48
RREESSUULLTTSS
3.1 Taxonomic details of the seven plant species 3.1.1 Asclepias curassavica L.
Asclepias curassavica L. is known as Kammalchedi in Malayalam
and Kakatundi in Hindi. It is distributed throughout Kerala.
Erect herb. Leaves lanceolate, opposite. Flowers bright, orange red
coloured, moderate sized, in umbellate cymes. Calyx 5-partite. Corolla
rotate deeply 5-lobed; lobes valvate or slightly overlapping; corona of 5
erect lobes attached to the staminal column. Stamens adnate near the base
of the corolla, the filaments connate in a tube; pollen masses solitary in
each anther locus, pendulous; caudicles folded and curved. Ovary of 2
distinct carpels; styles free below, connected above; style apex columnar,
truncate or depressed at tip. Fruit of 2 smooth usually beaked and stalked
follicles, often more or less covered with hairs. Seeds flattened, winged
ending in a silky coma.
Flowering and Fruiting: Throughout the year.
3.1.2 Calotropis gigantea (L.) R.Br.
Calotropis gigantea (L.) R.Br. is known as Erukku in Malayalam and
Tamil; Arkah in Sanskrit and Madar in Hindi. It is distributed throughout
Kerala.
Erect milky shrub, very pale in colour. Leaves large, opposite, sessile,
ovate or obovate, cordate at base. Flowers large, in umbellate pedunculate
cymes; pale purple in colour. Calyx-5-lobed; lobes broadly ovate,
glandular within. Corolla broadly campanulate or subrotate, divided more
49
than halfway down in 5 valvate lobes; corona scales 5, fleshy adnate to and
radiating from the large staminal column, with an upcurved involute spur.
Stamens 5, filaments connate into a staminal column; pollinia 2 per anther,
pendulous. Ovaries 2, free; ovules many, on marginal placenta; style
slender with a depressed pentagonal style-apex. Fruit of 2 large fleshy
follicles, seeds with an abundant white silky hairs.
Flowering and Fruiting: Throughout the year.
3.1.3 Gymnema sylvestre (Retz.) R.Br.
Gymnema sylvestre (Retz.) R.Br. is known as Chakkarakkolli in
Malayalam; Gudmar or Merasimgi in Hindi; Sirukurumkay or
Sakkaraikkolli in Tamil and Mesamgi or Madhunasini in Sanskrit. It is
distributed throughout Kerala.
Twining shrubs or undershrubs; tender parts pubescent. Leaves
opposite, elliptic to ovate or obovate, base truncate or obtuse, apex
shortly acuminate. Flowers small, in crowded axillary umbellate cymes,
greenish-yellow. Calyx 5-partite. Corolla subrotate campanulate; lobes
subvalvate; corona 5 fleshy processes adnate to the tube. Staminal
column arising from the base of the corolla; pollen-masses erect, attached
to the horny pollen-carriers by very short caudicles. Ovary of 2 carpels;
styles free near the top; style-apex conical. Follicles slender.
Flowering and Fruiting: August-December
3.1.4 Holostemma ada-kodien Schult.
Holostemma ada-kodien Schult. is known as Adapatian or
Atakotiyan in Malayalam; Palaikkirai in Tamil; Jivanti or Arkapushpi in
Sanskrit and Chirvel or Charivel in Hindi. It is distributed in Kollam,
Thiruvananthapuram, Palakkad, Thrissur, Malappuram and Idukki
districts of Kerala.
50
Twining shrub. Leaves opposite, cordate. Flowers large, purple, in
few-flowered axillary cymes. Calyx 5-partite. Corolla thick, subrotate,
deeply 5-lobed, the lobes overlapping to the right; corona affixed to the base
of the staminal column, annular, fleshy. Stamens adnate to the base of the
corolla-tube, the filaments connate in a 10-winged column; pollen-masses
pendulous, clavate, elongate, compressed, attached by long caudicles to the
hard brown linear pollen-carriers. Ovary of 2 distinct carpels; style slender,
style-apex oblong, 5-winged. Fruit of 1-2 thick lanceolate broad follicles.
Seeds ovoid, flattened, winged, ending in a white silky coma.
Flowering and Fruiting: July-December
3.1.5 Pergularia daemia (Forssk.) Chiov.
Pergularia daemia (Forssk.) Chiov. is known as Velipparutti in
Malayalam and Tamil; Kurutakah or Uttamarani in Sanskrit and Utaran,
Sagovani or Jutak in Hindi. It is distributed in Thrissur, Idukki, Palakkad,
Kollam and Kozhikode districts of Kerala.
Pubescent or tomentose twining undershrub. Leaves opposite,
cordate, covered with soft hairs. Flowers medium-sized, greenish-white, in
axillary racemose or corymbose, pedunculate cymes; pedicels slender.
