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Spermicidal activity of Azadirachta indica (neem) leaf extract
Contraception 68 (2003) 225–229
Original research article
Spermicidal activity of Azadirachta indica (neem) leaf extract B. Khillare*, T.G. Shrivastav Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi, India-110067 Received 21 March 2003; received in revised form 5 June 2003; accepted 9 June 2003
Abstract The present study was carried out to evaluate the effective concentration of aqueous extract of old and tender Azadirachta indica (neem) leaves to immobilize and kill 100% human spermatozoa within 20 s. Sander–Cramer test was used to study the spermicidal activity of neem leaf extract. Under the test conditions, minimum effective spermicidal concentrations for tender and old leaf extracts were 2.91 ⫾ 0.669 mg/million sperm and 2.75 ⫾ 0.754 mg/million sperm, respectively. The effect of extracts on morphology and viability of sperm was also studied and no change was observed in morphology of head, mid-piece and tail and no viable sperm seen. The leaf extracts were found to be water soluble and carbohydrate in nature. The effect of different concentrations of extracts (old and tender) on percentage motility of the sperm was also studied. With an increase in concentration, there is a linear decrease in percentage motility, becoming zero at a 3-mg dose within 20 s. © 2003 Elsevier Inc. All rights reserved. Keywords: Azadirachta indica; Leaf extract; Spermicidal; Sperm viability; Sperm morphology
1. Introduction Several potential approaches for induction of infertility have been investigated over a long period, including chemical, hormonal and immunological approaches. However, no suitable method has emerged that is effective and free from side effects. Medicinal plant products have a long history of indigenous use in India as well as other countries, but no systematic investigation has been carried out, except for studies of Triptergium wilfordi in China and neem oil in India, which aimed to find the potentiality for antifertility/ spermicidal activity. The neem tree, Azadirachta indica. A. Juss (Nimba), is indigenous to the Indian subcontinent and its medicinal properties have been described in Ayurveda. The bark and leaves are used as bitter tonic and astringent. It is considered as an antiseptic, a blood purifier and useful in skin disorders. Its fruit is an emollient and purgative. Azadirachta indica is also considered beneficial in the treatment of a wide range of disorders, such as coughing, nausea, vomiting, fever, jaundice, gonorrhea, urinary tract infection, intestinal worm infestation and leprosy [1–5].
* Corresponding author. Tel.: ⫹91-11-26107773; fax: ⫹91-116101623. E-mail address: [email protected] (B. Khillare). 0010-7824/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0010-7824(03)00165-3
The juice of fresh green leaves of Azadirachta indica was believed to suppress “Kam Vasana” (desire for sex). It was consumed by sanyasees in shrines and the pupils studying in Gurukul for the same purpose. Deshpande et al. [6] have studied the antifertility activity of Azadirachta indica leaves in male mice. Freshly prepared water extract of crushed green leaves of Azadirachta indica was orally fed to mice every day for 1 month to study its effect on male reproduction function. It was observed that control mice showed 100% fertility rate. In Azadirachta indica-treated animals, the antifertility effect was 80%. The study revealed that after 45 days of withdrawal of treatment, the percentage of pregnancies was 100%. Thus, Azadirachta indica leaves have shown reversible male antifertility activity. The powder of Azadirachta indica leaves is reported to cause histological and biochemical changes in the testes of rats [7,8]. Histological and biochemical changes in the testes of rats treated with the leaf powder of Azadirachta indica were studied. The pattern of recovery was also studied at 8, 16 and 24 days after withdrawal of the treatment. In the treated rats, a general reduction in the diameter of seminiferous tubules, nuclei of the germinal elements and mass atrophy of the spermatogenic elements has been observed. The Leydig cells were found to be atrophic. Biochemically, a decrease in the protein content and the activity of acid phosphatase and an increase in the total free sugar, glycogen,
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cholesterol contents and the activities of alkaline phosphatase and lactate dehydrogenase have been observed. A gradual recovery was observed in both the histological and biochemical parameters after 8, 16 and 24 days of cessation of the treatment. The results suggest a possible reversible antiandrogenic property of the leaves of Azadirachta indica in male albino rats. Besides all the above preliminary studies, there is no documented evidence referring to the spermicidal activity of the leaves extract of Azadirachta indica. It was, therefore, our interest to investigate the spermicidal activity of the neem leaves. The present study was carried out to evaluate the in vitro spermicidal activity of old and tender Azadirachta indica (neem) leaf extract against human spermatozoa. Study has been approved by the local clinical ethics committee.
spermicidal agent required to kill 100% sperm within 20 s. Test ingredients of various concentrations (1 mg, 2 mg, 3 mg, 4 mg, . . . 50 mg) were mixed with sperm suspension containing 1 million sperm. The mixture was observed under microscope for 20 s at 10⫻ and read for motile sperm. If any motile sperm were seen, the concentration was recorded as a “fail.” Two-hundred-fifty microliters of buffer were added to all the mixtures that passed the test and incubated at 37°C for at least 60 min. The solution was slowly vortexed and observed again after 60 min for presence of any motile sperm. The concentration at which it was tested was recorded as effective if both tests indicated absence of motile sperm. The end point was the lowest concentration of the neem leaf extract that caused complete immobilization of all the sperm within 20 s of mixing. The dose- and time-dependent study for spermicidal activity was done using the above test. The effect of different concentrations of both old and tender extracts on percentage motility was studied.
