ELSEVIER

Mechanism of Action of NIM-76: A Novel Vaginal Contraceptive from Neem Oil S.K. Sharma, M. SaiRam, G. Ilavazhagan, Kumar Devendra, S.S. Shivaji,” and W. Selvamurthy

The present study was undertaken to elucidate the mecha- nism of spermicidal action of NIM-76, a fraction isolated from neem oil. The spermicidal activity of NIM-76 was confirmed using a fluorescent staining technique. NIM-76 was found to affect the motility of the sperm in a dose- dependent manner. Supplementation of pentoxifylline, which is known to enhance the motility of the sperm, could not prevent the spermicidal action of NIM-76. There was a gradual leakage of cytosolic LDH from the sperm in the presence of NIM-76. Electron microscopic studies re- vealed the formation of pores and vesicles over the sperm head, indicating the damage to the cell membrane. Mem- brane fluidization studies did not reveal any significant change in the fluidity of sperm cell membrane struc- ture. 0 1996 by S.K. Sharma et al. CONTRACEPTION 1996; 54:373-378

KEY WORDS: vagina, NIM-76, contraceptive, mechanism

Introduction

T he necessity to develop a safe and effective pre- coital spermicidal contraceptive to control pregnancy and population growth still exists.

Although many types of spermicidal contraceptives are available, they have side effects and are not easily accepted. As a result, there is a growing interest in the search for contraceptives of natural origin. In our laboratory, we have been working on the develop- ment of contraceptives from fractions of neem oil. Since neem is a native plant of India and is known to the population for its hygienic and medicinal value from time immemorial, any product developed from it is likely to find acceptability.

Defense Institute of Physiology and Allied Sciences, Lucknow Road, Timarpur, Delhi-l 10054, India; *Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad-500007, India

Submitted for publication April 12, 1996 Revised August 2, 1996 Accepted for publication August 9, 1996 Name and address for correspondence: Dr. S.K. Sharma, Defense Institute of

Physiology and Allied Sciences, Lucknow Road, Timarpur, Delhi-l 10054, India. Fax: 91-011-2932869

0 1996 by SK Sharma et al.

Neem oil extracted from the seeds of Azadirechta indica has been widely studied for its contraceptive potential. l3 However, it also possesses antiimplanta- tion and abortifacient effects when tested in ani- mals.4j5 In this regard, efforts have been made in our laboratory to isolate the active fraction that has only spermicidal activity. During this process, an active fraction, termed NIM-76, was isolated from the neem oil by the process of hydrodistillation. Analysis by gas chromatography-mass spectroscopy (GC-MS) re- vealed that NIM-76 consists of predominantly sulfur- containing compounds, and higher-chain fatty acids and their esters.6 This fraction kills all sperm in vitro in less than 20 set at a concentration of 5 mg and 25 mg/ml for rat and human sperm, respectively.6 In ad- dition, NIM-76 did not possess abortifacient or anti- implantation activity, thereby making it a highly de- sirable vaginal contraceptive.7 Two other fractions, DNM-5 and DNM-7, isolated from neem oil were found to have anti-implantation and abortifacient ef- fects, respectively, in rats when administered orally (unpublished data). In the present study, we report on the experiments conducted to elucidate the spermi- tidal action of NIM-76.

Materials and Methods All of the experiments were conducted on human sperm. Semen was collected from donors after 2-3 days of abstinence from sexual activity. Samples showing sperm count less than 20 million/ml and motility less than 50% were rejected. Semen was al- lowed to liquefy in a CO, incubator at 37 * 1°C for 30 min and the active sperm were concentrated by the swim-up method using Ham’s F-10 medium. NIM-76 was added to saline containing 1% ethanol and soni- cated until a perfect emulsion was formed.