Calyx 5-partite, lobes acute. Corolla-tube short, campanulate or funnel-
shaped; lobes 5, ovate, spreading, ciliate; corona membranous, annular, the
lobes truncate or dentate. Stamens adnate to the corolla tube, filaments
connate in a column; pollen-masses pendulous, attached in pairs to the
shining horny pollen-carriers. Ovary of 2 distinct carpels; styles slender.
Fruit of 2 lanceolate, echinate, often recurved follicles with soft spines.
Seeds ovate, minutely pubescent, margined, ending in a silky white coma.
Flowering and Fruiting: September-May
51
3.1.6 Tylophora indica (Burm.f.) Merr.
Tylophora indica (Burm.f.) Merr. is known as Vallippala in
Malayalam; Naippalai, Nanjaruppan or Kagittam in Tamil; Lataksiri in
Sanskrit and Antamul in Hindi.This is distributed in Palakkad and
Thiruvananthapuram districts of Kerala.
Slender climber. Leaves opposite, glabrous, ovate, base rounded to
subcordate, apex acute, Flowers in umbellate or racemose pedunculate cymes.
Calyx 5- partite, lobes ovate or lanceolate. Corolla greenish-yellow with
purple center; corona single, free at apex arching over gynostegium. Anthers
small with a membranous appendage; pollen masses small, horizontal.
Carpels free; style-apex pentagonal or 5-lobbed. Ovary obconical. Follicle
cylindric, produced into an acute, slender beak. Seeds ovate, flat.
Flowering and Fruiting: June-November
3.1.7 Wattakaka volubilis (L.f.) Stapf.
Wattakaka volubilis (L.f.) Stapf. is known as Vattakkakkakodi in
Malayalam; Kodippalai or Kurinja in Tamil; Hemajivanti or Svarnaparna
in Sanskrit and Nakchhikni in Hindi. It is distributed in Kozhikode,
Kottayam, Kasargode, Kollam, Kannur, Pathanamthitta, Palakkad,
Thiruvananthapuram, Thrissur and Malappuram districts of Kerala.
Stout twiner. Leaves obovate, base cordate or rounded, apex acuminate,
chartaceous, glabrous. Flowers in axillary many-flowered umbellate cymes.
Peduncles longer than the petioles. Pedicels slender. Calyx-lobes 5,
oblong, ciliate, glandular. Corolla greenish, rotate, lobes 5, ovate; corona
5, membranous, adnate at the base of staminal column, fleshy. Carpels
globose; stylar apex convex. Follicles stout, woody, oblong, glabrous
when ripe. Seeds comose, obovoid.
Flowering and Fruiting: January-August
52
Plate 3.1 Habit of plants
A Asclepias curassavica L. B Calotropis gigantea (L.) R.Br.
C Gymnema sylvestre (Retz.) R.Br. D Holostemma ada-kodien Schult.
53
Plate 3.2 Habit of plants
E Pergularia daemia (Forssk.) Chiov. F Tylophora indica (Burm.f.) Merr.
G Wattakaka volubilis (L.f.) Stapf.
54
3.2 Phytochemical studies 3.2.1 Total ash
The total amount of minerals present in a plant material is called
the “ash content”. Ash is the inorganic residue remaining after water
and organic matter have been removed by heating. The total ash values
of the leaves and the stems of seven plant species were determined
(Table 3.1).
Table 3.1 Ash values of leaf and stem
Sl. No Name of the plant
Ash value of leaf in
percentage
Ash value of stem in
percentage 1 Asclepias curassavica L. 11.01 ± 0.07 6.26 ± 0.16
2 Calotropis gigantea (L.) R.Br. 11.76 ± 0.09 7.92 ± 0.10
3 Gymnema sylvestre (Retz.) R.Br. 7.09 ± 0.08 4.32 ± 0.03
4 Holostemma ada-kodien Schult. 10.06 ± 0.12 7.88 ± 0.08
5 Pergularia daemia (Forssk.) Chiov. 12.84 ± 0.21 6.41 ± 0.13
6 Tylophora indica (Burm.f.) Merr. 13.08 ± 0.24 9.82 ± 0.09
7 Wattakaka volubilis (L.f.) Stapf. 9.49 ± 0.06 6.22 ± 0.05
The leaf extracts showed the total ash values which ranged between
7.09 ± 0.08% - 13.08 ± 0.24%. The highest ash value among the seven
leaf extracts was 13.08 ± 0.24% in Tylophora indica (Burm.f.) Merr. and
the lowest ash value was 7.09 ± 0.08% in Gymnema sylvestre (Retz.)