2. Materials and methods 2.5. Sperm viability test 2.1. Semen samples The experiment was conducted on human sperm. Semen was collected from apparently normal males visiting our infertility clinic, who had 2–3 day’s abstinence from sexual activity. Samples having a volume ⱖ2 mL, motility ⱖ50%, sperm concentration ⱖ20 million/mL or more, more than 50% with normal morphology, viability ⬎50%, total leucocyte ⬍1 million/mL were used. Semen samples were collected in a dry and clean beaker and stored at 37°C in an incubator for half an hour.
Sperm were mixed with old and tender leaf extract separately for 20 s. Sperm viability was checked using EosinNigrosin technique given below. Unstained spermatozoa were counted as live and stained were counted as dead spermatozoa [10]. 2.6. Eosin-Nigrosin staining technique 2.6.1. Reagents Eosin Y (10 g/L, i.e., 1% in distilled water); Nigrosin (100 g/L, i.e., 10% in distilled water).
2.2. Chemicals Chemicals such as Eosin Y, Nigrosin and buffer salts were of analytical grade and were procured from E. Merck (India) Limited. 2.3. Preparation of neem leaf extract
2.6.2. Procedure One drop of above-treated semen mixed with 2 drops of 1% Eosin Y. After 30 s, 3 drops of 10% Nigrosin solution added and mixed. A drop of the semen-Eosin-Nigrosin mixture placed on a clear microscope slide; allowed to dry and observed under microscope.
An aqueous extract was prepared from Azadirachta indica (neem) leaves, collected from National Institute of Health and Family Welfare campus, New Delhi, India. Aqueous extract of both old and tender leaves were prepared separately. Neem leaves were ground in a mixer and filtered with a gauze. The filtrate was washed with chloroform in 1:1 proportion, centrifuged at 3000 rpm for 20 min. The pallet was discarded and the supernatant frozen at ⫺20°C for lyophilization. The extracts were lyophilized separately.
2.7. Sperm morphology
2.4. Test for spermicidal activity
The effectiveness of neem leaves extract during storage time was studied. The extract was stored in a refrigerator for 4 years and the spermicidal activity of neem leaves extract was studied using Sander–Cramer test for both old and tender neem leaf extracts.
The spermicidal activity was determined using a modified version of the protocol originally described by Sander and Cramer [9], which measures the minimum concentration of
Sperm morphology of treated sperm was studied under the microscope using Eosin Y and Nigrosin staining method as described above. A drop of semen treated with neem leaf extract both old and tender leaves extract was examined separately at 400⫻ under phase contrast microscope to record any change in morphology of the sperm. 2.8. Stability test
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Table 1 Minimum effective concentration (MEC) of aqueous extract of tender neem leaf required to kill 1 million sperm in 20 s Sample no.
Semen sample sperm count (mill/mL)
Amount of semen taken containing 1 million sperm (L)
% Motility
MEC (mg)
1 2 3 4 5 6 7 8 9 10 11 12
100 40 60 48 70 48 40 80 60 110 110 70
10.0 25.0 16.6 20.8 14.2 20.8 25.0 12.5 16.6 9.09 9.09 14.2
70 60 80 60 70 70 70 70 70 50 80 70
3 3 4 2 2 2 2 3 3 2 4 3
2.9. Chemical nature of extracts
3. Results
The chemical nature of ingredients of old and tender leaf extracts was studied for the presence of lipid, protein and carbohydrate. It has been observed that the ingredient was carbohydrate in nature. The details of carbohydrate estimation by Fehling’s test are given below.
3.1. Spermicidal activity
2.9.1. Preparation of Fehling’s solution Fehling’s solution was prepared by mixing copper sulfate solution and alkaline tartrate solution. Copper sulfate solution was prepared by dissolving 34.65 g copper sulfate in distilled water and made up to 500 mL. Alkaline tartrate solution was prepared by dissolving 125 g of potassium hydroxide and 173 g Rochelle salt in distilled water and made up to 500 mL. To prevent deterioration, these solutions were preserved separately in a rubber-stoppered bottle and mixed in equal volumes when needed for use. 2.9.2. Procedure To the warm Fehling’s solution, an extract was added and the mixture was heated after each addition [11].
