1JISC'l1SSIOg.{

5.0 DISCUSSION

Neem seed extracts have been extensively studied in the last decade for their

antifertility activity. Although, neem oil have been reported to have spermicidal

(Riar et ai., 1991) and anti-implantation (Sinha ef ai., 1984) effects, more recent

investigations have shown that the antifertility activity after intrauterine

administration in rats (Upadhyay ef al., 1990) and in bonnet monkeys (Upadhyay et

al., 1994) is mediated through activation of cellular immunity in the female

reproductive tract.

Present study describes the identification and chracterization of the active fraction

from neem (AzadiracJua indica) seeds, responsible for long term and reversible

blocking of fertility after a single intrauterine administration in adult female Wistar

rats. Since the study deals with the purification and characterization of the active

fraction responsible for immunocontraceptive activity, it is distinct from the

previous studies (Upadhyay ef aI, 1990, 1994 a), where total neem oil was reported

to have this activity. The active fraction has been identified to be a mixture of six

components, which comprises of saturated, mono and di-unsaturated free fatty acids

and their methyl esters (Figure 40). A single administration of 100 J.LI of this

fraction per uterine horn was found to be sufficient to block fertility for a minimum

period of 95 days or more in rodents. The effect is seen upto 5 % concentration of

the fraction in peanut oil. At lower concentration (l %) of the fraction, the

antifertility activity was observed in less number of animals.

In order to identify and characterize the active principle/s from the crude plant

extracts showing biolgical activity of interest, there are two approaches (Chaudhary,

1980) which could be followed. One approach is to isolate major pure compounds

from plants and test them for biological activity observed with crude plant extract.

Hexane fraotion 1+' NS-1001H ,

Neem aeeda I

Solvllnl

Ethanol fraotion 1-' I NS-1001E ,

Solvent plUflt/on/ng wllh aq. m8thllllOl

Residue 1-' I NP-200/R

I NC-300/1 (-)

Aq. methanol extraot (+' I NP-200 ,

I Flash column chromatography

NC-300/2 1+' NC-300/3 (-)

FI.sh 001. clYom.t I I I Prllp. HPLC I t

I I I NF-4001H NF";400fM Ni-400/1

(-) (-) (-)

PrIlPIU.tlvtl HPLC

I NC-400f1 NC-400f2 NC-400/3

QC nc I I

NG-500/1 NG-500/2 NT-S'OO/1

(+' Aotlve (-) Inaotlve

Water fraotion 1-' I NS-100/W ,

I NC-300/4 (-)

Ni-400/2 (-)

I NC-400f4

NT-500f2

Figure 40: Flow diagram for isolation of active fraction ane! its constituents from neem seed extract.

Discussion

Discussion 59

For example, nimbidin isolated from neem oil was tested and found to have

anti-arthritic and anti-inflammatory activities (Pillai and Santhakumari, 1981) which

are observed with present in total crude oil (Okpanyi and Ezekuwu, 1981). The

other approach is to go for further fractions, identify in which fraction the

biological activity resides and enhance activity by further isolation of the active

principle. In case of intrauterine antifertility activity, pure neem compounds such as

nimbin, nimbidin and azadirachtin were found to be inactive, an activity guided

fractionation, starting from neem seeds was carried out.

Present study for the first time, reports the identification, isolation and

characterization of the active fraction for antifertility activity observed after

intrauterine administration. For this purpose, an activity guided fractionation of

neem seed oil/extract was carried out. the biological assay on each fraction took

minimum 45 days, therefore simultaneous fractionation by various methods was done.

Initially, studies were carried out with mechanically expressed oil from neem seeds.

The oil was fractionated by silica gel column chromatography, solvent partitioning,

saponification and neutralization. Although none of the fractions obtained by these

separation techniques was completely active as an antifertility agent, but it is clear

that this activity was present in constituents of low to intermediate polarity. A

number of variables are involved in the process of mechanical expression of seeds,

such as pressure applied, heat generated due to friction in the rollers etc., which are

diificult to control in a reproducible manner. As a result, the quantitative and

qualitative composition might differ from batch to batch. Consequently, the

biological activity observed with these batches and their fractions is also inconsistent.

