Neem Capsules for Psoriasis Natural Treatment - Neem Benefits
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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.