Chapter II
Phytochemistry of Liv-Pro-08
2.1. Introduction
Medicinal and aromatic plants are a gift of nature is being used against
various infections and diseases in the world since past history. Natural products have
been proven to be the richest source of medicinal compounds. Although many drugs
are made by synthetic chemistry, most of the core structures or scaffolds for
synthetic chemicals are based upon natural products (Drews, 2000). As, historically,
plants and their products form the basis of medicines and also in present days.
Several compounds, which are pharmaceutically and medicinally important, derived
from plant sources. However, the medicinal value of plant depends on the nature of
plant constituents present in it, which is known as active principal or active
constituents. Active constituents are those chemical substances, which are solely
responsible for therapeutic activity of plant. Plants produce primary and secondary
metabolites, which encompass a wide array of functions (Croteau et al., 2000).
Primary plant metabolites are simple molecules or polymers of simple
molecules synthesized by plants, generally do not possess therapeutic as such but
essential for the life of plants and contains high-energy bonds. It includes amino
acids, simple sugars, nucleic acids and lipids are compounds that are necessary for
cellular processes (Keeling et al., 2006). Plants can manufacture many different
types of secondary metabolites, which have been subsequently exploited by humans
for their beneficial role in a disease array of application (Balandrin et al., 1985). The
secondary metabolites are involved in basic metabolic processes are not directly
essential for photosynthetic or respiratory metabolism in plants. Now, many plant
secondary metabolites have been identified to have health promoting and beneficial
effects on human health and new roles of such compounds on human health are
being found. Plants and plant products are subject to wide variation in their
phytochemical profile due to their variety climate conditions, maturity, post harvest
processing, storage, stability etc... It is extremely important to standardize the
ingredients and formulations based on marker compounds specific to each plant
followed by validation for their efficacy
Phytochemicals are naturally occurring, biologically active chemical
compounds in plants. Phytochemicals are protective and disease-preventing
43
particularly for some forms of infectious diseases such as HIV and degenerative
diseases like anti-inflammatory diseases etc. The most important action of these
chemicals with respect to human beings is somewhat similar in day today function
as antioxidants. Plants are natural reservoir of medicinal agents almost free from
side effects caused by synthetic chemicals (Rizvi et al., 2009). Despite the
remarkable progress in synthesis organic chemistry over 25% of prescribed medium
in industrialized countries derived directly or indirectly from plants (Newman et al.,
2000). Modern medicines have little to offer for alleviation of hepatic diseases and
it‟s chiefly the plant based preparations which are employed for the treatment of
liver disease (Karan et al., 1999). Many traditional plants are used for the treatment
of liver problems. So, there is the worldwide trend go back to traditional medicinal
plant (Mithra et al., 2000 and Sun et al., 2007).
Phytochemistry is rapidly expanding area with the new technique for the
analysis of organic plant components (Harbrone, 1973). In order to discover new
bioactive compound extracts of simultaneously evaluated by chemical screening
(Wink, 1999). Modern methods of extractions are based on chemical polarity and
solubility because an aqueous extract/organic extract might be prepared and further
analysis are required for cleanup and purifications followed by bio-separation of
components within the fraction by chromatography methods but final analysis of
Mass Spectroscopy or NMR of the selected sample is necessary in order to
unambiqusly identified the compound of interest (Kaufmann, 1999).
Phenolics are compounds possessing one or more aromatic rings with one or
more hydroxyl groups. They are broadly distributed in the plant kingdom and are the
most abundant secondary metabolites of plants. Plant phenolics are generally
involved in defense against ultraviolet radiation or aggression by pathogens,
parasites, and predators as well as contributing to plant color. Phenolics are
widespread constituents of plant food (fruits, vegetables, cereals, olive and legumes
& chocolate etc) and beverages, (tea, coffee, beer & wine etc) and partially
responsible for the overall organoleptic properties of plant foods. Natural phenolics
are of interest from many view points (antioxidants, astringency, bitterness,
browning reactions, color etc). Selection of the proper analytical strategy for
44
studying phenolics in plant material depends on the purpose of the study as well as
the nature of the sample and the analyse.
Plant phenolics include phenolic acids, flavonoids, etc... flavonoids are the
most abundant polyphenols in our diet. Flavonoids comprise a large group of plant
secondary metabolite characterized by a diphenyl propane structure (C6-C3-C6).
They are widely distributed throughout the plant kingdom and are commonly found
in fruits, vegetables and certain beverages. Numerous preclinical and some clinical
studies suggest that flavonoids have potential for the prevention and treatment of
several diseases (Maron, 2004; Neuhouser, 2004).
In recent times focus on plant research has increased all over world and large
body of evidence has been collected to show the immense potential of medicinal plants
used in traditional systems. Various plants have been screened and studied using
modern scientific approaches the results revealed the potentials of medicinal plants in
the field of pharmacology. The herbal drugs are believed to be harmless and free from
serious adverse reactions, as they are obtained from nature and are easily available.