R.Br. The ash values of all stem extracts ranged between 4.32 ± 0.03% -
9.82 ± 0.09%. The highest ash value of stem extract is 9.82 ± 0.09% in
Tylophora indica (Burm.f.) Merr. and the lowest 4.32 ± 0.03% in
Gymnema sylvestre (Retz.) R.Br. In both the leaf and stem extract the
highest quantity of ash was observed in the same plant Tylophora indica
(Burm.f.) Merr. and the lowest value was reported in Gymnema sylvestre
55
(Retz.) R.Br. This percentage revealed the total mineral content within
the plant material.
3.2.2 Ash analysis for minerals
Mineral content is the measure of the amount of specific inorganic
components present within a material such as calcium, iron, potassium,
magnesium, sodium and zinc. The solution of each plant extract was
used for the estimation of calcium, iron, potassium, magnesium, sodium
and zinc content. Estimation of minerals had been carried out by ICP-
AES.
Table 3.2 Percentage of Ca, Fe, K, Mg, Na and Zn in leaf extracts
Sl. No. Name of plant Ca Fe K Mg Na Zn
1 Asclepias curassavica L. 1.855 0.043 2.358 0.548 0.410 0.009
2 Calotropis gigantea (L.) R.Br. 2.004 0.039 2.276 0.918 1.221 0.007
3 Gymnema sylvestre (Retz.) R.Br. 1.596 0.025 1.296 0.818 0.243 0.005
4 Holostemma ada-kodien Schult. 1.688 0.066 2.296 0.559 0.142 0.008
5 Pergularia daemia (Forssk.) Chiov. 1.705 0.094 2.247 0.566 0.130 0.006
6 Tylophora indica (Burm.f.) Merr. 1.936 0.042 2.312 0.507 0.330 0.008
7 Wattakaka volubilis (L.f.) Stapf. 2.140 0.028 1.817 0.305 0.152 0.003
All the extracts showed the presence of calcium, iron, potassium,
magnesium, sodium and zinc. Both leaf and stem extracts of all the
seven plants showed comparatively higher concentration of calcium and
potassium and lower concentration of iron, sodium and magnesium and
very low concentration of zinc. Among the leaf extracts the quantity of
calcium ranged from 1.596% to 2.140%. Wattakaka volubilis (L.f.)
Stapf. leaf extract showed highest concentration of calcium (2.140%)
and the lowest amount of calcium was observed in Gymnema sylvestre
56
(Retz.) R.Br. (1.596%). Potassium content ranged from 1.296% to
2.296%. 2.296% of potassium was observed in Holostemma ada-kodien
Schult. and the lowest amount 1.296% in Gymnema sylvestre (Retz.)
R.Br. The iron content ranged from 0.025% - 0.094%. Iron was highest
in Pergularia daemia (Forssk.) Chiov. (0.094%) and lowest (0.025%) in
Gymnema sylvestre (Retz.) R.Br. Sodium ranged from 0.130% to
1.221%. The highest amount (1.221%) was observed in Calotropis
gigantea (L.) R.Br. and Pergularia daemia (Forssk.) Chiov. showed
lowest amount (0.130%). Magnesium content ranged from 0.305% to
0.918%. Highest amount 0.918% was observed in Calotropis gigantea
(L.) R.Br. and the lowest amount 0.305% in Wattakaka volubilis (L.f.)
Stapf. and zinc ranged between 0.003% to 0.009%. The highest quantity
was seen in Asclepias curassavica L. and the lowest in Wattakaka
volubilis (L.f.) Stapf. (Table 3.2; Fig. 3.1).
Table 3.3 Percentage of Ca, Fe, K, Mg, Na and Zn in stem extracts
Sl. No. Name of plant Ca Fe K Mg Na Zn
1 Asclepias curassavica L. 1.627 0.044 1.270 0.138 0.228 0.006
2 Calotropis gigantea (L.) R.Br. 1.132 0.026 1.854 0.422 0.565 0.008
3 Gymnema sylvestre (Retz.) R.Br. 1.091 0.037 0.807 0.257 0.123 0.004
4 Holostemma ada-kodien Schult. 0.993 0.049 2.185 0.375 0.255 0.005
5 Pergularia daemia (Forssk.) Chiov. 0.783 0.027 2.041 0.209 0.258 0.008
6 Tylophora indica (Burm.f.) Merr. 1.128 0.050 1.139 0.175 0.259 0.014
7 Wattakaka volubilis (L.f.) Stapf. 1.789 0.026 1.037 0.227 0.320 0.006
57
Fig. 3.1 Concentration of Ca, Fe, K, Mg, Na, Zn in leaf extracts
58
Fig. 3.2 Concentration of Ca, Fe, K, Mg, Na, Zn in stem extracts
59
Fig. 3.3 Na/K ratio of leaf and stem extracts of the seven plants
A.C. Asclepias curassavica L. C.G. Calotropis gigantea (L.) R.Br. G.S. Gymnema sylvestre (Retz.) R.Br. H.A. Holostemma ada-kodien Schult. P.D. Pergularia daemia (Forssk.) Chiov. T.I. Tylophora indica (Burm.f.) Merr. W.V. Wattakaka volubilis (L.f.) Stapf.