Motility Pattern Motility pattern of human sperm was studied with the Hamilton Thorn Motility Analyzer. The sperm

ISSN OOIO-7824/96/$15.00 PII SOOlO-7824(96)00204-i

374 Sharma et al Contraception 1996;54:373-378

were incubated in media containing different concen- trations of NIM-76, i.e., 10, 25, 50, 100, 200, and 1000 pg/ml, and the motility patterns were recorded. The parameters included percentage of motile sperm; pro- gressively motile sperm; path velocity; sperm with rapid medium, and slow velocity; percentage of static sperm; mean linearity; mean straightness; mean lat- eral head displacement of sperm; mean beat fre- quency; mean length, width, and area of sperm head, etc.

Calcium Supplementation This study was conducted by adding l-2 mM calcium to human sperm suspended in the Ham’s F-10 me- dium containing 100 ug/ml NIM-76 in a microfuge tube and incubated at 37 2 1°C in a COZ incubator for 1 h. Various motility parameters were then recorded.

Pentoxifylline Supplementation This study was conducted by adding 1.5 mg/ml pen- toxifylline to the medium containing 100 ug/ml NIM-76 and human sperm. Incubations were carried out in a CO2 incubator at 37 + 1°C for 1 h and the motility patterns were recorded after every 30 min.

Enzyme Leakage Sperm were exposed to NIM-76 (100 ug/ml) in Ham’s F-10 medium for 60 min and, at different intervals, the sperm were pelleted by centrifugation. Lactate de- hydrogenase (LDH) activity was estimated in the su- pernatant using an enzymatic kit (Ranbaxy, India).

Sperm Viability Test Sperm were incubated with NIM-76 and, at specific times, aliquots of 50 pl of the sperm suspension were taken and mixed with 10 ul each of fluorescein diac- etate (FDA; 5 mg/ml) and ethidium bromide (2 mg/ ml]. A drop of the mixture was placed on a slide and observed under dark field using a Nikon fluorescent microscope. Viable sperm emitted green fluorescence as against red fluorescence for dead cells.

Cell Culture Studies NIM-76 at a final concentration of 100 pg/ml was added to a confluent layer of vero cells grown in DMEM in 24-well culture plates. After overnight in- cubation, the cells were washed with sterile DMEM medium. The cells were then stained either with 0.1% trypan blue or FDA (5 mg/ml) and observed un- der light field or UV field, respectively.

Membrane Fluidization Studies The study was carried out using purified plasma membrane as earlier described by Jagannadham et al.* Briefly, to 100 ul of 0.1 M HEPES buffer, pH 8.5, con- taining 200 ug of plasma membrane, 25 ul of different concentrations of NIM-76 was added, followed by the addition of 3 ml of 12 mM pyrene. For control, only NIM-76 was added to pyrene without plasma mem- brane. The degree of difference in ratio of fluorescence (470:372] of the control and the test was calculated to determine the extent of membrane fluidization.

Ultrastructural Studies Human sperm were exposed to NIM-76 at a concen- tration of 5 mg/ml for 20 set in a test tube and fixed with 3% gluteraldehyde overnight. The next day, sperm were washed with phosphate buffered saline (PBS) three times, followed by distilled water two times, and finally suspended in distilled water. A uni- form film of these sperm was made on a clean glass slide and air dried. Sputter coating was done by a Jeol sputter coater and finally, scanning was done on a Jeol Scanning Electron Microscope.

Results

Motility Patterns The effect of different concentrations of NIM-76 on percentage mean motility and progressive motility is shown in Figure 1 (a,b). Although NIM-76 is effective even at as low as 10 ug/ml concentration, the effect is quite slow and it took about 2 h to kill all the sperm. With increase in concentration, i.e., 50, 100, 200, and 1000 pg/ml, there is a linear decrease in percentage of motile as well as progressively motile sperm with time. In addition, it was found that with increase in concentration or with increase in time at a specific concentration, there was a decrease in percentage of rapid, medium, and slow moving sperm, mean track speed, progressive velocity, mean linearity, and later- al head displacement, and an increase in percentage of static sperm.

The effect of NIM-76 on mean progressive velocity (MPV) and mean head size (MHS) is shown in Figure 2. There was a rapid fall in MPV within the first 10 min of exposure to NIM-76 (100 ug/ml). Later, there was slow and steady decrease in MPV with time; whereas, there was a slow decrease in MHS with time in the presence of NIM-76.