MEC (mg) mean ⫾ SD
2.91 ⫾ 0.669
The spermicidal activity of graded doses of neem leaf extract was studied in vitro using human semen. The results of Sander–Cramer test showed potent spermicidal activity of Azadirachta indica leaves extract. The minimum effective concentration of aqueous neem leaf extract required to kill 1 million sperm in 20 s was around 2.91 ⫾ 0.669 and 2.75 ⫾ 0.754 for tender and old leaf extract, respectively. Both old and tender leaves show similar potentiality and the minimum effective concentrations are almost the same (Tables 1 and 2). 3.2. Sperm motility and viability The effect of different concentrations of extract (old and tender) on percentage motility of 1 million sperm after exposure of different doses for 20 s is shown in Figs. 1 and 2. The results revealed that with an increase in concentration, there is linear decrease in percent motility. Approxi-
Table 2 Minimum effective concentration (MEC) of aqueous extract of old neem leaf required to kill 1 million sperm in 20 s Sample no.
Semen sample sperm count (mill/mL)
Amount of semen taken containing 1 million sperm (L)
% Motility
MEC (mg)
MEC (mg) mean ⫾ SD
1 2 3 4 5 6 8 9 10 11 12
100 40 60 48 70 48 80 60 110 110 70
10.0 25.0 16.6 20.8 14.2 20.8 12.5 16.6 9.09 9.09 14.2
70 60 80 60 70 70 70 70 50 80 70
3 3 4 2 2 3 3 3 3 4 3
2.75 ⫾ 0.754
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This study demonstrates that aqueous extracts of old and tender leaves of Azadirachta indica have potent spermicidal effect. Its minimum effective concentration attains 100% immobilization and killing of sperm within 20 s.
4. Discussion
Fig. 1. Effect on percent motility of 1 million sperm after exposure to different doses of aqueous extract of tender neem leaf for 20 s.
mately 3 mg of extract is required to immobilize and kill 100% of 1 million sperm in 20 s. The viability of sperm was studied using Eosin Y and Nigrosin stains. It was found that crude extract kills 100% of 1 million sperm within 20 s. 3.3. Sperm morphology The morphological study of sperm was done using Eosin-Nigrosin stain and no morphological changes were found in the sperm head, mid-piece or tail when compared with untreated sperm. 3.4. Chemical nature Using Fehling’s test, the nature of the old and tender leaf extract was found to be carbohydrate in nature. 3.5. Stability It was also observed that the activity of both the old and tender leaf extracts remains the same, and the effectiveness does not change with storage time of 4 years.
Fig. 2. Effect on percent motility of 1 million sperm after exposure to different doses of aqueous extract of old neem leaf for 20 s.
In the present study, we have described a two-step procedure to prepare aqueous extract from the leaves of Azadirachta indica for checking spermicidal activity. Using neem leaf, two studies have been carried out. One is suggestive of reversible antifertility effect when fresh green leaf extract was orally fed to male mice for 30 days [6]. Another is suggestive of reversible antiandrogenic effect on the histological and biochemical parameters of testis of adult albino rats [7,8], when 100 mg of dried leaf powder, suspended in 1 mL of distilled water per day, was given for a period of 24 days. The results of the present study revealed that the aqueous extract of old and tender neem leaves is a potent spermicide, which is demonstrated through dose-dependent study on the effect of motility of spermatozoa and then confirmed by viability test. No morphological changes were found in the sperm head, mid-piece and tail when compared with untreated sperm. Therefore, 100% killing of sperm may be due to blockage of some biochemical pathway like energy utilization, which would require further investigation. The potency of lyophilized aqueous extract of old and tender leaves remains the same and the effectiveness does not change with storage time of 4 years. The proposed spermicide can be a potent vaginal contraceptive and can be formulated as cream or a pessary. Carbohydrates possessing a free or potentially free aldehyde or ketone group have the property of readily reducing the ions of certain metals such as copper, bismuth, mercury, iron and silver. The most widely used tests for sugar are based on this property. For example, when blue cupric hydroxide suspended in an alkaline medium is heated, it is converted into insoluble black cupric oxide. However, in the presence of reducing agents such as certain sugars, the cupric hydroxide is reduced to insoluble yellow or red cuprous oxide. Based on the above property, the chemical nature of old and tender leaf extracts was studied using Fehling’s test. The production of yellow or brownish-red cuprous oxide indicated that reduction has taken place. The difference in color of cuprous oxide precipitate under different conditions was apparently due to differences in the size of the particles, the more finely divided precipitates having yellow color while the coarser ones were red. This test was found to be positive, indicating that the old and tender leaf extracts are carbohydrate in nature. The neem oil, also known as oil of Margosa, is believed to have medicinal properties, such as antibacterial [12], antifungal [13] and antidiabetic [14].