60
Fig. 3.4 Ca/Mg ratio of leaf and stem extracts
A.C. Asclepias curassavica L. C.G. Calotropis gigantea (L.) R.Br. G.S. Gymnema sylvestre (Retz.) R.Br. H.A. Holostemma ada-kodien Schult. P.D. Pergularia daemia (Forssk.) Chiov. T.I. Tylophora indica (Burm.f.) Merr. W.V. Wattakaka volubilis (L.f.) Stapf.
61
In the stem extracts the percentage of calcium ranged between 0.783%
to 1.789%. It was highest in Wattakaka volubilis (L.f.) Stapf. (1.789%) and
lowest in Pergularia daemia (Forssk.) Chiov. (0.783%). Potassium ranged
between 2.185% and 0.807%. Iron ranged between 0.026% and 0.050%,
sodium ranged between 0.123% and 0.565%, magnesium ranged between
0.138% and 0.422% and zinc ranged between 0.004% and 0.014%. Potassium
was highest in Holostemma ada-kodien Schult. (2.185%) and lowest in
Gymnema sylvestre (Retz.) R.Br. (0.807%). The highest sodium content
(0.565%) was seen in Calotropis gigantea (L.) R.Br. and lowest (0.123%) in
Gymnema sylvestre (Retz.) R.Br. The magnesium content was highest
(0.422%) in Calotropis gigantea (L.) R.Br. and lowest (0.138%) in Asclepias
curassavica L. Zinc was highest in Tylophora indica (Burm.f.) Merr.
(0.014%) and lowest in Gymnema sylvestre (Retz.) R.Br. (0.004%) (Table
3.3; Fig. 3.2).
The mineral contents were more in leaves than in stem. The
percentage of iron, magnesium, sodium, and zinc were low in all the
fourteen samples. All were rich in potassium and poor in sodium on
comparison (Fig. 3.3). Higher concentration of calcium and potassium
were detected in all the samples. The ratio of calcium and magnesium is
shown in Fig. 3.4.
3.2.3 Extractive values
The extractive value gives an idea about the nature of chemical
constituents present in the crude drug. It is useful for the estimation of
chemical constituents which are soluble in that particular solvent. The
amount of sample soluble in a given solvent is an index of its purity.
62
Table 3.4 Water soluble extractive values of the plant materials
Sl. No Name of the plant Leaf
(% w/w) Stem
(% w/w) 1 Asclepias curassavica L. 16.93 12.15 2 Calotropis gigantea (L.) R.Br. 18.97 8.95 3 Gymnema sylvestre (Retz.) R.Br. 18.31 9.61 4 Holostemma ada-kodien Schult. 14.71 12.69 5 Pergularia daemia (Forssk.) Chiov. 25.18 11.29 6 Tylophora indica (Burm.f.) Merr. 15.08 8.97 7 Wattakaka volubilis (L.f.) Stapf. 19.87 9.98
The water soluble extractive values of the seven leaf samples ranges
between 14.71% and 25.18%. The lowest value (14.71%) was seen in
Holostemma ada-kodien Schult. and the highest value (25.18%) in
Pergularia daemia (Forssk.) Chiov. In the extractive values of the stem
samples the values ranges between 8.95% and 12.69%. The highest value
(12.69%) was observed in Holostemma ada-kodien Schult. and the lowest
value (8.95%) was seen in Calotropis gigantea (L.) R.Br. (Table 3.4).
Table 3.5 Alcohol soluble extractive values of the plant materials
Sl. No Name of the plant Leaf
(% w/w) Stem
(% w/w) 1 Asclepias curassavica L. 11.85 8.232 2 Calotropis gigantea (L.) R.Br. 7.24 6.713 3 Gymnema sylvestre (Retz.) R.Br. 12.81 6.752 4 Holostemma ada-kodien Schult. 8.51 7.194 5 Pergularia daemia (Forssk.) Chiov. 8.37 8.034 6 Tylophora indica (Burm.f.) Merr. 8.79 7.706 7 Wattakaka volubilis (L.f.) Stapf. 12.62 6.88
The alcohol soluble extractive values ranged between 7.24 % and
12.81 %. The lowest value (7.24 %) was seen in Calotropis gigantea
(L.) R.Br. and the highest value (12.81%) in Gymnema sylvestre (Retz.)
R.Br. In the extractive values of the stem samples the values ranged
63
between 6.71% and 8.23%. The highest value (8.23%) was observed in
Asclepias curassavica L. and the lowest value (6.71%) was seen in
Calotropis gigantea (L.) R.Br. (Table 3.5).