Also, the limited therapeutic options and disappointing therapeutic success of modern
medicine has increased the usage of alternative medicine including herbal preparations
(Stickel and Schuppan, 2007). The use of herbal resources in Ayurveda, Siddha and
Chinese medicine for the treatment of liver diseases, is experiencing in a great demand
in recent years even though is a quite an old approach (Eisenberg et al., 1993).
45
2.2. Materials and Methods
To
perform
the
objectives,
various
experimental
protocols
were
deduced for the present study entitled, “Phytochemical and Biochemical
characterization of Liv-Pro-08” were conducted by the following standard
procedures.
1. To Evaluate Qualitative and Quantitative determination of Phytochemical and
Biochemical constituents of Liv-Pro-08
2. Screening and Characterization of Liv-Pro-08 by HPTLC and GC-MS
2.2.1. Preparation of plant extract (Liv-Pro-08)
The seeds of Nigella sativa, Entada pursaetha and fruits of Ficus glomerata
were collected from Kolli Hills in Namakkal District, Tamilnadu, India. The
samples were shade dried at room temperature and then ground to a fine powder in a
mechanic grinder. The powdered material was then extracted using various solvent
extraction (petroleum ether, chloroform, methanol, ethanol and aqueous) in the ratio
1:10 using Soxhlet apparatus. After extracting all colouring material of the solvent
was removed by evaporating on water bath which give rise to a solid mass of the
extract.
2.2.2. Qualitative analysis
Plants are found to be the sources of many chemical compounds, most of
which account for their various uses by man. The medicinal values of the plants rely
in the presence of certain chemical substances that produce a definite physiological
effect on the human body. Phytochemical screening was done according to the
published standard method (Peach and Tracey, 1955) as given in appendix 1.
2.2.3. Quantitative analysis
The ethanolic extract of Liv-pro-08 was analysed for some of the
biochemical constituents by standard procedures.
Estimation of alkaloids: The alkaloid content of Liv-Pro-08 was determined by the
procedure as explained in appendix 2
46
Estimation of steroids: Zak‟s method was used to measure the steroid content and
procedure was given in appendix 3
Estimation of flavanoids: The flavanoid content of Liv-Pro-08 was determined by
the procedure as explained in appendix 4 (Cameron et al., 1943)
Estimation of phenols: Total phenol content was estimated as depicted in appendix
5 (Sadasivam and Manickam, 1992)
Estimation of carbohydrate: Total carbohydrate was estimated using Hedge and
Hofreiter method as explained in appendix 6
Estimation of starch: Starch content of Liv-Pro-08 was determined by the
procedure as explained in appendix 7
Estimation of protein: Total protein content was estimated as depicted in appendix
8 (Lowry et al., 1951)
Estimation of cholesterol: Total cholesterol of Liv-Pro-08 was determined using
Zak method in appendix 9
Estimation of triglycerides: Triglycerides was quantified as depicted in appendix 10
Estimation of free fatty acids: Estimation of free fatty acids of Liv-Pro-08 was
estimated as described in appendix 11
2.2.4. Screening and Characterization of Liv-Pro-08 by HPTLC and GC-MS
Modern methods of extraction are based on chemical polarity and solubility.
Thus an aqueous extract or organic extract might be prepared and further analysis is
required for purification followed by bio separation of compounds within a fraction
by Chromatographic methods. But final analysis by Mass Spectroscopy or High
Performance Thin Layer Liquid Chromatography of the selected sample is necessary
in order to unambiguously identify the compound of interest.
HPTLC analysis of Liv-Pro-08, Nigella sativa, Entada pursaetha & Ficus
glomerata- Flavonoid, Phenol & Saponin profile.
47
Sample preparation
The extract 50 mg was dissolved in ethanol and made up to 1 ml. This
solution was centrifuged and collected the supernatant liquid. This solution was used
as test solution for HPTLC analysis.
Sample and Reference standard application
5 µl of test solution and reference standard were loaded as 8mm band length
in the 5 x 10 Silica gel 60F254 TLC plate using Hamilton syringe and CAMAG
LINOMAT 5 instrument.
Spot development
The samples loaded plate was kept in TLC twin trough developing chamber
(after saturated with solvent vapour) with respective mobile phase (Flavonoid,
Phenol & Saponin) and the plate was developed in the respective mobile phase up to
90mm.
Photo-documentation
The developed plate was dried by hot air to evaporate solvents from the
plate. The plate was kept in photo-documentation chamber (CAMAG REPROSTAR
3) and captured the images at white light, UV 254 nm and UV 336nm.
Derivatizatation
The developed plate was sprayed with respective spray reagent (Flavonoid,
Phenol & Saponin) and dried at 120°C in Hot air oven. The plate was photodocumented in UV 366 nm mode using photo-documentation (CAMAG
REPROSTAR 3) chamber.
Scanning
Finally, the plate was fixed in scanner stage and scanning was done at
366nm. The peak table, Peak display and Peak densitogram were noted.
48
Analysis details
Mobile phase
Toluene-Chloroform-Acetone (4:2.5:3.5)
Spray reagent
1% ethanolic Aluminium chloride reagent and dried at 120°C for 10 mln.