Solvent extraction, directly from neem seeds was seleted as an alternative to

Discussion 60

mechanical expression. Neem seed contaiils constituents with a wide range of

solubility, including non-polar or weakly polars fats and glycerides, terpenoids with

intermediate polarity and highly polar polysaccharides. Since the presence of a high

proportion of fats or glycerides reduces penetration of plant materials by aqueous

or alcoholic solvents due to insolubility of the lipids in solvent of higher polarity,

defatting IS prerequisite for subsequent extraction. It is a well known fact that

optimum conditions for extraction of lipids differ from sample to sample due to the

differences in their chemical composition. As the use of elevated temperatures may

cause detrimental effects on some of the lipid components of the mixture, it is best

to employ extraction at room temperature (25-26°C). Morever, elevated

temperature can induce activation of certain lip~lytic enzymes (Iipases, upto 45°C),

which in turn can affect the chemical composition. The most reasonable approach

therefore, is repeated extractions at room temperature and storing the concentrated

extract at -25 to -30DC. The use of temperature below 20DC is not advisable owing

to the limited solubility of the lipids. Based on these criteria, neem seeds were

extracted with hexane, ethanol, and water in a sequential manner (Figure 1). Out

of the three broad fractions so obtained, hexane extract was found to be biologically

active (Figure 40). This observation was in conformity with the experiments on

subfractions of mechanically expressed neem oil, where the non-polar portion was

found to have significant biological activity. Hexane fraction was found to consist of

approximately 97.5 % of lipids in the form of glycerides of palmitic acid, oleic acid,

linoleic acid, stearic acid and myristic acid. Also, some weakly polar terpenoids such

as azdirone, azadiradione and gedunin have been reported from the hexane extract

of neem seeds.

Perhaps one of the major difficulties associated with any study of the chemistry of

simple and complex lipids has been that of the isolation and separation into

Discussion 61

individual components. Concomittently there has been the question of the proper

criteria for defination of the purity of the isolated lipid fractions. Often the

"homogeniety" or uniformity of a lipid fraction is defined on the basis of the

non-fatty skeleton, but there is no doubt that the fatty acid composition significantly

affects the physical and chemical properties of the fraction. Hence as used in

many instances, the term "pure" lipid is a peculiar one (Gurd, 1960). The closely

related solubility properties, the interassociative effects and the presence of

contaminants have all contributed to the problems of purification of lipids.

Several possible routes of fractionation have been explored e.g. complex formation,

solvent partitioning and chromatography. While none of the procedures can be

labelled as all-encompassing, it is reasonably certain that certain chromatographic

techniques play an important role in contributing to unders~nding of the lipid

structure and function.

In the second stage of fractionation, aqueous-methanol (1: 19) extract (NP-200) of

the hexane fraction was found to be biologically active (Figure 40). This observation

was supported by presence of activity in the last fraction obtained by column

chromatography of the hexane fraction and similarity in their TLC chromatogram.

The activity in aqueous-methanolic extract was further confirmed by experiments on

the two fractions obtained by its partitioning with petroleum ether (60-80° fraction).

Both the fractions were found to be biologically active indicating that the active

principle(s) was distributed between two phases.

For better resolution, the fraction NP-200 was resolved into four fractions by flash

column chromatography on silica gel. Different batches of the second fraction

obtained by this method were found to be biologically active. Flash chromatography

Discussion 62

IS a preparative air pressure/vaccum driven (Still el ai, 1978) technique with

moderate resolution. It is relatively faster method for the purification of the samples

with minimal sample loss and less risk of decomposition so often encountered with

conventional open column chromatography. The concept is exceptionally simple and

preparative separations are easy to perform using readily available and cheap

laboratory glassware. Flash chromatography, although occasionally used as a final

purification step in the separation of natural products, is most often employed for

the rapid preliminary fractionation of complex mixtures. It is therefore an important

step in isolation strategies involving combination of chromatographic methods.