3.2.4 Qualitative detection of phytoconstituents
Qualitative chemical tests were carried out to identify the various
major constituents present in the leaf and stem extracts of the seven
plants.
Table 3.6 Qualitative analysis of phytoconstituents in the leaf and stem extracts
Secondary metabolite
A.C. C.G. G.S. H.A. P.D. T.I. W.V.
L ─ ─ + ─ + + ─ Alkaloids
S ─ ─ + ─ + + ─ L + + + + + ─ +
Tannins S + + + + + ─ + L + + + + ─ + +
Saponins S + + + + ─ + + L + + + + + + +
Flavonoids S + + + + + + + L + + + + + + +
Sterols S + + + + + + + L + + + + + + +
Phenols S + + + + + + + L + + + + + + +
Terpenoids S + + + + + + +
+ Present - Absent L - Leaf S – Stem
A.C. Asclepias curassavica L. C.G. Calotropis gigantea (L.) R.Br. G.S. Gymnema sylvestre (Retz.) R.Br. H.A. Holostemma ada-kodien Schult. P.D. Pergularia daemia (Forssk.) Chiov. T.I. Tylophora indica (Burm.f.) Merr. W.V. Wattakaka volubilis (L.f.) Stapf.
Flavonoids, sterols, phenols and terpenoids were found to be
present in all the stem and leaf extracts. Alkaloids were found to be
64
present in the leaf and stem extracts of Gymnema sylvestre (Retz.) R.Br.,
Pergularia daemia (Forssk.) Chiov. and Tylophora indica (Burm.f.)
Merr. Similarly tannins were found to be present in the leaf and stem
extracts of all the plants except Tylophora indica (Burm.f.) Merr.
Saponins were found to be present in the leaf and stem extracts of all the
plants except Pergularia daemia (Forssk.) Chiov. (Tables 3.6).
3.2.5 Thin layer chromatography
The leaf and stem extracts of all the plants were subjected to TLC
and HPTLC analysis. TLC studies were done seperately for the detection
of flavonoids, phenols, sterols and terpenoids. The extract prepared for
the detection of flavonoids were tried with several solvent systems. A
suitable solvent system was identified with the help of NP/PEG reagent.
Butanol: Ethyl acetate: Acetic acid: Formic acid (30:10:10:4) was
selected as the best solvent system.
The extract prepared for the detection of phenols were subjected to
various solvent systems. After spraying with folin’s reagent and the plate
was heated thoroughly. A better solvent system was selected seeing the
maximum number of spots and the good separation of the compounds.
The solvent system Toluene: Ethyl acetate: Formic acid (50:30:4) was the
best solvent for the separation of components.
For the detection of sterols, the prepared extracts were subjected to
various solvent systems. The solvent system with best separation was
selected with the help of Liebermann-Burchard reagent. Hexane:
Diethylether: Acetic acid (8:20:1) was selected as the mobile phase since
that showed maximum separation.
For terpenoids, the solvent system selected was Toluene: Ethyl
acetate: Formic acid (50:30:4). By conducting TLC, a good solvent
65
system with maximum resolution of spots were selected to perform
HPTLC analysis.
3.2.6 High performance thin layer chromatography (HPTLC)
The solvent system which showed the maximum resolution of spots
was selected as the solvent system in HPTLC studies. In the HPTLC
studies the number of compounds separated, the Rf values and their
percentage were noted. HPTLC fingerprint profile was prepared for the
leaf and stem extracts of all the seven plants.
3.2.6.1 Phenols
a Leaf
The HPTLC fingerprint profile of the leaf extracts at 254 nm are
presented in Fig. 3.5 (1-7) where in each graph, different peaks obtained
were recorded and the corresponding Rf values, heights, percentage of
heights were given. In Table 3.7 the Rf value in different extracts
obtained, and the major peaks were summerized. One can easily come
across the common peaks representing common phenols by going
through the Fig. 3.5 (1-7) and Table 3.7. Densitometric scanning of all the
extracts showed different chemical components, which are expressed as
different peaks with specific Rf value. Each Rf value corresponds to a
chemical compound.
Among all the extracts Tylophora indica (Burm.f.) Merr. had
maximum number of peaks (17). Wattakaka volubilis (L.f.) Stapf. had
sixteen peaks. The minimum number of peaks was nine in Gymnema
sylvestre (Retz.) R.Br. The major peaks were with Rf value 0.38 – 0.39
and 0.78 – 0.79 in all the extracts. The chemical component of Rf value
0.38 – 0.39 was the same and was present in all the leaf extracts. The
chemical component of Rf value 0.78 – 0.79 is the same and is seen in all
66
the extract. The third major peak is with an Rf value of 0.32 to 0.33 was
seen in all the plants. But in Pergularia daemia (Forssk.) Chiov. it was
not the third major peak. Other chemical compounds of different Rf
values were present in the extracts among the seven plants. Some
compounds were present in all the extracts and certain compounds are
present in only certain species.