2.2.5. Gas Chromatography-Mass Spectroscopy
Mass Spectroscopy is frequently used in conjunction with GLC and the
combined operation provides to go for a qualitative and quantitative identification of
many structurally complex components which may present together in a particular
plant extract.
GC-MS plays a key role in the analysis of unknown components of plant
origin. GC-Ms ionizes compounds and measures their mass numbers. Ionization
method includes EI (Electron Ionization) and CI (Chemical Ionization). Typically,
the CI method is used. The EI method produces ions by colliding thermal electrons
emitted from a filament with sample gas molecules. This method provides high
stability in ionization and the obtained mass spectra show good reproducibility. The
EI method provides good results for quantitative analysis as well. Quantisation with
GC-MS, in which, only the ions specific to the compounds are measured, is highly
selective method without interfering components.
49
2.3. Results and Discussion
Plants are the best known sources of secondary metabolites which are
classified by their chemical structure and/or of physical properties in one or more of
the following groups: alkaloids, terpenoids, steroids, saponins, resins and peptides
(Drager, 2002; Breemen et al., 2001). Secondary metabolites are chemicals
produced by means of secondary reactions resulting from primary metabolism
carbohydrates, amino acids and lipids (Ting, 1982). Plants have an almost limitless
ability
to
synthesise
aromatic
substances
mainly
secondary
metabolites
(Mallikharjuna et al., 2007). These secondary metabolites are known to exhibit
diverse biochemical and pharmacological effects. The preliminary phytochemical
studies of Liv-Pro-08 with petroleum ether, chloroform, methanol, ethanol and
aqueous plant extracts of Nigells sativa, Entada pursaetha and Ficus glomerata
were performed to detect the presence of following phytochemical namely
alkaloids,
flavonoids,
phenols,
tannins,
saponins,
steroids,
glycosides,
carbohydrates and proteins are given in table 2.
Table 2: Preliminary Phytochemical Screening of Liv-Pro-08
Phytochemical
Constituents
Different Solvents
Alkaloids
Petroleum
ether
-
Flavonoids
-
-
-
+
+
Phenols
-
-
+
+
+
Tannins
+
+
-
+
-
Saponins
-
-
-
+
+
Glycosides
-
-
+
-
-
Steroids
+
+
+
+
+
Proteins
+
+
+
+
+
carbohydrates
+
+
+
+
+
Chloroform
Methanol
Ethanol
Aqueous
-
+
+
-
+: presence;
-: Absence; from the above results, it was inferred that more
secondary metabolites were present in ethanolic and aqueous extract of Liv-Pro-08;
therefore it is utilized for further experimental studies.
50
The qualitative data presented in the table 2 of Liv-Pro-08 reveals the
presence of wide range of secondary components alkaloids, flavonoids, phenols,
tannins, saponins, steroids, carbohydrates and proteins. The petroleum ether and
chloroform extracts showed the presence of tannins, steroids, proteins and
carbohydrates. The methanolic extract of Liv-Pro-08 exhibited the presents of
alkaloids, phenols, glycosides, steroids, carbohydrates and proteins, while the
ethanolic extract showed the presence of alkaloids, flavonoids, phenols, tannins,
saponins, steroids, carbohydrates and proteins. Flavonoids, phenols, saponins,
steroids, carbohydrates and proteins where present in the aqueous extract of Liv-Pro08. From the above table 2 results it was inferred that broad range of secondary
metabolites were present in the ethanolic extract of Liv-Pro-08. Hence, it is utilized
for experimental analysis.
Table 3 reveals the quantitative assessment of primary and secondary
metabolites (alkaloids, flavonoids, phenols, steroids, carbohydrates and proteins).
From the table alkaloids value is found to be 3.40 mg/g of ethanolic extract of LivPro-08. Alkaloids present in plants are known to have numerous beneficial
pharmacological effects (Harborne, 1973). Alkaloids are one of the most important
groups of secondary metabolites due to the great member of isolated products and
their pharmacological activity (Perez-Amador et al., 2007). Alkaloids are all
nitrogen heterocyclic mainly occurring as salts of common carboxylic acids. They
have restricted distribution and are readily affected by the plant growth location and
atmospheric condition (Perez-Amador et al., 2007). It has been recognized that
alkaloids show antioxidant activity and their effects on human nutrition and health
care is considerable (Kumpulainen and Salonen, 1999).
Table 3 predicts the level of flavonoid value 12.32 mg of rutin /g tissue.
Flavonoids represent the group of phytochemicals that are known to offer significant
health benefit to humans (Comalada et al., 2005 and Nair et al., 2004). Flavonoids
are concerned with the major important phenolic component presented in food
(Mervat and Taie, 2009). Flavonoids prevent oxidative damage and cell death by
several mechanisms such as, scavenging oxygen free radicals, protecting antioxidant
enzymes and lipid peroxidation (Roy et al, 2005). Flavonoids have been proven to
51
display wide range of pharmacological and biochemical actions such as antimicrobial, anti-thrombotic, anti-mutagenic and carcinogenic activities (Cook and
Samman, 1996) in food systems. Flavonoids can act as free radical scavengers and
terminate radical change reactions that occur during the oxidation of triglycerides.
