarcely clings on. The mosquito will have no other choice but to stay in flight or landing on the substrate (impregnated mosquito net). In order not to lose in mass quantities, it has been established that for a surface mosquito net of 0.0169 m2, you need a volume of 2 ml of organic solvent. The suitable organic solvent used herein to diluate the oil is the petroleum ether. With the help of a pipette, 2 ml of ether are removed and poured into a petri dish containing the mass of the corresponding active substance. After a complete dissolution of the oil in the ether, the plots of the nets were soaked. Then the nets were left to dry under the sun for a period of one hour so as to allow the plant extract to fully adhere on the and to completely let evaporate the ether. Meanwhile, the negative control was based solely on ether which is used to assess its influence on mosquitoes. Tests of insecticidal effect of oils on adults Anopheles gambiae Impregnated nets were hanged to the base of the cone using a transparent tape. A total of 13 cones were used including 10 reserved for the different concentrations of each type of extracted plants and 3 others like cones’ witnesses. One is impregnated with 2 deltamethrin 55 mg/m , actually used by the population and being distributed by the government to the population. This net was considered as a positive control to assess the effectiveness of the extracted oils from ripe seeds of Azadirachta indica and Khaya senegalensis. Another cone was equipped with mosquito nets soaked in ether only; this was to ensure the neutrality of ether on mosquitos during the test. After the distribution of cones and the fixing of nets, 30 unsexed adults anopheles were introduced into each cone with vacuum insects. The cone containing

mosquitoes are closed by a piece of cotton wool and these cones were suspended separately in the laboratory using a string to allow ventilation. After the introduction of the insects in the cones, the insecticide efficiency is measured by their Knock-down effect and they died after 24 hours of observation. In order to determine the Knock-down effect, three minutes after the reaction were observed on the mosquitoes. The number of mosquitoes shocked or stunned or dead were recorded in a table. Then every hour up to twelve hours, the last observation was made after the expiration of the last 12 hours. The test of each concentration was repeated three times to minimize errors. The averages of the three replicates were calculated. Data analysis According to Finney DJ (1971), a lethal concentration of 50 (LC50) and a lethal time of 50 (HL50) were obtained from the following formulas: LC50 = log10-1 (y-b / a); HL50 = log10-1 (y−b / a). The concentration-response graph for each exposure time is plotted against the logarithm of the concentrations by means of software.The right probit = f (log concentration) is plotted using the Microsoft Office Excel 2007 software; the equation of the line known as the correlation coefficient is also obtained with this software. Concentrations and times that kill 50% of mosquitoes (LC50 and HL50) were determined graphically. The regression lines have yielded correlation coefficients, which in turn helped to highlight the different correlations compared to the Long Lasting Insecticide treated nets. RESULTS Profit yield of the extraction of vegetable oils Mature seeds of A. indica and K. senegalensis showed relatively high yields of vegetable oils as weighing 1500g which gave of seeds 600g and 450g of vegetable oil,

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resulting 40% and 30% respectively for A. indica and K. senegalensis. Vegetable oils of A. indica and K. senegalensis are the most popular products in the Far North Cameroon, given their wide range of applications in the treatment of diseases such as malaria, helminthes infections, constipation, itching. Many people confirm that anointing of these two products provides temporary protection against the biting of insects especially mosquitoes. Depending on the selling period, the price for a liter of these plants is between 6000F and 13000F in Maroua town, Cameroon. Sellers are more and more opting for retail sale to allow easy access to all. Mortality rate of vegetable oils A .indica and K. senegalensis on Anopheles gambiae The data obtained helped to draw graphs on mortality rate by using time for each concentration of the vegetable oils and for each batch of mosquitoes. The graph on mortality rate versus time and concentration of the extract from Azadirachta indica on vegetable oil in anopheles is represented by figure 5. Figure 5. Curves of mortality rates over time to different concentrations of Azadirachta indica in Anopheles gambiae

Determination of lc50 of A. indica and K. senegalensis The determination of the extract of vegetable oil concentration that causes a mortality rate of 50% among adult anopheles

According to figure 5, the mortality rate increases with the concentration of the extract of Azadirachta indica oil. In fact, the correlation coefficients are positive (r = 0.9593). This study showed a dose- mortality of all oils tested, with an increase in larval mortality, when the dose increases. The activity of the oils could be due to the active substances contained in the plant material. Figure 6 below shows the evolution of the mortality rate depending on the time and the concentrations of Khaya senegalensis in Anopheles gambiae sl. The observation of figure 6 shows that mortality increases with the concentration of extracted oil of Khaya senegalensis. This observation is confirmed by the correlation coefficient r = 0.9572. It was found a very high mortality rate of mosquitoes in the active control of Insecticide treated nets, 100% after about 1 hour 30 min of exposure. This confirms the powerful effect of the insecticide Deltamethrin. Deltamethrin is effective at a very low dosage (55mg/m2) compared to lethal oils of Azadirachta indica and Khaya senegalensis. Figure 6. Mortality rate versus time for different concentrations of Khaya. senegalensis in Anopheles gambiae sl

was made from regression equation of mortality rates, expressed as probit value, and depending on the concentrations of extracts of vegetable oil in decimal logarithm value (Fig.7 and Fig.8).