Each extract possess a unique chemical profile. Each plant species
can be identified by going through its HPTLC fingerprint profile.
Eventhough the chemical identity of each spot was not revealed in the
profile it could be used as a marker to identify the plant from which the
drug was obtained.
b Stem
The HPTLC fingerprint profile of the stem extracts detected at 254
nm are presented in Fig. 3.6 (1-7). There in each graph, different peaks
obtained are recorded and the corresponding Rf values, heights,
percentage of heights are given. In Table 3.8, the Rf value in different
extracts obtained, and the major peaks were summerized. Common peaks
represent common phenols and could be easily observed in the Table 3.8.
An Rf value of 0.78 – 0.80 was present in all the extracts except
Holostemma ada-kodien Schult. and Pergularia daemia (Forssk.) Chiov.
But in those plants, it was the second major peak. As in the case of leaf
extracts, in the stem extracts also Tylophora indica (Burm.f.) Merr.
showed the maximum number of peaks - ten. In Wattakaka volubilis
(L.f.) Stapf. also ten peaks could be seen. The minimum number of peaks
(six) can be seen in Holostemma ada-kodien Schult. and Pergularia
daemia (Forssk.) Chiov.
67
Fig. 3.5 HPTLC profile of phenols in the leaf extracts
Asclepias curassavica L.
Calotropis gigantea (L.) R.Br.
(Contd …… Fig. 3.5)
68
Gymnema sylvestre (Retz.) R.Br.
Holostemma ada-kodien Schult.
(Contd …… Fig. 3.5)
69
Pergularia daemia (Forssk.) Chiov.
Tylophora indica (Burm.f.) Merr.
(Contd …… Fig. 3.5)
70
Wattakaka volubilis (L.f.) Stapf.
71
Table 3.7 Distribution of phenols in the leaf extracts 1 2 3 4 5 6 7 Rf A.C. C.G. G.S. H.A. P.D. T.I. W.V.
1 0.11
2 0.13
3 0.14
4 0.16
5 0.17
6 0.22
7 0.27
8 0.28
9 0.32
10 0.33
11 0.35
12 0.38
13 0.39
14 0.45
15 0.46
16 0.48
17 0.49
18 0.52
19 0.54
20 0.55
21 0.56
22 0.57
23 0.59
24 0.60
25 0.63
26 0.64
27 0.65
29 0.67
30 0.69
31 0.71
32 0.78
33 0.79
34 0.84
35 0.85
36 0.88
37 0.90
38 0.91
39 0.92
40 0.93
41 0.94
42 0.96
43 0.97
Number of Peaks 12 11 9 11 14 17 16 Major Peak 1 Rf 0.78 0.78 0.78 0.39 0.78 0.79 0.78 Major Peak 2 Rf 0.39 0.39 0.39 0.78 0.39 0.39 0.38 Major Peak 3 Rf 0.49 0.32 0.32 0.33 0.49 0.32 0.32
A.C. Asclepias curassavica L. C.G. Calotropis gigantea (L.) R.Br. G.S. Gymnema sylvestre (Retz.) R.Br. H.A. Holostemma ada-kodien Schult. P.D. Pergularia daemia (Forssk.) Chiov. T.I. Tylophora indica (Burm.f.) Merr. W.V. Wattakaka volubilis (L.f.) Stapf.
72
Fig. 3.6 HPTLC profile of phenols in the stem extracts
Asclepias curassavica L.
Calotropis gigantea (L.) R.Br.
(Contd …… Fig. 3.6)
73
Gymnema sylvestre (Retz.) R.Br.
Holostemma ada-kodien Schult.
(Contd …… Fig. 3.6)
74
Pergularia daemia (Forssk.) Chiov.
Tylophora indica (Burm.f.) Merr.
(Contd …… Fig. 3.6)
75
Wattakaka volubilis (L.f.) Stapf.