In order to resolve the active fraction from the previous stage (NC-300/2) into polar

and non-polar compounds, it was further fractionated by flash column

chromatography using hexane and methanol as the mobile phases into two fractions.

Both the fraction were inactive. Similar resolution was achieved by preparative

HPLC using RP, C-18 column and methanol as the solvent system. In biological

activity assay, one of the two fractions was inactive and the other one was active in

40.0% of the animals only. These observations point to the fact that the initially

active fraction lost its biological activity by further separation. It was concluded at

this stage that NC-300/2 was the last active fraction in the antifertility activity

guided fractionation and it could not be resolved further into biolgicaIly active

compounds.

Dose response study was performed with the last active fraction (NC-300/2). It was

active in 100% of the animals till 5% concentration and lost its activity at 1 %

concentration. In the experiments with different batches of the active fraction, it

was observed that in some cases, the fraction was active in majority of the animals

but not in all the animals tested. The observed behaviour is explained by the fact

Discussion 63

that intrauterine antifertility activity of neem extracts is mediated through

activation of local cell mediated immunity (Upadhyay ef ai, 1990; 1994a). Humoral

and cell mediated immune responses generated against an appropriate stimulus

differs from individual to individual. For example, the antibody levels after one year,

with a formulation of human chorionic gonadotropin (o:-ovine-B-hCG-TII Cholera

toxin chain B) and its boosters (Singh er ai, 1989) at the dose of 100 ug, were below

20 ng in 62 out of 88 cases studied. Since the biological activity was studied on an

outbred population of wi star rats, variation in individual responses of the animals

are expected.

Once a plant extract has shown interesting antifertility activity III preliminary

screening, it is generally thought that further fractionation will result in the activity

becoming more and more in one of the fractions. However, plants do not always

behave in this manner and can be divided into two types on the basis of

observations on the biological activities with the sub-fractions. Some plants will

demonstrate enhanced activity when further fractionation is carried out, while in

others, further fractionation would result in loss of biological activity. Garg ef al

(1978) have reviewed the results obtained after testing 210 extracts of di fferent parts

of 36 plants reported to produce contraceptive effect. In about 50% of the plants

tested, the activity increased in the fractions. In case of of Daucus carow seeds,

Po[ygonul1l hydropiper roots and Sap indus rr({o/iala seeds further extraction and

fractionation enhanced the anti-implantation activity. On the other hand BUiea

monOSpel171a behaved quite differently; while the alcoholic extract of the seeds

inhibited implantation in all the animals, its sub-fractions did not demonstrate activity

comparable to the total alcoholic fraction. The activity was either decreased or lost.

In another exeperiment, two fractions obtained by flash column chromatography

Discussion 64

(NF-400/H and NF-400/M) and HPLC (NH-4001l and NH-400/2) of the active

fraction were mixed together (NR-500) in the proportions, similar to their yield and

tested. The mixture regained activity in 50% of the animals. It is interesting but

perhaps not surprising that even if the different fractions are mixed together and

administered, the original activity does not reappear (Garg ef aI, 1978). This may

be because of synergism in the biological activity of the constituents, as they exist in

nature. The process of separation or puri fication can change the proportions of these

compounds in a qualitative or quantitative manner. As a result, even after

reconstitution, biological activity does not reappear to the same extent.

The antifertility activity with the active fraction was reversible in nature, since 8 out

of 12 animals delivered normal ups in period ranging from 95 days to 171 days.

The absence of any systemic toxic effect following the administration of the active

fraction is put in evidence by the effect seen following unilateral administration of

the fraction. Block in pregnancy was seen only in the active fraction treated horn,

while normal embryos were present on the contralateral horn. The administration

of the fraction did not have toxic effect on the embryo development on the

contralateral uterine horn. The unilaterally treated animals, as well as some of those

on long term fertility studies delivered normal pups ruling out the possibility of any

long lasting teratogenic effects.

In order to completely characterize the active fraction, it was (NC-300/2) resolved

into individual "pure" compounds by HPLC. HPLC finds application in the analytical

and preparative scale separation of samples which range from microgram to gram

quantities. On the whole, however, HPLC is commonly applied as a last step in

purification process and in this respect, the quantities involved tend to be at the

lower end of scale. The reason, why pre-purified samples and not crude

Discussion 65

chromatographed by HPlC IS that the sorbents are of small particle size and

consequently very expansive.