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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 8 | August 2014 | 312–322

Figure 7: Regression for determining the LC50 of the extract of Azadirachta indica vegetable oil on Anopheles gambiae

Apparently, the LC50 of A. indica and K.senegalensis are almost equal (table 1). However, they are very high in A. indica (2.77g/m2) and in K. senegalensis (2.79 g/m2) compared to that of the Insecticide treated nets (MILDA) with an LC50 of 2.30 × 10-4g/m2. There is no significant difference at α = 5 % between the LC50 of A. indica and K. senegalensis. Taken at this dosage,

Figure 8: Regression for determining the LC50 of the extract of Khaya senegalensis vegetable oil on Anopheles gambiae

deltamethrin is 4279 times more effective than A. indica and 4488 faster than K. senegalensis. LC50 obtained from anopheles adults which showed that two oil extracts are interesting in terms of toxicity. In reality, the lethal concentrations vary depending on the method of extraction and of the extract of the plant or plant part in question.

Table 1. LC50 of A. indica, K.senegalensis and Insecticide treated nets Insecticides

Regression equations

r

A. indica K. senegalensis MILDA

y=1,551x+4,313 y=1,860x+4,171 Y=6,739x+7,438

0,96* 0,97* 1***

CL50 (g/m2) 2,77a 2,79a 2,30 x10-4 d

Column LC50; values are followed by different letters are significantly different at the 5% level. Very highly significant (p ≤ 0.001). Highly significant (p ≤ 0.01).

Determination of hl50 A. indica, K. senegalensis and long acting insecticide treated nets (milda) The determination of hours from which the plant extract concentration induced 50 % mortality of Anopheles gambiae was performed. This parameter was obtained from the equation of regression mortality expressed in probit value based on exposure times in decimal logarithm (table 2). The HL50 obtained

revealed that Azadirachta indica provided HL50 smaller values about 11h56min42s than Khaya senegalensis (14H51min19s). A significant difference at the 5% level between HL50 A. indica and K. senegalensis was observed. This means that the vegetable oil from Azadirachta indica is higher than K. senegalensi toxicity. The values of correlation coefficient obtained show that there is a close correlation between the duration of exposure and the mortality rate (0.9 ≤ r ≤ 1).

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Table II. HL50 A. indica, K.senegalensis and Long Lasting Impregnated Nets Insecticides A.indica K. senegalensis MILDA

Régression equations y=1,451x+3,437 y=1,309x+3,466 Y=2,492x+5,847

r 0,98* 0,96** 1***

HL50 11h56min42sa 14H51min19sb 2h52mn28sc

Column of HL50; values followed by different letters are significantly different at the 5% level. Very highly significant (p ≤ 0.001) highly significant (p ≤ 0.01).

DISCUSSION We have noticed that mature seeds of A. indica and K. senegalensis showed relatively high yields of vegetable oils, 40% and 30% respectively. Meanwhile, Chougourou et al., (1997) have found rather a higher oil yield of 43.4 % with A. indica seeds. In fact, Azevodo et al., (2002) justifed this through the extraction efficiency of a plant which depends on several factors, namely the nature of the soil, climate and the harvest season. The repellency of the extracts of A. Indica and K.senegalensis on insects is increasingly confirmed by several researches. Fawrou et al., (2006) conducted a study on A. indica oil in pre-imaginal stages of Culex quinquefasciatus and confirmed that A. Indica oil was made effective at both larvae and nymph levels. Our results corroborate those of Tchoumbougnang et al., (2009) on the larvicidal activity of four plants cultivated in Cameroon (Cymbopogon citratus (DC.) Stapf, Ocimum canum Sims, Ocimum gratissimum L and Thymus vulgaris L) on Anopheles gambiae Giles 1902. Hence, the results of the tests performed on larvicidal activities on essential oils show a direct relationship between the percentage of the mortality of the larvae L4 and the concentration of essential oils. In addition, Chougourou et al., (2012) revealed that the bioactive substances such as azadirachtin, curcin, Ricin and thevetin are respectively present in A. indica seeds. Furthermore, Sharma et al., (1993) also showed that a mixture of oil Azadirachta indica in coconut oil has provided a 100% protection for 12 hours against anopheles bites and those against sandfly vectors of leishmaniasis, 100% overnight against Phlebotonus argentipes and 100% for 7 hours against Phlebotonus papatasis. These results show that the