76
Table 3.8 Distribution of phenols in the stem extracts
1 2 3 4 5 6 7 Rf A.C. C.G. G.S. H.A. P.D. T.I. W.V. 1 0.09
2 0.14
3 0.17
4 0.18
5 0.2
6 0.26
7 0.28
8 0.29
9 0.33
10 0.34
11 0.4
12 0.42
13 0.45
14 0.49
15 0.53
16 0.62
17 0.63
18 0.66
19 0.68
20 0.76
21 0.78
22 0.79
23 0.8
24 0.87
25 0.95
Number of Peaks 8 7 8 6 6 10 10 Major Peak1 Rf 0.80 0.40 0.79 0.79 0.40 0.79 0.78 Major Peak2 Rf 0.42 0.79 0.40 0.18 0.79 0.40 0.40 Major Peak3 Rf 0.29 0.78 0.33 0.40 0.33 0.33 0.33
A.C. Asclepias curassavica L. C.G. Calotropis gigantea (L.) R.Br. G.S. Gymnema sylvestre (Retz.) R.Br. H.A. Holostemma ada-kodien Schult. P.D. Pergularia daemia (Forssk.) Chiov. T.I. Tylophora indica (Burm.f.) Merr. W.V. Wattakaka volubilis (L.f.) Stapf.
77
3.2.6.2 Flavonoids
a Leaf
The chemoprofile of flavonoid enriched leaf extracts of all the 7
plants at 254 nm were prepared. These are shown in Fig. 3.7 (1-7). In the
chemoprofile each spot is having an Rf value, corresponding height and
precentage of area of each peak. Table 3.9 summarizes the distribution of
different Rf values among 7 leaf extracts, number of peaks in each extract
and the major peaks in each extract. We could observe the presence of
similar compounds in all the 7 extracts. In the flavonoid profile among all
the leaf extracts, Pergularia daemia (Forssk) Chiov. had maximum
number of peaks (Fig. 3.7 (1-7). The minimum number of peaks was six in
Calotropis gigantea (L.) R.Br. In Table 3.9, the Rf value in different
extracts obtained, the major peaks and the common peaks are summerized.
b Stem
The fingerprint profile of flavonoids in the stem extracts at 254 nm
were presented in Fig. 3.8 (1-7). Rf values, percentage of height obtained
and the area percentage of all the peaks are given. Number of spots with
Rf value and their distribution and the major peaks are given in table
3.10. There in each graph, different peaks obtained are recorded and the
corresponding Rf values, heights, percentage of heights are given. In
Table 3.10, the Rf value in different extracts obtained, and the major
peaks are summerized. Common peaks represent common flavonoids and
could be easily observed in the Table 3.10. In the stem extracts,
Gymnema sylvestre (Retz.) R.Br., Holostemma ada-kodien Schult. and
Tylophora indica (Burm.f.) Merr. showed the maximum number of peaks
(ten) (Table 3.10).
78
Fig. 3.7 HPTLC profile of flavonoids in the leaf extracts
Asclepias curassavica L.
Calotropis gigantea (L.) R.Br.
(Contd …… Fig. 3.7)
79
Gymnema sylvestre (Retz.) R.Br.
Holostemma ada-kodien Schult.
(Contd …… Fig. 3.7)
80
Pergularia daemia (Forssk.) Chiov.
Tylophora indica (Burm.f.) Merr.
(Contd …… Fig. 3.7)
81
Wattakaka volubilis (L.f.) Stapf.
82
Table 3.9 Distribution of flavonoids in the leaf extracts
1 2 3 4 5 6 7 Rf A.C. C.G. G.S. H.A. P.D. T.I. W.V. 1 0.13
2 0.17
3 0.18
4 0.2
5 0.22
6 0.23
7 0.24
8 0.25
9 0.28
10 0.3
11 0.33
12 0.35
13 0.37
14 0.41
15 0.44
16 0.45
17 0.48
18 0.5
19 0.51
20 0.56
21 0.58
22 0.6
23 0.61
24 0.65
25 0.66
26 0.7
27 0.71
28 0.74
29 0.75
30 0.76
31 0.77
32 0.78
33 0.81
34 0.83
35 0.84
Number of Peaks 9 6 7 8 10 8 7 Major Peak 1 Rf 0.77 0.83 0.84 0.66 0.76 0.78 0.77 Major Peak 2 Rf 0.51 0.70 0.35 0.75 0.66 0.48 0.58 Major Peak 3 Rf 0.83 0.56 0.65 0.50 0.71 0.60 0.50
A.C. Asclepias curassavica L. C.G. Calotropis gigantea (L.) R.Br. G.S. Gymnema sylvestre (Retz.) R.Br. H.A. Holostemma ada-kodien Schult. P.D. Pergularia daemia (Forssk.) Chiov. T.I. Tylophora indica (Burm.f.) Merr. W.V. Wattakaka volubilis (L.f.) Stapf.
83
Fig. 3.8 HPTLC profile of flavonoids in the stem extracts
Asclepias curassavica L.
Calotropis gigantea (L.) R.Br.
(Contd …… Fig. 3.8)
84
Gymnema sylvestre (Retz.) R.Br.
Holostemma ada-kodien Schult.
(Contd …… Fig. 3.8)
85
Pergularia daemia (Forssk.) Chiov.
Tylophora indica (Burm.f.) Merr.