Flavonoids consumed in large amounts in the daily diet are known to protect the
liver (Di Carlo et al., 1999).
Table 3: Quantitative Estimation of Phytochemical of Liv-Pro-08
phytochemicals
mg/g of ethanolic extract of
Liv-Pro-08
Alkaloids
3.40 ± 0.02
Steroids
28.53 ± 0.82
Phenols
31.76 ± 2.60
Flavonoids
12.32 ± 1.00
Carbohydrate
73.80 ± 3.54
Starch
28.85 ± 2.11
Glycogen
23.91 ± 1.20
Protein
86.43 ± 0.84
Triacylglycerol
23.10 ± 0.92
Free fatty acids
15.40 ± 0.47
Values are mean ± SD (n=3)
Table 3 presents the level of steroids 28.53 mg/g of ethanolic extract of LivPro-08. Steroids are biologically active compounds which are considered as a part of
plants defense system, and as such have been included in large group of protective
molecules found in plants indeed „phytoanticipins‟ or „phytoprotectants‟ (Morrissey
& Osbourn, 1999). During the last decade there has been an unprecedented
escalation of interest in the potential health benefits of phytosterols, in particular as
efficacious cholesterol lowering agents. Moreover phytosterols may possess
anticarcinogenic, anti-inflammatory and anti-oxidative activities (De Jong et al.,
52
2003). The results of the steroid content in Liv-Pro-08 reveals its potential effect on
stabilization of phospholipid bilayer in the cell membrane which emphasizes that the
sources focussed can act as an effective therapeutic agent against liver diseases. The
presence of polyphenols in foods has been widely reported, in fruits and several
beverages being the main sources of these compounds in the diet (Urquiaga and
Leighton 2000). Although several classes of phenolic molecules such as quercetin
can be found in most plant foods (wine, tea, cereals, legumes, fruit juices, etc.),
others are found only in a specific type of food (flavanones in citrus fruit,
isoflavones in soya, phloridzin in apples, etc.). However, in nature, it is common for
several types of polyphenols to be found in the some food products.
The best described property of almost every group of flavonoids is their
capacity to act as antioxidants. Quercetin, kaempferol more in myricetin, rutin by
activity as antioxidants exhibited beneficial effects. They have also been suggested
to play the protective role in liver diseases cataracts and cardiovascular diseases.
Quercetin and silybin acting as free radical scavengers where shown to exert the
protective effect in liver reperfusion ischemic tissue damage (Hillwell, 1994). The
presence of significant level of flavonoids in the Liv-Pro-08 ayurvedic formulation
signifies the medicinal validity of these plants. Plants from the genus Ficus are rich
sources of prenylatted flavonoids and isoflavonoids, lignans, terpenoids, alkaloids
and coumarins (Kuo and Li, 2000 and Amooru et al., 2005). Hence the
quantification result signifies the Liv-Pro-08 holds to be protective and effective
therapeutic agent for treating liver disorders.
Phenolic content of Liv-Pro-08 was identified to be 31.76 mg of gallic acid/g
tissues. Phenolic compounds constitute one of the main classes of secondary
metabolites which are synthesized by plants due to plant adaptation in response to
biotic and abiotic stresses (Pitchersky and Gang, 2000). Among the important
constituents in plants, phenolic compounds are mainly involved in cell defense
system against free radicals (Szeto et al., 2002). The antioxidant capacity of the
phenolic compounds is mainly due to the redox properties, which allow them to act
as reducing agents, hydrogen donors, singlet oxygen quenchers (Marimuthu et al.,
2008). Phenolic compounds have an important role in stabilizing lipid peroxidation
53
and are associated with antioxidant activity (Cakir et al., 2003 & Gulcin et al.,
2003). The notable amount of phenol in Liv-Pro-08 enhances the therapeutic values
which seem to be a positive indication of reclamation and restriction of hepatic cell
damage indicated due to reactive oxygen species.
Table 3 reveals the amount of carbohydrates, proteins and lipids of Liv-Pro08. The estimation of carbohydrates plays an important part in both pure and applied
plant physiology. The term “total available carbohydrate” may be defined as
including all those carbohydrates which can be used in the plant body as a source of
energy or as building material, either directly or indirectly after having been broken
down by enzymes. In most ordinary, higher green plants the bulk of available
carbohydrate is composed of sugars, fructosans, dextrin and starch, whereas
hemicelluloses and cellulose act merely as structural materials and as such cannot
further be utilized in the same way as the former. Proteins play critical roles in cell
biology, they have many potential therapeutic uses in preventing and curing diseases
and disorders. Carbohydrate and starch content of Liv-Pro-08 are 73.8 g/100g and
28.85 g/100g respectively. Liv-Pro-08 possesses high protein content and very low
fat (86.43, 15.4 g/100g). Therefore, biochemistry and medicine are intimately
related health depends on harmonious balance of biochemical reactions occurring in
the body, disease reflects abnormalities in biomolecules in biochemical reactions
(Robert, 2006).