vegetable oil from Azadirachta indica has a residual effect. In addition, Dua et al., (1994) showed that with cream Azadirachta indica, two grams per person provides protections of 80–90 % against Aedes, Culex and Anopheles. Sharma et al., (1994) in their work showed that Azadirachta indica oil combustion of 1% in kerosene in the usual lamps provided 100% protection against anopheles’ bites. The chemical compositions of several plants give their insecticidal properties. Several other effects were observed on insects; and the study of the chemical composition of Khaya senegalensis revealed the presence of alkaloids, terpenoids, phenolics and flavonoids that variously affect the existence or the development cycle of pests (Boeke et al., 2004). Essential oils further demonstrate their multiple activities, anti-oxidant properties that come from the multitude and diversity of their chemical constituents (Saotoing, 2005). Similar studies conducted by Njan Nlôga et al., (2007) showed the insecticidal effect of six plants in the Northern part of Cameroon. These plants were: Ocimium canum, Plectrancthus glandulosus, Laggera pterodonta, Eucalyptus camaldulensis, Hyptis spicigera, Pittosporum viridiflorum. Their LC50 ranged from 11.95 to 71 mg/m². Moreover, previous work Traboulsi (2002) had demonstrated the insecticidal activity of four medicinal plants harvested in Lebanon (Myrtus communis L., Lavandula stoechas L., Origanum L. and Mentha microphylla syriacum K.Koch) on larval Culex pipiens molestus Forskal. LC50 obtained were between 16 and 89mg/l. The studies conducted by Gauvin et al., (2002) on the composition of the extracts from the seeds of Azadirachta indica showed that this oil contains a mixture of more than 100 compounds. Among these, the azadirachtin (C35H14O16) is one of the

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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 8 | August 2014 | 312–322

most important bio-insecticides (Mordue and Blackwell 1993; Schmutterer, 1990; Zongo et al., 1993). Furthermore, these same authors have demonstrated that azadirachtin is not the only active and responsible insect for mortality tested substance, but it acts synergically with a mixture of several substances in the extract. CONCLUSION The results of this study show that vegetable oils extracted from Azadiratchta indica and Khaya senegalensis have an insecticidal effect on mature Anopheles of Maroua. LC50 obtained are 2.77g/m² for Azadirachta indica and 2.79 g/m² for Khaya senegalensis. The HL50 obtained are 11 hours 56 min 42 seconds for Azadirachta indica and 14 heures 51 minutes 19 seconds for Khaya senegalensis. However, these two vegetable

REFERENCES

oils have much lower insecticidal effect than that of deltamethrin (2.30 x10-4g/m2) used for Long Lasting Insecticide Treated Nets. Azadiratchta indica is the plant which is widely spread in the Northern region Cameroon. People know that this plant has an insecticidal effet but do not know the lethal dosage of toxicity. In the future work, we will tray to characterise the bio-active substances contained in the oil in order to formulate it. ACKNOWLEDGEMENT We are grateful to the authorities of the University of Maroua from whom we received funding for research and the publication of this article.

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Source of Support:

University of Maroua,

Mann. (2014). Ethnobotanical study of medicinal plants used for the treatment of malaria in nupeland, north central Nigeria. Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 4 | April 2014 | 112–126. ISSN 2277-4289 | www.gjrmi.com | International. Yann L’hote., (2000). Climatologie. In : Atlas de la province de l’Extrême-Nord Cameroun. Eds. Seignobes C. et IyebiMandjek O., IRD. MINREST, Paris, 27–33. Yengue J.L et Yann C., (2002). L’arbre et la ville dans la région de Maroua (Extrême-Nord Cameroun), sciences et changements planétaires/sécheresse, 13(3): 155–163. Zongo, J.O., C. Vincent et R.K. Stewart. (1993). Effects of neem seed kernel extracts on egg and larval survival of the sorghum shoot fly, Atherigona soccata Rondani (Dipt.: Muscidae). J. Appl. Entomol. 115 : 363–369.