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Upadhyay et al. [15] observed a long-term contraceptive effect of single intra-vas administration of neem oil in male rats. The antifertility effect was observed for 8 months and was found to be an alternative approach to vasectomy. No inflammatory/obstructive changes in epididymis and vas deferens were observed. There was no change in blood testosterone levels. Purified neem seed extract called praneem was reported to cause termination of pregnancy by bleeding and a decrease in progesterone levels in rats, baboons and monkeys [16]. It was also reported to abrogate pregnancy in primates [17]. The volatile fraction of neem oil (NIM-76) inhibited spermatozoal activity in vitro at the concentration of 0.25 mg/mL and 25 mg/mL for rat and human spermatozoa, respectively [18]. The spermicidal activity of NIM-76 was investigated in vivo in rats, rabbits and rhesus monkey. It acts mainly by spermicidal effect [19]. A vaginal contraceptive has been developed from NIM-76 by Sharma et al. [20]. Two neem oil derivatives, viz., sodium nimbinate [21] and sodium nimbidinate [22], have been found to possess weak spermicidal action in vitro. In view of this, a study was undertaken by Sinha et al. in 1984 [23] on the role of neem oil as a spermicidal agent. Indigenously available neem oil in its natural form was tested for its spermicidal activity (in vitro and in vivo). Undiluted neem oil was found to possess strong spermicidal action (within 30 s) against rhesus monkey and human spermatozoa in vitro. Whereas 3 mg of neem leaf extract, when treated with human spermatozoa, kills 100% of sperm within 20 s. The time taken by sperm to travel to the oviduct from the cervix in women is 5– 68 min, when all conditions are favorable in the fertile period [24]. Therefore, neem leaf extract is more advantageous than the undiluted neem oil, which takes 30 s to kill the sperm. Praneem polyherbal cream has been developed by Garg et al. [25], which has synergistic spermicidal concentration for praneem (25%), reetha saponins (0.05%) and quinine hydrochloride (0.34%) and was found at this concentration to result in 100% immobilization of sperm within 20 s [26]. Until now, neem seed extract or oil or oil components were studied as an effective contraceptive that is hydrophobic in nature. The neem leaf extract, which is hydrophilic in nature, mixes immediately with water as well as body fluids and kills sperm within 20 s; with its use, a more potent vaginal contraceptive may be developed.
Acknowledgments We are thankful to Mr. R. K. Sharma for his technical help.
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References [1] Chopra RN, Nayar SL, Chopra IC. Glossary of Indian medicinal plants. New Delhi: CSIR, 1956 p. 31–2. [2] Lakshmipathi A. One hundred useful drugs. 3rd ed. Madras: Arogya Ashram Samithi, 1973. p. 85–7. [3] Kirtikar AR, Basu BD. Indian medicinal plants, Vol 1. Allahabad, India: Lalit Mohan Basu, 1935. p. 536 – 42. [4] Satyavati GV, Raina MK, Sharma M. Medicinal plants of India, Vol 1. New Delhi: Indian Council of Medical Research, 1976. p. 112–7. [5] Thakur RS, Singh SB, Goswami A. Azadirachta indica A. Juss. A review. Curr Res Med Aromatic Plants 1981;3:135– 40. [6] Deshpande VY, Mendulkar KN, Sadre NL. Male antifertility activity of Azadirachta indica in mice. J Postgrad Med 1980;26:167–70. [7] Joshi AR, Ahamed RN, Pathan KM, Manivannan B. Effects of Azadirachta indica leaves on testis and its recovery in albino rats. Indian J Exp Biol 1996;34:1091– 4. [8] Aladakatti RH, Nazeer Ahmed R, Ahmed M, Ghosesawar MG. Sperm parameters changes induced by Azadirachta indica in albino rats. J Basic Clin Physiol Pharmacol 2001;12:69 –76. [9] Sander FV, Cramer SD. A practical method of testing the spermicidal action of chemical contraceptives. Hum Fertil 1941;6:134 – 8. [10] Eliasson R. Supravital staining of human spermatozoa. Fertil Steril 1977;28:1257. [11] Hawk PB. Hawk’s physiological chemistry. New York: McGraw-Hill Book Company, 1965. p. 83–1322. [12] Singh N, Sastri MS. Anti-microbial activity of neem oil. Indian J Pharmacol 1981;13:102. [13] Kher A, Chaurasia SC. Anti-fungal activity of essential oils of three medicinal plants. Indian Drugs 1977;15:41–2. [14] Sharma MK, Khare AK, Feroz H. Effect of neem oil on blood levels