Preliminary phytochemical investigation of the six active EtOAc and n-BuOH
fractions guided by review of literatures of its genus showed that phenolic components
constitute the major components of it. The total phenolic, tannin, flavonoid and flavonol
contents of the six active EtOAc and n-BuOH fractions were identified (Abdl-Hameed,
2009). A number of ficus species are used as food & for medicinal properties in
Ayurvedic and Traditional Chinese Medicine (TCM) especially amongst people where
these species grow. These uses, however, originated and are most widely found in the
Middle East & India (Lansky et al., 2008).
Over the past decade evidence has accumulated that plant polyphenols and
especially, flavonoids are a most important class of antioxidant defense. With several
endogenous antioxidants they play a role in optimum protection from oxidative stress
54
caused by the increase in the level of reactive oxygen species (ROS) in the human
organism. Under oxidative stress conditions, ROS may be very damaging and play a
causative role in aging and several degenerative diseases, for example heart disease,
atherosclerosis, cataracts, cognitive dysfunction, hepatotoxicity, inflammation and
cancer. This pathological process is caused by ROS attack on cell membrane lipids,
proteins in tissues or enzymes, carbohydrates and DNA. The documented flavonoids
antioxidant action includes suppressing ROS formation, direct ROS scavenging and
upregulating or protecting antioxidant defense (Pietta, 2000).
The results of the present study showed that the extract of Liv-Pro-08, which
contain highest amount of secondary compounds (phenols, flavonoids, steroids and
saponins) play an important role in human health. Therefore, it would be interesting
to know more about the possible pharmacological effects of this formulation.
2.3.2. High Performance Thin Layer Chromatography
Modern chromatographic techniques like HPLC and HPTLC were used to
judge the authenticity of traditional recommendation (Khan et al., 2009). HPTLC is
an invaluable quality assessment tool for the evaluation of botanical materials. It
allows for the analysis of a broad number of compounds both efficiently and cost
effectively (Arokiyaraj et al., 2008).
Preliminary qualitative phytochemical screening of Liv-Pro-08, Nigella
sativa, Entada pursaetha and Ficus glomerata, the results showed the presence of
phenolics, flavonoids and saponins in three samples but the Liv-Pro-08 formulation
showed the presence of flavonoids. HPTLC finger print profile of the extract for
flavonoids had been developed. R f values and the relative percentage of the
separated compounds were recorded. Analysis of flavonoids in Liv-Pro-08 and
phenolics, flavonoids and saponins in Nigella sativa, Entada pursaetha and Ficus
glomerata were recorded in Table 4-7. Fluorescent zone was detected in UV after
and before derivatization in the chromatogram at UV 366 mode in sample track and
reference (Quercetin, Naringenin, Rutin and Saponin) track, plate 2 & 3 which
confirmed the presence of flavonoids as compared with quercetin and naringenin in
the given plant extract (Liv-Pro-08). The Rf value of Liv-Pro-08 was found to be
55
0.24 peak of 5 in the sample. Nigella sativa, Entada pursaetha and Ficus glomerata
shows the peak values in table 4, 5 & 6. Three flavonoid compounds, six phenolic
compounds & three saponin compounds were identified in ethanolic extract of
Nigella sativa, whereas in Entada pursaetha extract two flavonoids, one phenolic
compound and one saponin compound were identified, while four flavonoids, five
phenolics and six saponin compounds were determined in Ficus glomerata ethanolic
extract. The highest peak of the respective flavonoid, phenol and saponin also given
in the plate 4-9.
Thus the HPTLC finger print profile of the major chemical constituents in
the ethanolic extract along with their RF values and percentage proportions were
recorded which would serve as a reference standard for the scientist who engaged in
research on the medicinal properties of this plant. The phytochemical evaluation is
one of the tools for the quality assessment, which includes preliminary
phytochemical screening; chemo profiling and marker compound analysis using
modern analytical techniques. In the last two decades HPTLC has emerged as an
important tool for the qualitative, semi-quantitative and quantitative phytochemical
analysis of herbal drugs and formulations. The major advantage of HPTLC is that
several samples can be analysed simultaneously using a small quantity of mobile
phase (Modi et al., 2008).