Conflict of Interest: None Declared

Cameroon

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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 8 | August 2014 | 323–330 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Review Article EVALUATION OF PUMSAVANA VIDHI IN THE CONTEXT OF PRECONCEPTION AND PRE NATAL DIAGNOSTIC TECHNIQUE ACT (1994) Vaikos C D1* 1

Associate Professor and Head of the Department, Department of Rachana Sharir, Government Ayurveda College, Nanded, Maharashtra, India *Corresponding Author: E-mail: vaikosc@gmail.com

Received: 13/06/2014; Revised: 25/07/2014; Accepted: 31/07/2014

ABSTRACT Pumsavana vidhi is the procedure applied for the procreation of ideal and healthy progeny which contains pre-and post conception gender selection method (described in Ayurvedic sciences for the parents who want to have desired gender of child). There is a tendency among Indians and Asian parents to have male child. Because of this tendency the male female ratio in India has lowered which is 1000:940 in 2011which is very bad. Pumsavana fulfils the desire of expected gender. There is a question among most of the Ayurvedic practitioners that in the light of Pre Conception and Pre Natal Diagnostic Technique Act 1994 the practice of Pumsavana process is legal or not? There are two main objects/parts of this act 1) prohibition of sex selection by any means before or after conception, 2) regulation of pre-natal diagnostic techniques to prevent their misuse for sex determination leading to female foeticide; and, for matters connected therewith or incidental thereto. Gender selection means any sort of Procedure, Technique, Test, Administration, Prescription and Provision of anything for the purpose of ensuring or increasing the probability that an embryo will be of a particular sex. The Pumsavana vidhi (meant for desired sex i.e. sex selection) and the first object of PCPNDT Act are in line with each other and needs detailed analysis to cross check with the legal aspects there as to encourage or discourage the practice. This review article discusses the act & the procedure to. KEYWORDS: conception, diagnostic techniques, Pumsavana vidhi, progeny, PCPNDT Act

Cite this article: Vaikos C D (2014), EVALUATION OF PUMSAVANA VIDHI IN THE CONTEXT OF PRECONCEPTION AND PRE NATAL DIAGNOSTIC TECHNIQUE ACT (1994), Global J Res. Med. Plants & Indigen. Med., Volume 3(8): 323–330

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INTRODUCTION A specific procedure performed by couple to get the desired progeny is termed as Pumsavanavidhi / karma (measure which helps procreating male progeny) (P V Tivari, 2006). The intrinsic desire to have son (putraisana) is reflected in the literature from the very beginning as the son is supposed to make one immortal (P. V. Tivari, 2006). A very scientific description is given in Vedas that male seed is responsible for the birth of the son. Besides, coitus on even or odd days to get son or daughter is also mentioned (P. V. Tivari, 2006). In Ayurvedic classics, in addition to influence of days it is also mentioned that deposition of sukra over chandramukhi / candramasi and gauri nadi of vaginal canal is responsible for the birth of son and daughter respectively (Brahmashankar Mishra, 1933). The Pumsavana vidhi / karma done at appropriate time is so powerful that it can alter even destiny. It is to be done on pushya nakshtra immediately after conception or missed period and before clear manifestation of body parts of the foetus (which is visible in third month of pregnancy (P.V. Sharma, 2005 & 2011; Atrideo Gupta, 1993). As per Ayurvedic classics this procedure is done in two stages (1) before conception (2) after conception. Desired gender of fertilized ovum is thus achieved by administering Pumsavana before the foetus is manifested (P.V. Sharma, 2011). The Pre- Conception & Pre- Natal Diagnostic Techniques Act, (1994) was enacted and brought in to operation from 1st January 1996 in order to check female foeticide and to control the declining sex ratio.The declining sex ratio of girls and women would lead to serious socio-cultural problems like violence and population imbalance. The issue of survival of girl child is critical one, deep rooted in society which needs systematic efforts in mobilizing the community. The main purpose of the act is to ban and prevent the use of sex selection techniques before and after conception as well as to prevent the misuse of such diagnostic techniques and to regulate