of normal hyperglycemic and diabetic animals. Nagarjun 1983;26: 247–50. [15] Upadhyay SN, Dhavan S, Talwar GP. Effect of neem (Azadirachta indica) oil in male rats by single intra-vas administration—an alternative approach to vasectomy. J Androl 1993;14:275– 81. [16] Talwar GP, Shah S, Mukherjee S, Chabra R. Induced termination of pregnancy by purified extract of Azadirachta indica (Neem). Am J Reprod Immunol 1997;37:485–91. [17] Mukherjee S, Lohiya NK, Pal R, Sharma MG, Talwar GP. Purified neem (Azadirachta indica) seed extract (Praneem) abrogates pregnancy in primates. Contraception 1996;53:375– 8. [18] Riar SS, Devkumar C, Bardhan J, et al. Volatile fraction of neem oil as spermicide. Contraception 1990;42:79 – 87. [19] Riar SS, Devkumar C, Sawhney RC, et al. Antifertility activity of volatile fraction of neem oil. Contraception 1991;44:319 –27. [20] Sharma SK, Sairam M, Ilavazagan G, Kumar D, Shivaji SS, Selvamurthi W. Mechanism of action of NIM-76. A novel vaginal contraceptive from neem oil. Contraception 1996;54:373– 8. [21] Sharma VN, Saksena PK. Spermicidal action of sodium nimbinate. Indian J Med Res 1959;47:322– 4. [22] Sharma VN, Saksena PK. Sodium nimbidinate. In vitro study of its spermicidal action. Indian J Med Res 1959;13:1038 –24. [23] Sinha KC, Riar SS, Tiwari RS, et al. Neem oil as a vaginal contraceptive. Indian J Med Res 1984;79:131– 6. [24] Knobil E, Neill JD. The physiology of reproduction, Vol 1. New York: Raven Press, 1994. p. 139. [25] Garg S, Taluja U, Upadhayay SN, Talwar GP. Studies on the contraceptive efficacy of praneem polyherbal cream. Contraception 1993; 48:591– 6. [26] Garg S, Doncel G, Chabra S, Upadhyay SN, Talwar GP. Synergistic spermicidal activity of neem seed extract, reetha saponin and quinuine hydrochloride. Contraception 1994;50:185–90.
Original research article
Spermicidal activity of Azadirachta indica (neem) leaf extract B. Khillare*, T.G. Shrivastav Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi, India-110067 Received 21 March 2003; received in revised form 5 June 2003; accepted 9 June 2003
Abstract The present study was carried out to evaluate the effective concentration of aqueous extract of old and tender Azadirachta indica (neem) leaves to immobilize and kill 100% human spermatozoa within 20 s. Sander–Cramer test was used to study the spermicidal activity of neem leaf extract. Under the test conditions, minimum effective spermicidal concentrations for tender and old leaf extracts were 2.91 ⫾ 0.669 mg/million sperm and 2.75 ⫾ 0.754 mg/million sperm, respectively. The effect of extracts on morphology and viability of sperm was also studied and no change was observed in morphology of head, mid-piece and tail and no viable sperm seen. The leaf extracts were found to be water soluble and carbohydrate in nature. The effect of different concentrations of extracts (old and tender) on percentage motility of the sperm was also studied. With an increase in concentration, there is a linear decrease in percentage motility, becoming zero at a 3-mg dose within 20 s. © 2003 Elsevier Inc. All rights reserved. Keywords: Azadirachta indica; Leaf extract; Spermicidal; Sperm viability; Sperm morphology
1. Introduction Several potential approaches for induction of infertility have been investigated over a long period, including chemical, hormonal and immunological approaches. However, no suitable method has emerged that is effective and free from side effects. Medicinal plant products have a long history of indigenous use in India as well as other countries, but no systematic investigation has been carried out, except for studies of Triptergium wilfordi in China and neem oil in India, which aimed to find the potentiality for antifertility/ spermicidal activity. The neem tree, Azadirachta indica. A. Juss (Nimba), is indigenous to the Indian subcontinent and its medicinal properties have been described in Ayurveda. The bark and leaves are used as bitter tonic and astringent. It is considered as an antiseptic, a blood purifier and useful in skin disorders. Its fruit is an emollient and purgative. Azadirachta indica is also considered beneficial in the treatment of a wide range of disorders, such as coughing, nausea, vomiting, fever, jaundice, gonorrhea, urinary tract infection, intestinal worm infestation and leprosy [1–5].