Table 4: Peak Values of Flavonoids in Liv-Pro-08 Sample
Track
Peak
Rf
Height
Area
Assigned substance
Liv-Pro-08
5
0.24
199.8
5943.4
Flavonoid 1
Liv-Pro-08
6
0.37
64.5
1150.9
Unknown
Liv-Pro-08
7
0.41
209.3
7143.6
Unknown
Liv-Pro-08
8
0.44
195.9
7363.8
Unknown
Liv-Pro-08
9
0.60
85.2
4394.5
Unknown
Liv-Pro-08
10
0.72
175.4
16993.2
Unknown
QUER
NGN
1
2
0.28
0.44
441.9
671.0
26465.1
16716.5
Quercetin standard
Naringenin standard
56
Plate 2: Chromatogram of Flavonoids in Liv-Pro-08
Before Derivatization
After Derivatization
Plate 3: Quercetin and Naringenin Standard Peak and Liv-Pro-08 Peak Baseline and
Densitogram display (Scanned at 254nm)
(a): Baseline display-Quercetin (Std)
(b): Densitogram display- Quercetin (Std)
57
(c): Baseline display-Naringenin (Std)
(d): Densitogram display- Naringenin (Std)
(e): Baseline display-Liv-Pro-08 (sample)
(f): Densitogram display- Liv-Pro-08 (sample)
Table 5: Peak Values of Flavonoids in Nigella sativa (1), Entada pursaetha (2) &
Ficus glomerata (3) Samples
Track
RUT
Sample 1
Sample 1
Sample 1
Sample 2
Sample 2
Sample 3
Sample 3
Sample 3
Sample 3
Peak
1
9
10
11
2
3
1
2
7
9
Rf
0.45
0.60
0.74
0.82
0.33
0.56
0.07
0.16
0.38
0.56
Height
59.3
299.2
54.0
31.4
49.0
203.2
410.2
205.4
66.2
103.7
58
Area
1559.2
13761.5
1701.2
857.0
1725.9
8023.3
12143.3
5517.0
2558.5
4951.7
Assigned substance
Rutin standard
Flavonoid 1
Flavonoid 2
Flavonoid 3
Flavonoid 1
Flaovnoid 2
Flavonoid 1
Flavonoid 2
Flavonoid 3
Flavonoid 4
Plate 4: Chromatogram of Flavonoids in Nigella sativa (1), Entada pursaetha (2)
and Ficus glomerata (3)
Before derivatization
After Derivatization
Plate 5: Rutin Standard and Ethanolic Extract of Nigella sativa, Entada pursaetha
and Ficus glomerata Peak Baseline and Densitogram Display (Scanned at 254nm)
(a): Baseline display of Rutin
(b): Densitogram display of Rutin
59
(c): Baseline display of Nigella sativa
(d): Densitogram display of Nigella sativa
(e): Baseline display of Entada pursaetha
(f): Densitogram display of Entada pursaetha
(g): Baseline display of Ficus glomerata
(h): Densitogram display of Ficus glomerata
60
Table 6: Peak Values of Phenols in Nigella sativa (1), Entada pursaetha (2) &
Ficus glomerata (3) Samples
Track
Peak
Rf
Height
Area
Assigned substance
QUE
1
0.78
380.9
9481.8
Quercetin standard
Sample 1
2
0.07
182.1
3644.0
Phenolic 1
Sample 1
5
0.36
162.2
11394.6
Phenolic 2
Sample 1
6
0.47
257.3
13542.9
Phenolic 3
Sample 1
7
0.55
116.1
5253.8
Phenolic 4
Sample 1
8
0.64
150.5
8809.7
Phenolic 5
Sample 1
9
0.73
459.7
19858.2
Phenolic 6
Sample 2
2
0.76
92.0
1746.3
Phenolic 1
Sample 3
1
0.09
253.7
15133.0
Phenolic 1
Sample 3
2
0.21
380.2
11783.6
Phenolic 2
Sample 3
4
0.28
398.2
16012.1
Phenolic 3
Sample 3
8
0.69
293.3
15278.4
Phenolic 4
Sample 3
9
0.75
72.4
886.5
Phenolic 5
Plate 6: Chromatogram of Phenols in Nigella sativa (1), Entada pursaetha (2) and
Ficus glomerata (3)
Before derivatization
After Derivatization
61
Plate 7: Quercetin Standard and Ethanolic Extract of Nigella sativa, Entada
pursaetha and Ficus glomerata Peak Baseline and Densitogram Display (Scanned at
254nm)
(a): Baseline display of Quercetin (std)
(b): Densitogram display of Quercetin (std)
(c): Baseline display of Nigella sativa
(d): Densitogram display of Nigella sativa
(e): Baseline display of Entada pursaetha
(f): Densitogram display of Entada pursaetha
62
(g): Baseline display of Ficus glomerata
(h): Densitogram display of Ficus glomerata
Table 7: Peak Values of Saponins in Nigella sativa (1) seed, Entada pursaetha (2)
& Ficus glomerata (3) Samples
Track
Peak
Rf
Height
Area
Assigned substance
SAP
1
0.17
201.6
6742.8
Saponin standard
Sample 1
1
0.13
80.8
2364.6
Saponin 1
Sample 1
2
0.17
48.0
724.5
Saponin 2
Sample 1
3
0.32
625.9
40594.4
Saponin 3
Sample 2
1
0.43
15.8
521.6
Unknown
Sample 2
2
0.70
20.2
495.3
Unknown
Sample 2
3
0.92
288.0
12249.9
Saponin 1
Sample 3
1
0.03
144.0
1896.8
Saponin 1
Sample 3
2
0.05
478.4
12837.4
Saponin 2
Sample 3
3
0.14
522.8
30152.2
Saponin 3
Sample 3
6
0.38
360.4
24093.3
Saponin 4
Sample 3
8
0.67
170.3
8949.8
Saponin 5
Sample 3
9
0.78
79.2
2833.6
Saponin 6
63
Plate 8: Chromatogram of Saponins in Nigella sativa (1), Entada pursaetha (2) and
Ficus glomerata (3)
Before derivatization
After Derivatization
Plate 9: Saponins Standard and Ethanolic Extract of Nigella sativa, Entada
pursaetha and Ficus glomerata Peak Baseline and Densitogram Display (Scanned at
254nm)
(a): Baseline display of Saponin (std)
(b): Densitogram display of Saponin (std)
64
(c): Baseline display of Nigella sativa
(d): Densitogram display of Nigella sativa
(b)
(c)
(e): Baseline display of Entada pursaetha
(f): Densitogram display of Entada pursaetha
(g): Baseline display of Ficus glomerata