them. Therefore it is necessary to eliminate the discrepancy and ambiguity of its use and to evaluate and study the legal aspects of Pumsavana Vidhi in the context of first part of PCPNDT act.1994 and to decide how it can be used in present social condition in the light of The Pre-conception and Pre-natal Diagnostic Techniques (Prohibition of Sex Selection) Act and to create awareness among Ayurvedic practitioner before using it. REVIEW OF LITERATURE Gender selection Gender selection means choosing the sex of a future child either before or after conception. In India it has been used to promote the birth of a male child, which exacerbates discrimination against female and thus contributes to decline in sex ratio. The efforts to affect the gender of a child prior to conception are of two types "biologic" and "symbolic." Biologic methods are concerned with the behaviour during copulation, the timing of copulation, and the diet of the female. Symbolic methods, in contrast, are mystical. Pre –Conception Sex Selection Biologic methods Aristotle, Greek philosopher Anaxagoras and Hindu Tantrik texts have different views on Gender selection (Own D Jones, 1992). Aristotle states that strong vigour of male child increases the possibility of male child during copulation while the Greek philosopher Anaxagoras suggests copulation by sleeping on left side (as sperm from right side) is responsible for male child. According to Hindu Tantric texts at the moment of orgasm, if the "solar breath," (inhaling through right nostril) is taken, a male child could be expected and if "lunar breath," (inhaling through left nostril) is taken, a female child could be expected). Hindus consider sun to be masculine, while the moon to be feminine, fiery and cooling in energy. In the middle ages, when a woman desires to have a male child, it was advised to

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drink a concoction of wine and lion's blood (in proper proportions) and then copulate under a full moon while an abbot prayed for a boy (presumably from a safe distance)? The role of nutrition is important in influencing gender. Dietetic theories suggested eating various combinations of fish, seeds, sugars, peas, lettuce, cheese, salt, sweets, and even the testes of certain animals will give birth to male child (Own D Jones, 1992). Gender of child depends on the emission of semen during coitus by couple. Rabbi Isaac (Fred Rosner, 1979) in the Babylonian Talmud states that if a woman "emits her semen" first she bears a male child and if the man emits first she bears a female. Shettles (online document) explains that male-desiring couples should have sex closest to the time when a woman's egg is released (ovulation). This way, the speedy male sperm could get to the egg sooner than the female one. The Y chromosome apparently also enjoys an advantage over its counterpart when the sperm is discharged as close as possible to the opening of the cervix. This is achieved through rear entry intercourse (man enters woman from behind). Parents desiring a girl, on the other hand, are encouraged to have sex in the missionary position (face to face, man on top) about two to four days before ovulation so that by the time the ova comes, only the heartier, more resilient X sperm will remain in the woman's reproductive tract. Symbolic Methods Symbolic methods (Own D Jones, 1992) for male child involves, hanging of one's trousers on the appropriate bedpost (Pennsylvania) and keeping poppies or sugar on the windowsill for female (Czechoslovakia and Hungary). or to conceive a male: women should dress like a man before intercourse (Palau Islands); a man should take an axe to bed with a woman while singing a prescribed song (Spessart Mountains of Germany); a young boy should be present in bed during intercourse (Yugoslavia); and the man should bite the woman's right ear before his orgasm

(Italian Province of Modena) (Own D Jones, 1992). Modern Methods The gender of embryo is determined by X or Y chromosome. The sperm bears XY chromosome and is of two types. The sperm which contains X chromosome is termed as gynogenic sperm and those which contain Y chromosomes are termed as androgenic sperm. The X or Y chromosome fertilizes the ovum which bears X chromosome. The fertilization by Y chromosome results in male and those by X chromosome results in female. The Sex chromosome was discovered in 1924 (Theophilus S. Painter, 1924). MicroSort (MicroSort Technology, 1996) a method that involves separation of male sperm from the female -- is the current gold standard. The technique involves separation of X- and Ybearing sperm using laser light, dye, and a machine called a flow cytometer. Once the sperms are divided, the specimens are inserted back into the woman through artificial means, such as in vitro fertilization. Other methods or techniques Other methods practised are (1) timing of intercourse during the woman's menstrual cycle (Loras) (intercourse on odd or even days during ovulations period ) (2) the time of artificial insemination, (3) the provision of acidic (or alkaline) environments for sperm, (4) the degree of penetration, (5) a woman's diet. Intercourse on odd or even days, artificial insemination done during ovulation period will give expected gender. The androgenic sperm survive more in alkaline environment of female genital tract. The manipulation of sodium, potassium, and magnesium ions in a woman's body is important to obtain the expected gender (Stolkowski J, Choukroun, 1986). The intervention of ions in ovarian metabolism is obtained by controlling the diet of the woman, the decisive factor being the ratio of K++ Na+ to Ca2+ Mg2+ in the daily diet. High values of this ratio lead to male, and low values to female. Using this method since 1970 in 47 births, only