HPlC has been employed for isolation of Azadirachtin H and I (Govindachari el

al., 1991) and for quantitative determination of Azadirachtins in insecticidal

formulations (Hull ef al., 1993). The use of high performance liquid chromatography

(HPlC) in the area of lipid analysis is still relatively uncommon and TlC or GC

are the most commonly used techniques but it was used in the present study for

collecting the samples in sufficient quantity for characterization, biological testing

and for the high degree of resolution required.

For present study, the reversed phase columns with bonded type stationary phase

(C-18 and C-8) were used. The majority of lipid separations carried out using

reversed phase mode are for the separation of molecular species of lipids within a

single lipid class, where the separation is dependent on the fatty acyl or alkyl chain

length. The actual choice of the mobile phase is determined by the nature of

sample and the column used. Since the C-8, RP column is known to give best

separation with acetonitrile:water solvent system, this combination was used in a

ratio of 67:33. For both analytical and preparative scale HPlC, a number of solvent

systems were tried to optimise the separation and the best combinations were

selected. The simultaneous separation and quantitation of lipids by HPlC has

always eluded the chromatographer because of limitations in using UV and RI

detectors. The absorbance due to double bonds contained in the fatty acid chains,

in the 200-215 nm region, is dependent on the concentration and degree of

unsaturation of the molecules, and direct quantitation of a complex mixture is not

possible with UV detection. The highest sensitivity of UV detector, which is most

commonly used detector for HPlC, is towards compounds with conjugated double

Discussion 66

bonds and aromatic ring systems, I;either of which are common in naturally occuring

lipids. Chemical derivatization to form compounds which do absorb in the UV

region is a way of extending the use of this detector to other lipids (Sewell, 1992).

This approach was not feasible in this study, since the individual compounds from

the active fraction were required in the pure form for the purpose of

characterization. Ideally, the detector should be able to respond to all molecules

with equal sensitivity and should not be affected by changes in mobile phase

composition as in gradient elution. The total fraction NC-300/2, when scanned for

UV absorption, was found to lack any sharp, well defined maxima. As a result RI

detector was used in the present study. The RI detector is universal, i.e. it will

respond to any molecule that has a different RI to that of the mobile phase. The

response of detector is proportional to this difference so that, its sensitivity may be

enhanced by changing the mobile phase. The RI detector is very sensitive to

uncompensated changes in ambient temperature and changes in mobile phase

composition and is not really practical to use with gradient elution. Thus, in a true

sense, use of RI detector in isocratic mode changes the reversed phase

chromatography to normal phase partitioning.

By virtue of their carboxyl groupings, all fatty acids show absorption in the far UV

region. "Saturated acids" e.g. palmitic acid have a broad but weak absorption band

(Pitt and Mortan, 1957) with a maxima near 215 nm ; stronger absorptions begin

at 185 nm, rising steeply on the short wavelength side as far as the measurement

extended. The absorption of saturated fatty acids at wavelengths greater than 220

nm can usually be neglected.

For the fraction NT-5001l and NC-400/4, broad absorption with maxIma over a

range of 205-225 nm and 200-230 nm respectively are observed. Monoenoic acids,

Discussion 67

such as oleic acid containing a carboxyl group and an ethylenic linkage distant

therefrom, have no well defined maxima but show intense absorption as in case of

NT-50012. Polyunsaturated acids with isolated double bonds (separated by one

another by at least one methylene group), exhibit selective weak absorption in the

more accessible region of the spectrum, but end absorptions i.e. the skirt of the

absorption band which goes well below 200 nm. Although the significance of

absorption around 210 nm is not quite clear, it can be said as a generalisation that

the presence of polyunsaturated acids in fats greatly increases the intensity of

absorption around and just below 220 nm without producing a maximum. In brief,

above 220 nm, saturated fatty acids are. practically transparent and unsaturated

acids increase the unselective end absorption (Pitt and Morton, 1957).