(h): Densitogram display of Ficus glomerata
65
Phenolic acids scavenge the free radical and play an important role in the
prevention and therapy of diseases (Bala et al., 2006). Flavonoids can exercise their
antioxidant activity in several ways, antiradical, anti-lipoperoxidation and activities
of metal chelation (Bombardelli and Morazzoni, 1993). Flavonoids are powerful
antioxidants against free radicals, because they act as “radical scavengers”. This
activity is attributed to their hydrogen-donating ability. Indeed, the phenolic groups
of flavonoids serve as a source of readily available “H” atoms such that the
subsequent radicals produced can be delocalized over the flavonoid structure (Burda
and Oleszek, 2001; DiMajo et al., 2005). The variety of flavonoids occurring in
plant materials is usually large, the components of flavonoid fractions come from
different classes of aglycone, mono and polyglycoside, or acylated compound and
differ from each other in polarity, molecular weight and chromatographic and
spectrophotometric properties. Every plant has an original and unique flavonoid
profile, which makes quantification difficult (Olszewska, 2007). Rao et al., reported
that the qualitative phytochemical results showed the presence of phenolics,
flavonoids, steroids & terpenoids and quantitative HPTLC determination shows the
presence of gallic acid and ellagic acid in ethanolic extract of Ficus glomerata (Rao
et al., 2008)
The HPTLC results confirm that the flavonoid was present in the sample
Liv-Pro-08 is a considerable amount. Phenols, flavonoids & saponins were present
in all the three samples (Nigella sativa Entada pursaetha & Ficus glomerata). Since
phenols, flavonoids & saponins are gaining importance in therapeutics. Hence, it
may be confirmed that the plant may possess hepatoprotective and antioxidant
activity.
2.3.3. Gas Chromatography-Mass Spectroscopy analysis of Liv-Pro-08
The hyphenated technique, GC-MS is a useful tool in modern food, medicine
and biological result aiming at the separation and identification of components of
organic mixtures (Delazar et al., 2004).
66
Analysis of herbal extracts by GC-MS generates a large number of unknown
compounds that need to be identified. In principle, MS can used to elucidate the de
novo structure of unknown compounds beginning with the determination of
molecular formula of the molecular ion, using accurate mass measurements that
have errors less than a few parts per million (ppm). However, even when mass
measurements are obtained with errors below 3 ppm and the isotopic distribution of
the monoisotopic peak is being used to eliminate many of the possible molecular
formula, additional information (i.e., literature information on possible degradation
products of the major components in the herbal extract) is needed to elucidate the
chemical structure of an unknown compound (Lopez-Avila et al., 2009).
Constituents of the extract were identified by comparison of their mass
spectral pattern and retention time with those of standard samples. The components
identified from the extract, their retention time and percentage compositions are
summarized in figure 15 and table 8.
Figure 15: Phyto-Components Identified in Ethanolic Extract of Liv-Pro-08
RT: 0.00 - 38.32
NL:
1.19E9
TIC MS
EM214
25.86
100
90
80
Relative Abundance
70
60
50
40
30
21.68
20
10
4.57
9.50
5
10
15.79
17.85
28.84
0
0
15
20
Time (min)
67
25
30
36.02
35
The solvent extraction of Liv-Pro-08, more than 20 components was
identified in the extract, which represented about 50.32% of the fatty acid and transdihydrocarvone shown in table 8. Linoleic acid (50.32%), 9, 12-octadecadienoic
acid, trans-dihydrocarvone, phenols are the major components. The fatty acid
composition of solvent extraction of Nigella sativa determined by the present
investigation is similar to literature (Houghton et al., 1995). phytochemical
composition of the Liv-Pro-08 listed in table 8. More than 20 components
constituting cholesteroyl-2,4-Dihydroxycinnamate (2.45%), stigmast-5-en-3-ol, (3a)
(2.45%), phenol (2.27%), benzoicacid 4-hydroxy (2.27%), 3-trt-Butylcatachol
(0.61%),
3-hydroxy-4-methoxy
acetophenone
(0.61%),
2,4-Dihydroxy-3-
methylacetophenone (0.61%). Vanillic acid (4-hydroxy-3-hydroxy benzoic acid) is a
phenolic derivative known to possess antimicrobial (Delaquis et al., 2005) and
hepatoprotctive activities (Singh et al., 2006). The present study shows the presence
of 4-hydroxy benzoic acid and other phenolic components. The extract is
characterized by a large amount of phenylpropanoids and steroids. These results are
nearly similar to the qualitative results obtained from other investigations (Mozaffari
et al., 2000).