Since NIM-76 kills human sperm within 20 set at 25 mg/ml concentration, it is difficult to determine the mode of action of NIM-76 during this time. Therefore, the studies were conducted at a lower con- centration to study its spermicidal action. Of all the

Contraception 1996;54:373-378

Mechanism of Action of NIM-76 375

1203 I^ 1 I

0 15 30 45 60

Time (minutes)

40 ug/ml -m- 50 ug/ml * 100 ug/ml ‘“200 ug/ml

Figure 1. Effect of different concentrations of NIM-76 on the motility and progressive motility of human spermato- zoa.

concentrations tested, it was found that at 100 pg/ml concentration, sperm become totally immotile in 1 h; hence, this concentration was selected for further ex- periments.

Calcium Supplementation This study was done to determine whether the NIM- 76 kills the sperm by depleting intracellular calcium. It was found that calcium supplementation even at the 2 mM concentration did not help in maintaining motility in the presence of NIM-76 (Figure 3 a,b).

Pentoxifylline Supplementation The effect of pentoxifylline on motility of sperm in the presence of NIM-76 is shown in Figure 4 (a,b). It is evident that the presence of pentoxifylline marginally delayed the spermicidal action of NIM-76 on the sperm but it did not result in the complete protection of the sperm.

+ MPV 50

0 0 5 10 15 20 25 30 35 40 45 50 55 68

Time (min)

Figure 2. Effect of NIM-76 (100 pg/ml) on mean progres- sive velocity (MPV) and head size (HS) of human spermato- zoa.

LDH Leakage Studies In control sperm, there was no significant leakage of LDH extracellularly. The presence of 1% ethanol re- sulted in about 40% increase over control at 1 h. However, in the presence of 100 ug/ml NIM-76, there was a progressive increase in extracellular LDH with time (Figure 5).

Cell Culture Studies The effect of NIM-76 ( 100 pg/ml) on vero (monkey kidney) cell line is shown in Figure 6. All of the cells remained viable even after exposure overnight as re- vealed by vital staining with trypan blue/fluorescein diacetate and neutral red uptake, indicating that NIM-76 did not damage the normal cells. Similar re- sults were obtained with L- 132 (human fetal lung) cell line and peritoneal macrophages (data not shown).

Membrane Fluidization Studies The fluorescence emission intensity of the eximer peak of pyrene at 470 nm has been used to monitor the membrane fluidity since the eximer formation is related to lateral mobility of pyrene molecules in the lipid bilayer. Our experiments using purified plasma membrane did not show any significant change in E/M ratio in the presence of NIM-76 (Figure 7).

Ultrastructural Studies Figure 8 (a-c) shows the effect of NIM-76 on human sperm at the ultrastructural level (scanning electron microscopy]. In the presence of NIM-76, there was

376 Sharma et al.

4 iC

120

100

:’ 3 60

-ij 60

& 40 a?

20

____-.

0 00 15 30 45 t

-rime <minutes)

- Control -2mMCa

- NIM-76 (1 OO( ug/ml) - NIM-76 + 2 mM Ca

Figure 3. Effect of calcium on the spermicidal action of NIM-76.

formation of pores and vesicles all over the sperm head (Figure 814, while in the PBS control (Figure 8a) and in the presence of 1% ethanol (Figure 8b), there was no such effect and the surface of the head re- mained smooth with intact membrane.

Discussion Neem oil has been reported to have anti-flagellate action and spermicidal activity.‘r” However, it also has anti-implantation and abortifacient effects. In or- der to circumvent this problem, we isolated a fraction called NIM-76 from neem oil that has high spermi- tidal activity without anti-implantation or abortifa- cient effects, which is highly desirable. Further, re- peated vaginal applications of NIM-76 in rabbits did not cause any significant irritation as evidenced by oedematous thickening of submucosal layer, infiltra- tion of leukocytes, vascular congestion, etc.6

The present observation that NIM-76, even at con-

Contraception 1996;54:373-378

4 0 15 30 45 60

Time (minutes)