In high resolution NMR spectrum, the actual value of chemical shift in long chain

fatty compounds depend somewhat on the solvent, concentration, temperature and

configuration or conformation of the compound. Since protons in different

environments have different chemical shifts, the NMR spectra of a given fatty acid

will have peaks at different positions corresponding to the degree of shielding of

each proton or group of protons (Hopkins, 1958). In the simplest case, i.e. a straight

chain saturated acid, there are essentially four peaks representing terminal methyl

protons centered at 0.9 ppm, the a-methylene protons (adjacent to carboxyl) centered

at 2.3 ppm, the remaining (isolated) methylene protons of the chain centered at 1.3

ppm and the acid (carboxyl) protons at very low field region. The position of the

acid proton peak is affected by the degree of dilution in the solvent, as a result of

hydrogen bonding. A fatty acid methyl ester as in the case of NG-500/1 and

NG-500/2 gives a single sharp peak at 3.7 ppm representing the -COOCH3 protons.

In olefinic acids or esters, the protons attached to the double bond carbons gives a

Discussion 68

distinctive band. The position of the double bond in monoenoic fatty acid has

little effect on the olefinic proton signal, unless the double bond linkage begins at

the first, second, or third carbon from either end of the chain. When the double

bond is near the center of the chain, this band appears at S.3S ppm (NG-SOO/2 and

NT-SOOI2. The methylene groups in the 0: position to the double bond carbons give

peak at about 2.0 ppm. The methylene group in the B position to a double bond

carbon atoms give a signal at slightly lower field than the totally isolated methylene

groups of the chain. This results in a slight splitting of the large methylene peak in

the spectrum, observable under conditions of high resolution. The NMR spectra of

dienoic acids are similar to those of monoenoic acids only when the two double

bonds are isolated from each other i.e. if there are two or more methylene groups

between the olefin groups. In NC-400/2, the olefinic groups are separated by one

methylene group and the grouping is referred to as "methylene-interrupted". The

protons of the double bond carbons have the usual chemical shift but the diallylic

methylene protons appear at 2.8 ppm as a characteristic triplet.

Elctron ionisation (EI) is the most widely used technique in the structure elucidation

of lipids by mass spectroscopy. In the EI spectrum of fatty acid methyl ester of

saturated fatty acid (NT-5001l), base peak at mlz 74 arises through the six centre

McLaffarty rearrangement, which results in proton transfer and cleavage of bond

linking the C-2 and C-3 carbons. The relative abundance of the m/z 74 ion

decreases with increasing unsaturation (NT-SOO/2) of alkyl chain as a result of

competitive fragmentations. The mass spectra of unsaturated fatty acid methyl ester

differ from their saturated counterparts by exhibiting an M-32 (loss of methanol) ion

rather than M-3l (loss of methoxy radical). Other high mass ions, occuring at M-31

and M-43 in the spectrum of saturated fatty acid methyl ester (at 227 and 239 in

NG-SOO/l), corresponds to the loss of methoxy and propyl groups respectively.

Discussion 69

The molecular ion peak of a straight chain monocarboxylic acid is weak, but usually

discernible. In long chain acids (NC-400/2, NT-500/l, NT-500/2 and NC-400/4) the

spectrum consists of two series of peaks resulting from cleavage at each C-C bond

with retention of charge either on oxygen containing fragment (m/e 45,59,73, ... ):>r

on the alkyl fragment (m/e 29,43,57,71,85, .... ).Besides the McLafferty

rearrangement peaks, the spectrum of a long chain fatty acid resembles the series

of "hydrocarbon" clusters at an interval of 14 mass units. In each cluster, however

is a prominent peak at CnH2n_102. In case of polyunsaturated fatty acids, it is not

possible to deduce the position of unsaturation, owing to the migration of the

double bonds during the ionisation process. Derivatisation of the double bonds in

case of NC-400/2 and NT-500/2 was performed for this purpose. Nature of double

bonds in NT-500/2, NC-400/2 and NG-500/2 was determined by their coupling

constants (1) In NMR spectra. The double bonds are of cis type in all the three

compounds.