Hypolipidemic property may be due to the presence of oleic acid, an
unsaturated fatty acid. The low degree of fatty acid unsaturation in biomembranes of
long-lived animals may confer advantage by decreasing their sensitivity to lipid
peroxidation. The present study represents the comprehensive analysis will be
helpful in utilizing the abundant source of this plant in the production of
pharmaceutical and other industrial products. Monoterpenes are natural plant
products found in the essential oils of many commonly consumed fruits and
vegetables and have been widely used as flavor and fragrance additives in food and
beverages. Monoterpenes have been shown to possess antitumorigenic activities
(Kelloff et al., 1996).
68
Table 8: Phytocomponents Identified in Ethanolic Extract of Liv-Pro-08
No.
RT
Name of compounds
Molecular
formula
MW
Peak
Area %
1
7.98
2,3-Dihydro-3,5-dihydroxy-6methyl-4H-pyran-4-one
C6H8O4
144
0.73
2
14.99
2',4'-Dihydroxy-3'methylacetophenone
C9H10O3
166
0.61
3
14.99
3-Hydroxy-4methoxyacetophenone
C9H10O3
166
0.61
4
15.79
3-Cyano-6-hydroxy-4-methyl-2(1H)-pyridone
C7H6N2O2
150
2.27
5
15.79
1,2,3,4-Tetrahydropyrrolo[1,2c]pyrimidine-1,4-dione
C7H6N2O2
150
2.27
6
15.79
Benzoicacid 4-hydroxy
C7H6O3
138
2.27
7
15.79
Phenol (CAS)
94
2.27
8
16.50
R-(-)-2-Octyl-n-octyl carbonate
286
1.03
9
21.68
Pentadecanoic acid, 14-methyl-,
methyl ester (CAS)
C17H34O2
270
7.41
10
21.68
Eicosatetraenoic acid, methyl ester
(CAS)
C20H32O2
304
7.41
11
22.84
Pentadecanoic acid, 2,6,10,14tetramethyl-, methyl ester
C20 H40O2
312
7.20
12
25.49
Trans-Dihydrocarvone
152
50.32
13
25.87
9,12-Octadecadienoic acid (Z,Z)-,
methyl ester (CAS)
294
50.32
C6H6O
69
C17H34O3
C10H16O
C19H34O2
14
25.87
7,10-Hexadecadienoic acid, methyl
ester (CAS)
C17H30O2
15
26.80
Cholesteryl - 2,4Dihydroxycinnamate
C36H52O4
16
26.80
stigmast-5-en-3-ol, (3a)
C29H50O
266
50.32
548
2.45
414
2.45
Nickavar et al. (2003) reported that the presence of linoleic acid, oleic acid,
palmitic
acid
and
phenyl
propanoid
compounds
(monoterpenoid
and
dihydrocarvone) were identified in the ethanolic extract of Nigella sativa by GC-MS
analysis (Nickavar et al., 2003). Black cumin was rich in linoleic acid, which has a
beneficial effect on blood lipids, lowering blood pressure and serum cholesterol
(Cheikh Rouhou et al., 2006, 2007). Their unique fatty acid composition, relatively
high polyphenol content and quality and hence high protection against
oxidative stress, relatively good shelf life, and other desirable physicochemical
characteristics lead to more diverse and novel applications of Nigella sativa
seed and oils in the food, pharmaceutical, cosmetic and non-food industries (Cheikh
Rouhou et al., 2006, 2007).
Sterol profile of black cumin lipid fraction was widely investigated for
Turkish and German origins (Ramadan and Morsel, 2004). Phytosterols are of a
great interest due to their antioxidant activity and impact on health. The oxygenated
monoterpenes carvone and dihydrocarvone are potential inhibitors of bacterial
(Helader et al., 1998), fungal growth (Smid et al., 1995) as well as protective
insect repellents in Nigella sativa (Salom et al., 1996). The study reported by Nzowa
et al reveals that, two triterpenoid saponins were identified from the seed kernals of
Entada rheedii (Nzowa et al., 2010). Some of the researchers reported that the
presence of alkaloids, saponins and fatty acids in Nigell sativa have been intensively
investigated (Atta-ur-Rahman et al., 1985). Nigella sativa seed oil could be
considered as a potential source of natural phenolic compounds. Although their
participation in conferring specific flavour to oil (Caponio et al., 1999), phenolic
compounds may have a positive effect in the prevention of coronary heart disease
and cancer (Owen t al., 2000; Tuck & Hayball, 2002).
70
The results obtained based on the phytochemical and biochemical study
outlays an interesting and wide array of secondary compounds (Alkaloids, Steroids,
Phenols and Flavanoids). The plant sources (Nigella sativa, Entada pursaetha and
Ficus glomerata) possess a good store of biochemical constituents which provides
the basics of nutritive potential. The parallel characterization of phytochemical
compounds by HPTLC confirmed the presence of phenols, flavonoids and saponins
as secondary compound which is of medicinal and therapeutic value. The combined
synergetic effect of the three plant species as Liv-Pro-08 ayurvedic formulation
holds rich source of valuable and medicinally important phytochemicals. The prime
phytochemical potential indulged in the Liv-Pro-08 has been successfully explored
as an outcome of this study.
71