* Corresponding author. Tel.: ⫹91-11-26107773; fax: ⫹91-116101623. E-mail address: [email protected] (B. Khillare). 0010-7824/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0010-7824(03)00165-3
The juice of fresh green leaves of Azadirachta indica was believed to suppress “Kam Vasana” (desire for sex). It was consumed by sanyasees in shrines and the pupils studying in Gurukul for the same purpose. Deshpande et al. [6] have studied the antifertility activity of Azadirachta indica leaves in male mice. Freshly prepared water extract of crushed green leaves of Azadirachta indica was orally fed to mice every day for 1 month to study its effect on male reproduction function. It was observed that control mice showed 100% fertility rate. In Azadirachta indica-treated animals, the antifertility effect was 80%. The study revealed that after 45 days of withdrawal of treatment, the percentage of pregnancies was 100%. Thus, Azadirachta indica leaves have shown reversible male antifertility activity. The powder of Azadirachta indica leaves is reported to cause histological and biochemical changes in the testes of rats [7,8]. Histological and biochemical changes in the testes of rats treated with the leaf powder of Azadirachta indica were studied. The pattern of recovery was also studied at 8, 16 and 24 days after withdrawal of the treatment. In the treated rats, a general reduction in the diameter of seminiferous tubules, nuclei of the germinal elements and mass atrophy of the spermatogenic elements has been observed. The Leydig cells were found to be atrophic. Biochemically, a decrease in the protein content and the activity of acid phosphatase and an increase in the total free sugar, glycogen,
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cholesterol contents and the activities of alkaline phosphatase and lactate dehydrogenase have been observed. A gradual recovery was observed in both the histological and biochemical parameters after 8, 16 and 24 days of cessation of the treatment. The results suggest a possible reversible antiandrogenic property of the leaves of Azadirachta indica in male albino rats. Besides all the above preliminary studies, there is no documented evidence referring to the spermicidal activity of the leaves extract of Azadirachta indica. It was, therefore, our interest to investigate the spermicidal activity of the neem leaves. The present study was carried out to evaluate the in vitro spermicidal activity of old and tender Azadirachta indica (neem) leaf extract against human spermatozoa. Study has been approved by the local clinical ethics committee.
spermicidal agent required to kill 100% sperm within 20 s. Test ingredients of various concentrations (1 mg, 2 mg, 3 mg, 4 mg, . . . 50 mg) were mixed with sperm suspension containing 1 million sperm. The mixture was observed under microscope for 20 s at 10⫻ and read for motile sperm. If any motile sperm were seen, the concentration was recorded as a “fail.” Two-hundred-fifty microliters of buffer were added to all the mixtures that passed the test and incubated at 37°C for at least 60 min. The solution was slowly vortexed and observed again after 60 min for presence of any motile sperm. The concentration at which it was tested was recorded as effective if both tests indicated absence of motile sperm. The end point was the lowest concentration of the neem leaf extract that caused complete immobilization of all the sperm within 20 s of mixing. The dose- and time-dependent study for spermicidal activity was done using the above test. The effect of different concentrations of both old and tender extracts on percentage motility was studied.
2. Materials and methods 2.5. Sperm viability test 2.1. Semen samples The experiment was conducted on human sperm. Semen was collected from apparently normal males visiting our infertility clinic, who had 2–3 day’s abstinence from sexual activity. Samples having a volume ⱖ2 mL, motility ⱖ50%, sperm concentration ⱖ20 million/mL or more, more than 50% with normal morphology, viability ⬎50%, total leucocyte ⬍1 million/mL were used. Semen samples were collected in a dry and clean beaker and stored at 37°C in an incubator for half an hour.
Sperm were mixed with old and tender leaf extract separately for 20 s. Sperm viability was checked using EosinNigrosin technique given below. Unstained spermatozoa were counted as live and stained were counted as dead spermatozoa [10]. 2.6. Eosin-Nigrosin staining technique 2.6.1. Reagents Eosin Y (10 g/L, i.e., 1% in distilled water); Nigrosin (100 g/L, i.e., 10% in distilled water).
2.2. Chemicals Chemicals such as Eosin Y, Nigrosin and buffer salts were of analytical grade and were procured from E. Merck (India) Limited. 2.3. Preparation of neem leaf extract
2.6.2. Procedure One drop of above-treated semen mixed with 2 drops of 1% Eosin Y. After 30 s, 3 drops of 10% Nigrosin solution added and mixed. A drop of the semen-Eosin-Nigrosin mixture placed on a clear microscope slide; allowed to dry and observed under microscope.
An aqueous extract was prepared from Azadirachta indica (neem) leaves, collected from National Institute of Health and Family Welfare campus, New Delhi, India. Aqueous extract of both old and tender leaves were prepared separately. Neem leaves were ground in a mixer and filtered with a gauze. The filtrate was washed with chloroform in 1:1 proportion, centrifuged at 3000 rpm for 20 min. The pallet was discarded and the supernatant frozen at ⫺20°C for lyophilization. The extracts were lyophilized separately.