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7 of them failed to produce the expected sex. (6) low or high sperm count within the female tract, (7) Another method of trying to conceive a child of a particular sex is to inject the woman with antibodies against androgenic (i.e., male-determining, Y-bearing) sperm or gynogenic (i.e., female-determining, Xbearing) sperm. (8) There has also been speculation "about the eventual development of a sex-selection pill which might, for instance, alter the ratio of androgenic and gynogenic sperm produced by the man, or induce the woman's immune system to selectively attack and destroy sperm of one or the other sort." (9) Development of a diaphragm which would allow only one type of sperm to pass through has also been suggested. (10) Currently the most promising methods of sex pre-selection are those involving the separation of androgenic and gynogenic spermatozoa followed by artificial insemination of a woman or in vitro fertilization with sperm of predominantly one type. Such methods increase the probability of conceiving a child of a particular sex. Post conception Sex Selection Post fertilization procedures (Loras) involve determining the sex of the child conceived through some form of prenatal diagnosis, such as amniocentesis or ultrasound, and then aborting a child of an unwanted sex. (1) Yet other methods which may be used for determining sex include removing chorionic placental tissue via the cervix and identifying Y-specific DNA, measuring the level of testosterone (a hormone produced more in male than in female foetuses) in maternal blood or saliva, determining fetal hormone levels in amniotic fluid, and assessing fetal blood cells in maternal blood. (2) In vitro fertilization (IVF) with pre implantation diagnosis also offers an opportunity for sex selection: the developing cells can be tested for sex, and only those embryos implanted which are of the desired sex. (3) It is likewise speculated that the experimental procedures of parthenogenesis and the fusion of ova could be used as sex

selection technologies. (Loras college health ethics online document). Ayurvedic methods – Pre Conception Sex Selection According to Charaka the women (who expect a son according to her desire) should perform the rites along with her husband and should ask the priest to sit to the south side of fire. She and her husband must sit to the west side of fire following the instructions of priest. In order to fulfil the son desire of women, the Priest pointing towards the ‘Prajapati‟ should perform the desire fulfilling rites in the genital parts of expectant women with the recitation of mantras as „Vishnur Yonim‟. Then he should cook the bolus in an earthen vessel, process it with ghee and offer as oblation to the fire for three times according to prescribed method. The priest should process and purify the water with sacred mantras according to own tradition and religion and give it to women saying any work associated with water should be done using this water only. On completion of rite the women should walk around the fire with her husband with right leg first, keeping in mind that the fire should be on right side. Then after the Brahmanas have invocated blessings and holy reading, the man should take the processed ghee first and then women without leaving any portion. Then the couple should join together to have sexual intercourse for eight successive nights with a desire of son that they want. The women who expects (a) son of sky complexioned with red eyes, broad chest and long arms or (b) black complexioned with black, soft and long hairs, white eyes-teeth, vigorous and self restraint sons then they should observe the same procedure to procreate the son they want. The women belonging to sudra clan should offer only salutation to Gods, fire, Brahmans, preceptor, ascetics, and accomplished persons. The women expecting son of particular country, state, nation etc should be advised to travel around these localities mentally and use diet, behaviour, regimens and apparels customary to the people living in those locality (P.V.Sharma, 2011).

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As per Sushrut Samhita, the women after getting free from menstruation (on fourth day) and desiring a birth of a son should clean herself by bath, wear nice garment and ornament and appear before her husband after auspicious hymns have been chanted. It is very probable that the child born is like the man whom a woman shall see first just after she cleans herself (on fourth day). Therefore she should first glance at her husband. The husband should observe celibacy for a month and take milk, ghee and rice. The wife should also observe celibacy for a month and should take diet consisting of profuse oil, black gram. The couple should be anointed with ghee and oil. At night with words of love and encouragement the husband should win the affection of wife and have intercourse with her. For the want of son, the intercourse should be done on fourth, sixth, eighth, tenth, or twelfth night. Those who are willing the birth of daughter should have intercourse on odd nights beginning with fifth, seventh, ninth and eleventh night only. The intercourse on thirteenth and rest of nights are always to be deprecated (R.V. Patwardhan, 1943). Post Conception Sex Selection Embryo (one month) formed after intercourse is genderless mass. It is neither male nor female. Desirable gender can be established in such embryo by vedic rites properly performed and expected result can be certainly obtained on performing the action with favourable and excellent time and place otherwise not. Hence the pregnant women should undergo Pumsavana before the gender of embryo is established and before the signs and symptoms of gender in pregnant women appear. As the women becomes pregnant, immediately any one of the four herbal drugs (viz. Lakshmana, young buds of banyan, Sahadeva or Vishvadeva) is crushed in milk and three to four drops is administered through the right nostril of conceived women if she desires male child and if she desires female same drops is administered through left nostrils. Various formulations are used in pushya nakshatra to ensure the gender of