The compound NC-400/1 was found to be a mixture of methyl ester of hexadecanoic

acid (NG-500/I, Methyl palmitate) and cis-9-octacecenoic acid (NG-500/2, Methyl

oleate). The structure of the compound NC-400/2 is proposed to be cis-9,cis-I2-

octadecadienoic acid (Linoleic acid). The NC-400/3 was also a mixture of

hexadecanoic acid (NT-500/l, Palmitic acid) and cis-9-octadecenoic acid (NT-500/2,

Oleic acid). The data on compound NC-400/4 was suggestive of octadecanoic acid

(Stearic acid). Major limitation with HPLC on reversed phase is that it can not

resolve critical pairs such as oleic acid and palmitic acid, which differ by a small

difference in chain length and unsaturation. This explains the elution of two pairs,

(NT-500/l, NT-500/2 and NG-500/ I, NG-500/2) as single peaks.

Thus the active fraction obtained by antifertility activity guided fractionation of

Discussion 70

neem seed extract is a mixture of saturated, monounsaturated and polyunsaturated

fatty acids and their methyl esters. However, in view of very complicated nature of

lipid fraction in hand, the possibility may not be ruled out that other minor

compounds which might have eluded final purification steps, may also contribute

directly to the observed biological activty or act in a synergistic manner to enhance

the degree of activity of reported compounds. It was not possible to identify a single

compound as the active principle. This observation is similar in nature to the earlier

reports with neem extracts. The bioactivity guided fractionation, isolation and

identification of active compounds have been reported in case of neem bark

constituents, which have inhibitory effects on both the pathways of compliment

activation as well as production of oxygen radicals by activated polymorphonuclear

leukocytes (Van Der Nat ef ai, 1991; 1989). In this case, the active

immunomodulatory fraction have been identified as a mixture of gallic acid,

gallectocatechin, catechin and epicatechin alongwith some polysaccharides .. The only

report about active fraction for antifertility activity with neem extracts is by Riar et

af (1991) wherein the volatile fraction (NIM-76) obtained by steam distillation of

neem oil has been shown to have spermicidal activity. The fraction NIM-76 was

found to be a mixture of 25 components by GC-MS.

Intrestingly, alkaline hydrolysate of peanut oil was also found to have biological

activity in 57.1 % of the animals. In the present study, refined peanut oil was used

as a control in all the experiments. Peanut oil consists of glycerides of palmitic,

stearic, arachidic, lignoceric, oleic and linoleic acid (Merck Index, 1989) and the

process of refining is used to remove the free fatty acids (The Wealth of India,

1985). Alkaline hydrolysis of refined peanut oil causes the breakdown of glycerides,

thus liberating the free fatty acids. Although quantitative composition of the active

fraction identified from neelll and alkaline hydrolysis of peanut oil is not identical,

Discussion 71

but the marked similarity in their qualitative composition explains the results

obtained with neem seed extract.

There have been several reports of immunomodulatory effects of various saturated,

monounsaturated and polyunsaturated fatty acids such as linoleic acid and stearic

acid (Tebby and Buttka, 1990; Calder et aI, 1989). Studies on the

immunomodulatory properties of neem oil (Upadhyay et ai,1992) following

intraperitoneal administration in mice have shown enhanced MHC-II expression and

phagocytic activity of peritoneal macrophages. Neem oil treatment also induced the

production of gamma interferon. Spleen cells of neem oil treated animals showed

a significantly higher lymphocyte proliferative response to in-vitro challenge with

Con-A or tetanus toxoid than that of the controls. Pre treatment with neem oil,

however did not augment the anti-1T antibody response. The study indicates that

neem oil acts as a non-specific immunostimulant and that it activates selectively

the cellular immune mechanism to elicit an enhanced response to subsequent

mitogenic or antigenic challenge. Although the exact mechanism of action with the

compounds of the active fraction identified is yet to be established, the presence

of immunomodulatory activities in the fatty acids explains observation of cell

mediated antifertility activity observed with the active fraction obtained from neem

seeds.