2.7. Sperm morphology
2.4. Test for spermicidal activity
The effectiveness of neem leaves extract during storage time was studied. The extract was stored in a refrigerator for 4 years and the spermicidal activity of neem leaves extract was studied using Sander–Cramer test for both old and tender neem leaf extracts.
The spermicidal activity was determined using a modified version of the protocol originally described by Sander and Cramer [9], which measures the minimum concentration of
Sperm morphology of treated sperm was studied under the microscope using Eosin Y and Nigrosin staining method as described above. A drop of semen treated with neem leaf extract both old and tender leaves extract was examined separately at 400⫻ under phase contrast microscope to record any change in morphology of the sperm. 2.8. Stability test
B. Khillare, T.G. Shrivastav / Contraception 68 (2003) 225–229
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Table 1 Minimum effective concentration (MEC) of aqueous extract of tender neem leaf required to kill 1 million sperm in 20 s Sample no.
Semen sample sperm count (mill/mL)
Amount of semen taken containing 1 million sperm (L)
% Motility
MEC (mg)
1 2 3 4 5 6 7 8 9 10 11 12
100 40 60 48 70 48 40 80 60 110 110 70
10.0 25.0 16.6 20.8 14.2 20.8 25.0 12.5 16.6 9.09 9.09 14.2
70 60 80 60 70 70 70 70 70 50 80 70
3 3 4 2 2 2 2 3 3 2 4 3
2.9. Chemical nature of extracts
3. Results
The chemical nature of ingredients of old and tender leaf extracts was studied for the presence of lipid, protein and carbohydrate. It has been observed that the ingredient was carbohydrate in nature. The details of carbohydrate estimation by Fehling’s test are given below.
3.1. Spermicidal activity
2.9.1. Preparation of Fehling’s solution Fehling’s solution was prepared by mixing copper sulfate solution and alkaline tartrate solution. Copper sulfate solution was prepared by dissolving 34.65 g copper sulfate in distilled water and made up to 500 mL. Alkaline tartrate solution was prepared by dissolving 125 g of potassium hydroxide and 173 g Rochelle salt in distilled water and made up to 500 mL. To prevent deterioration, these solutions were preserved separately in a rubber-stoppered bottle and mixed in equal volumes when needed for use. 2.9.2. Procedure To the warm Fehling’s solution, an extract was added and the mixture was heated after each addition [11].
MEC (mg) mean ⫾ SD
2.91 ⫾ 0.669
The spermicidal activity of graded doses of neem leaf extract was studied in vitro using human semen. The results of Sander–Cramer test showed potent spermicidal activity of Azadirachta indica leaves extract. The minimum effective concentration of aqueous neem leaf extract required to kill 1 million sperm in 20 s was around 2.91 ⫾ 0.669 and 2.75 ⫾ 0.754 for tender and old leaf extract, respectively. Both old and tender leaves show similar potentiality and the minimum effective concentrations are almost the same (Tables 1 and 2). 3.2. Sperm motility and viability The effect of different concentrations of extract (old and tender) on percentage motility of 1 million sperm after exposure of different doses for 20 s is shown in Figs. 1 and 2. The results revealed that with an increase in concentration, there is linear decrease in percent motility. Approxi-
Table 2 Minimum effective concentration (MEC) of aqueous extract of old neem leaf required to kill 1 million sperm in 20 s Sample no.
Semen sample sperm count (mill/mL)
Amount of semen taken containing 1 million sperm (L)
% Motility
MEC (mg)
MEC (mg) mean ⫾ SD
1 2 3 4 5 6 8 9 10 11 12
100 40 60 48 70 48 80 60 110 110 70
10.0 25.0 16.6 20.8 14.2 20.8 12.5 16.6 9.09 9.09 14.2
70 60 80 60 70 70 70 70 50 80 70
3 3 4 2 2 3 3 3 3 4 3
2.75 ⫾ 0.754
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This study demonstrates that aqueous extracts of old and tender leaves of Azadirachta indica have potent spermicidal effect. Its minimum effective concentration attains 100% immobilization and killing of sperm within 20 s.
4. Discussion
Fig. 1. Effect on percent motility of 1 million sperm after exposure to different doses of aqueous extract of tender neem leaf for 20 s.
mately 3 mg of extract is required to immobilize and kill 100% of 1 million sperm in 20 s. The viability of sperm was studied using Eosin Y and Nigrosin stains. It was found that crude extract kills 100% of 1 million sperm within 20 s. 3.3. Sperm morphology The morphological study of sperm was done using Eosin-Nigrosin stain and no morphological changes were found in the sperm head, mid-piece or tail when compared with untreated sperm. 3.4. Chemical nature Using Fehling’s test, the nature of the old and tender leaf extract was found to be carbohydrate in nature. 3.5. Stability It was also observed that the activity of both the old and tender leaf extracts remains the same, and the effectiveness does not change with storage time of 4 years.