-7!s---_------ --- \

60 60

40 40

20 20

-. 0 0

0 0 15 15 30 30 45 45 c c 3

- Control -a+ PP (1.5/ml)

- NIM-76 (100 ug/ml> -c PP + NIM-76

Figure 4. Effect of pentoxifylline on the spermicidal ac- tion of NIM-76.

centrations as low as 10 ug/ml, causes decrease in the percentage of motile sperm, percentage of progres- sively motile sperm, and mean track velocity, and an increase in percentage of static sperm indicates its strong spermicidal action. The spermicidal action of NIM-76 on sperm was also confirmed by fluorescent double-staining using fluorescein diacetate and ethid- ium bromide. Interestingly, our studies revealed that NIM-76 did not damage the normal cells.

Calcium is essential for motility of sperm because it activates the ATPase of contractile proteins such as spermosin and plactin and thus generates contraction and relaxation needed to propel the sperm,l’ besides its involvement in acrosome reaction. Since calcium supplementation did not relieve the sperm from the spermicidal action of NIM-76, it was evident that NIM-76 did not cause any depletion of intracellular calcium.

c-AMP is known to be a key element for sperm movement. l2 Inhibitors of phosphodiesterase (i.e., en- zymes capable of cleaving the nucleotide) can lead to

Contraception 1996;54:373-378

Mechanism of Action of NIM-76 377

0 15 30 45 60

Time (min)

Figure 5. Effect of NIM-76 (100 pg/ml) on the leakage of LDH extracellularly from human spermatozoa.

A

la

Figure 6. Effect of NIM-76 (100 pg/ml) on vero cell line. A: Control; B: NIM-76-treated.

q Test ..I ! W Control

40 50 100

Concentration of NIM-76 (ug/ml)

Figure 7. Effect of NIM-76 (100 pg/ml) on the fluidization of sperm plasma membrane.

increase in the concentration of c-AMP in the sperm. l3 Pentoxifylline is one such inhibitor which can be used as a motility stimulant. Although addi- tion of pentoxifylline marginally delayed the spermi- tidal action of NIM-76, no complete protection was seen, indicating that NIM-76 may not involve the role of C-AMP.

Since there was considerable leakage of cytoplas- mic LDH extracellularly in the presence of NIM-76, it indicates the possible damage of the plasma mem- brane of the sperm. As there is no change in the flu- idity of the membrane, it is speculated that the integ- rity of the membrane (protein-protein interaction, protein-lipid interaction, etc.) might have been al- tered, which is not only important for sperm metabo- lism but also for the fertilization process, i.e., capaci- tation, acrosome reaction, and successful union of male and female gametes. Studies with scanning elec- tron microscopy showed the formation of pores and vesicles on sperm head in the presence of NIM-76, indicating damage to the cell membrane. Similar ef- fects, i.e., vacuolation, vesiculation, and disruption of membrane, were also observed by Dhar et a1.14 after exposure of sperm to saponins isolated from Sapindus mukorossi. Wilborn et all5 also reported similar types of vesiculation and disruption of membrane af- ter incubating sperm with the spermicide nonoxynol- 9. The acrosome region of the sperm head is sur- rounded by five membranes. Formation of vesicles on any of these membranes leads to the detachment of all of the membranes and sperm become immotile and lose fertilizing capacity. These changes are due to stretching, loosening, and breakdown of membrane

378 Sharma et al. Contraception 1996;54:373-378

Figure 8. Effect of NIM-76 on the morphology of human spermatozoa (scanning electron micrograph). A: Control; B: 1% ethanol (vehicle); C: NIM-76 (5 mg/ml).

and even ultimate removal of the acrosome, as ob- served by Zaneveld and Gould. l6 The shrinkage in the size of the head (Figure 2) confirms the possible re- moval of the acrosome region and leakage of cytoplas- mic contents results in a reduction in the size of the

head. These results indicated that NIM-76 damaged the sperm cell membrane structure resulting in the death of sperm.

It is concluded that NIM-76 selectively kills the sperm without affecting the normal cells, which makes it a highly desirable vaginal contraceptive agent for the future.

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