embryo for e.g. 1) paste or solution of two young healthy undamaged clean leaf buds of banyan tree collected from north - east branch, two excellent black gram and powder of two white mustard seeds are crushed and mixed in curd. The paste or solution thus prepared is to be taken on Pushya nakshatra. 2) Drinking of processed milk made using the paste of Jivaka, rsabhaka, apamarga and saireyaka. 3) Kudyakitak (a type of insect) or Matsayka (a small fish) is given to drink in a handful of water in pushya star. 4) Drinking of milk, curd or solution made by heating the metallic (silver, gold or iron) image of beloved man and dissolving it in milk, curd or water, on Pushya star will give male gender to conceived women. 5) In Pushya star she should inhale the steam coming from Sali Rice while being cooked. 6) liquid solution prepared by mixing flour in water and pour a drop of liquid using cotton swab in the right nostril of pregnant women (P.V. Sharma, 2011) Conception and Pre Technique Act (1994)

Natal

Diagnostic

Prior to the elimination of female foetus, the sex of the foetus is to be determined by amniocentesis, chorion villus biopsy and now by the most popular technique, ultrasonography. Once the sex of the foetus is determined, if it is a female foetus, it is often aborted under the supervision of medical practitioner. The continuous demand and easy money has lead to medical practitioners colluding with parents and relatives in doing sex determination tests. As a consequence sex determination centres have mushroomed in all parts of the country including small districts and villages. The increase in female foeticide has seen the proportionate decrease in female sex ratio and if this decline is not checked the very delicate equilibrium of nature can be permanently destroyed. The issue of female foeticide is one that needs to be addressed urgently. While it is a social problem requiring changing the mindset of people, yet all possible efforts need to be made at every level. Here the law can be used as an effective tool to create fear in the minds of those who resort to sex-

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determination tests or those who carry out such techniques (GOI, 2006). Legal Initiatives With the rise of pre-natal diagnostic techniques especially amniocentesis, the government in 1978 issued a directive banning the misuse of amniocentesis in government hospitals/laboratories. Thereafter due to the relentless efforts of activists, a law to prevent sex determination tests was passed in Maharashtra known as the Maharashtra Regulation of Pre-natal Diagnostic Techniques Act, 1988. Finally after intensive public debate all over India the Parliament enacted the PreNatal Diagnostic Techniques (Regulation and Prevention of Misuse) Act on 20th September 1994 (hereinafter referred to as the PNDT Act) to provide for the regulation of: the use of prenatal diagnostic techniques for the purpose of detecting genetic or metabolic disorders or chromosomal abnormalities or certain congenital mal-formations or sex-linked disorders; for the prevention of the misuse of such techniques for the purpose of pre-natal sex determination leading to female foeticide; and for matters connected there with or incidental thereto. This Act came into force in 1996. Amendments to the Act of 1994 During the course of the implementation of the said Act, certain inadequacies and practical difficulties in the administration of the Act came to the notice of the Government. At the same time techniques have been developed to select the sex of the child before conception which may further declines sex-ratio. After detailed deliberations, the PNDT Act has been amended and the Act has come into force with effect from January 2003. The main purpose is to ban the use of sex-selection techniques before or after conception as well as the misuse of pre-natal diagnostic techniques for sex selective abortions and to regulate such techniques. To make this clear, the long title of the Act has been suitably amended to read: “The Pre-conception and Pre-natal Diagnostic Techniques (Prohibition of Sex

Selection) Act.”. The amended Rules have come into effect from 14th Feb, 2003 Sex selection "refers to means whereby the sex of an offspring can be chosen." This includes the use of reproductive technologies which attempt to achieve the conception of a child of a particular sex (sex preselection) and the abortion of a child of an unwanted sex. Sex selection includes: Procedure, Technique, Test, Administration, Prescription and Provision Of anything for the purpose of ensuring or increasing the probability that an embryo will be of a particular sex (GOI, 2006). DISCUSSION The methods described in Ayurveda and other medical science for selection of gender (particularly male child) includes pre conception and post conception methods. The act prohibits the method or techniques adopted for the selection of gender before or after conception. As mentioned above the act possesses two parts, (1) pre conception methods for expected gender (2) post conception technique for desired male child. Gender selection includes any Procedure, Technique, Test, Administration, Prescription and Provision of anything for the purpose of ensuring or increasing the probability that an embryo will be of a particular gender. Admission to life should not be conditional upon a child being of a particular gender. The PCPNDT Act prohibits all possible activities done by Ayurvedic methods to ensure and to increase the probability that the embryo or ‘Garbh‟ is of a particular gender (especially that the embryo or ‘Garbh‟ is male) the Pumsavana Vidhi is also prohibited as it ensures and increases probability that the embryo is of a desired gender. No one can select gender nor follow the pre conception procedures like, performing ritual rite for the want of son, diet that promotes and ensures the birth and development of son or selecting odd or even days for coitus to ensure the gender of embryo. Post conception procedures are also banned as they are done under the supervision of a trained Vaidya which is illegal and is liable