Fig. 2. Effect on percent motility of 1 million sperm after exposure to different doses of aqueous extract of old neem leaf for 20 s.
In the present study, we have described a two-step procedure to prepare aqueous extract from the leaves of Azadirachta indica for checking spermicidal activity. Using neem leaf, two studies have been carried out. One is suggestive of reversible antifertility effect when fresh green leaf extract was orally fed to male mice for 30 days [6]. Another is suggestive of reversible antiandrogenic effect on the histological and biochemical parameters of testis of adult albino rats [7,8], when 100 mg of dried leaf powder, suspended in 1 mL of distilled water per day, was given for a period of 24 days. The results of the present study revealed that the aqueous extract of old and tender neem leaves is a potent spermicide, which is demonstrated through dose-dependent study on the effect of motility of spermatozoa and then confirmed by viability test. No morphological changes were found in the sperm head, mid-piece and tail when compared with untreated sperm. Therefore, 100% killing of sperm may be due to blockage of some biochemical pathway like energy utilization, which would require further investigation. The potency of lyophilized aqueous extract of old and tender leaves remains the same and the effectiveness does not change with storage time of 4 years. The proposed spermicide can be a potent vaginal contraceptive and can be formulated as cream or a pessary. Carbohydrates possessing a free or potentially free aldehyde or ketone group have the property of readily reducing the ions of certain metals such as copper, bismuth, mercury, iron and silver. The most widely used tests for sugar are based on this property. For example, when blue cupric hydroxide suspended in an alkaline medium is heated, it is converted into insoluble black cupric oxide. However, in the presence of reducing agents such as certain sugars, the cupric hydroxide is reduced to insoluble yellow or red cuprous oxide. Based on the above property, the chemical nature of old and tender leaf extracts was studied using Fehling’s test. The production of yellow or brownish-red cuprous oxide indicated that reduction has taken place. The difference in color of cuprous oxide precipitate under different conditions was apparently due to differences in the size of the particles, the more finely divided precipitates having yellow color while the coarser ones were red. This test was found to be positive, indicating that the old and tender leaf extracts are carbohydrate in nature. The neem oil, also known as oil of Margosa, is believed to have medicinal properties, such as antibacterial [12], antifungal [13] and antidiabetic [14].
B. Khillare, T.G. Shrivastav / Contraception 68 (2003) 225–229
Upadhyay et al. [15] observed a long-term contraceptive effect of single intra-vas administration of neem oil in male rats. The antifertility effect was observed for 8 months and was found to be an alternative approach to vasectomy. No inflammatory/obstructive changes in epididymis and vas deferens were observed. There was no change in blood testosterone levels. Purified neem seed extract called praneem was reported to cause termination of pregnancy by bleeding and a decrease in progesterone levels in rats, baboons and monkeys [16]. It was also reported to abrogate pregnancy in primates [17]. The volatile fraction of neem oil (NIM-76) inhibited spermatozoal activity in vitro at the concentration of 0.25 mg/mL and 25 mg/mL for rat and human spermatozoa, respectively [18]. The spermicidal activity of NIM-76 was investigated in vivo in rats, rabbits and rhesus monkey. It acts mainly by spermicidal effect [19]. A vaginal contraceptive has been developed from NIM-76 by Sharma et al. [20]. Two neem oil derivatives, viz., sodium nimbinate [21] and sodium nimbidinate [22], have been found to possess weak spermicidal action in vitro. In view of this, a study was undertaken by Sinha et al. in 1984 [23] on the role of neem oil as a spermicidal agent. Indigenously available neem oil in its natural form was tested for its spermicidal activity (in vitro and in vivo). Undiluted neem oil was found to possess strong spermicidal action (within 30 s) against rhesus monkey and human spermatozoa in vitro. Whereas 3 mg of neem leaf extract, when treated with human spermatozoa, kills 100% of sperm within 20 s. The time taken by sperm to travel to the oviduct from the cervix in women is 5– 68 min, when all conditions are favorable in the fertile period [24]. Therefore, neem leaf extract is more advantageous than the undiluted neem oil, which takes 30 s to kill the sperm. Praneem polyherbal cream has been developed by Garg et al. [25], which has synergistic spermicidal concentration for praneem (25%), reetha saponins (0.05%) and quinine hydrochloride (0.34%) and was found at this concentration to result in 100% immobilization of sperm within 20 s [26]. Until now, neem seed extract or oil or oil components were studied as an effective contraceptive that is hydrophobic in nature. The neem leaf extract, which is hydrophilic in nature, mixes immediately with water as well as body fluids and kills sperm within 20 s; with its use, a more potent vaginal contraceptive may be developed.
Acknowledgments We are thankful to Mr. R. K. Sharma for his technical help.
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