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for legal action or penalties against both the Vaidya and the couple by appropriate legal government authority. Though female foeticide or abortion is not done in Pumsavana vidhi but its misuse promotes the birth of male child more than female, which declines the gender ratio and creates social problems like violence and population imbalance and it intervenes with the natural process of child birth. Hence it is banned by PCPNDT Act. These procedures are done especially for the child with desired sex and therefore they are prohibited. The procedure mentioned in Ayurvedic sciences does not encourage female foeticide but it promotes the birth of a son in a family which in turn will lead to population imbalance.

CONCLUSION It is clear that the pumsavana vidhi is done after conception for the want of expected gender. The tendency of most Indian couple is towards having a male child which brought this practice of Pumsavana vidhi into the society. Though in ancient times, the practice was authorized, and enough references are available in Ayurveda which explains the antiquity & obviousness of the practice, now in this era, there are laws to monitor misuse of such practices based on the prevailing conditions in the society. Hence, it is better that practicing physicians of Indian medicine become aware of the details of the PCPNDT act (1994) and refrain from such practices so as to have a safer and ethical practice career.

REFERENCES Brahmashankar Mishra, (1933) Bhav prakash, 8th Edition, Published by Chaukhamba Sanskrit Sansthan, Gopal mandir Lane, Varanasi. Fred Rosner, (1979) The Biblical and Talmudic Secret for Choosing One's Baby's Sex, Israel Journal Of Medical Sciences, Sept;15(9): 784–7 Landrum B. Shettles, How to Choose the Sex of Your Baby [Online Document] http://www.webmd.com/baby/features/c hoosing-sex-of-your-child?page=3). Loras

College Health Ethics, [Online Document] (http://www2.loras.edu/~CatholicHE/Ar ch/Sexuality/sex_selection.html)

"MicroSort Technology."(2007) [Online Document] MicroSort. 1996. Genetics and IVF Institute. Feb.-Mar., Available at: http://www.microsort.net/process.php

GOI (2006), Ministry of Health and Family Welfare Government of India, Handbook on Pre- Conception & PreNatal Diagnostic Techniques Act, 1994 and Rules with Amendments, published by, chapter1, page 9 [Online Document] Available at http://aurangabad.nic.in/newsite/whats_ new/PC_AND_PNDT_ACT_1994.pdf Own D Jones, (1992) Sex Selection Regulating Technologies enabling the predetermination of child gender, Harvard journal of Law and Technology, Volume 6,.[Online Document] http://jolt.law.harvard.edu/articles/pdf/v 06/06HarvJLTech001.pdf) P. V. Tivari, (2006) Text Book of Prasuti Tantra and Stri Roga, Second Edition, Chaukhambha Visvabharti Publisher, Varanasi P.V. Sharma, (2005) Charak Samhita, english translation, Chaukhambha orientalia P.B.No 1032, Golghar Varanasi

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||


Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 8 | August 2014 | 323–330

P.V. Sharma, (2011) Charak Samhita, english translation, Chaukhambha orientalia P.B.No 1032, Golghar Varanasi

17(11):1061–7. http://www.ncbi.nlm.nih.gov/pubmed/6 947968

R.V. Patwardhan, (1943) Sushrut Samhita, Published by The Bombay Provincial Ayurveda Research Association, Poona Edition

Theophilus S. Painter, (1924) The Sex Chromosomes of Man, 58 AM. NAT.506. [Online Document] http://www.nasonline.org/publications/b iographical-memoirs/memoirpdfs/painter-theophilus-shickel.pdf

Stolkowski J, Choukroun J. (1981) Preconception selection of sex in man. Israel Journal of Medical Sciences, Nov Source of Support: NIL

Conflict of Interest: None Declared

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||


Call for Papers – Vol. 3, Issue 10, October 2014 Submit your manuscripts (Research articles, Review articles, Short Communications, Letters to the Editor, Book Reviews) to Global Journal of Research on Medicinal plants & Indigenous medicine – GJRMI Submit it online through www.gjrmi.com or mail it to submitarticle@gjrmi.com on or before September 10th 2014.

To advertise on the Flip book Cover page freely, write to chiefeditor@gjrmi.com or editorinchief@gjrmi.com Or Call - +919590574495


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