(Contd …… Fig. 3.8)
86
Wattakaka volubilis (L.f.) Stapf.
87
Table 3.10 Distribution of flavonoids in the stem extracts
1 2 3 4 5 6 7 Rf A.C. C.G. G.S. H.A. P.D. T.I. W.V.
1 0.16 2 0.17 3 0.18 4 0.19 5 0.21 6 0.22 7 0.23 8 0.24 9 0.25 10 0.26 11 0.27 12 0.31 13 0.34 14 0.36 15 0.37 16 0.38 17 0.39 18 0.4 19 0.41 20 0.47 21 0.48 22 0.5 23 0.51 24 0.52 25 0.54 26 0.59 27 0.61 28 0.62 29 0.65 30 0.66 31 0.67 32 0.68 33 0.71 34 0.73 35 0.75 36 0.77 37 0.79 38 0.81 39 0.82 40 0.83 41 0.84 42 0.85 43 0.87 44 0.88
Number of Peaks 6 6 10 10 8 10 7 Major Peak 1 Rf 0.25 0.84 0.85 0.81 0.83 0.77 0.79 Major Peak 2 Rf 0.41 0.73 0.79 0.79 0.68 0.84 0.73 Major Peak 3 Rf 0.54 0.22 0.59 0.65 0.61 0.48 0.83 A.C. Asclepias curassavica L. C.G. Calotropis gigantea (L.) R.Br. G.S. Gymnema sylvestre (Retz.) R.Br. H.A. Holostemma ada-kodien Schult. P.D. Pergularia daemia (Forssk.) Chiov. T.I. Tylophora indica (Burm.f.) Merr. W.V. Wattakaka volubilis (L.f.) Stapf.
88
3.2.6.3 Detection of quercetin in the stem and leaf extracts using HPTLC
To understand the chemical composition of the separated compounds,
we had to develop chromatograms of standard pure compounds along with
the plant extracts. Here, chromatogram of standard quercetin was
developed along with the flavonoid enriched extracts of leaf and stem.
The chromatogram obtained from quercetin is shown in Fig. 3.9. The Rf
value of quercetin is 0.81 at a λ max of 280 nm. Presence of a peak with
the same Rf (0.81) at the same λ max of 280 nm and the clear
superimpossibility confirmed the presence of quercetin in the extracts.
Quercetin was found to be present only in the leaf and stem extracts of
Holostemma ada-kodien Schult. The superimposed graph is shown in
Fig. 3.10.
Fig. 3.9 HPTLC chromatogram of quercetin (standard)
89
Fig. 3.10 Overlapping chromatographic comparison of leaf and stem extracts and quercetin standard
, ,
Stem extract 1 Asclepias curassavica L. 2 Calotropis gigantea (L.) R.Br. 3 Gymnema sylvestre (Retz.) R.Br. 4 Holostemma ada-kodien Schult. 5 Pergularia daemia (Forssk.) Chiov. 6 Tylophora indica (Burm.f.) Merr. 7 Wattakaka volubilis (L.f.) Stapf.
Leaf extract 8 Asclepias curassavica L. 9 Calotropis gigantea (L.) R.Br. 10 Gymnema sylvestre (Retz.) R.Br. 11 Holostemma ada-kodien Schult. 12 Pergularia daemia (Forssk.) Chiov. 13 Tylophora indica (Burm.f.) Merr. 14 Wattakaka volubilis (L.f.) Stapf.
15 quercetin standard
3.2.6.4 Detection of β-sitosterol in the stem and leaf extracts using
HPTLC Chromatogram was developed for all the 7 leaf and stem extracts
and the standard β-sitosterol on a single plate using the solvent system
Hexane: Di-ethylether: Acetic acid (8:20:1). Densitometric scanning
was done at λ max of 206 nm where it showed maximum absorption.
β-sitosterol showed a peak with Rf 0.25. In the chemoprofile of all 14
90
(7 leaf and 7 stem) samples, a peak with Rf 0.25 was observed which
showed the presence of β-sitosterol in the leaves and stems of
Asclepias curassavica L., Calotropis gigantea (L.)R.Br., Gymnema
sylvestre (Retz.)R.Br., Holostemma ada-kodien Schult., Pergularia
daemia (Forssk) Chiov., Tylophora indica (Burm.f.) Merr. and
Wattakaka volubilis (L.f.) Stapf. Results are presented in Fig. 3.11 (1-
7); Fig. 3.12 (1-7) and Fig. 3.13. The presence of β-sitosterol with an
Rf value 0.25 was confirmed by superimpossing HPTLC densitograms
obtained from all the extracts along with the standard pure compound
β-sitosterol. The superimposed graph is shown in Fig. 3.14.
Fig. 3.11 HPTLC fingerprint profile of methanolic leaf extracts
Asclepias curassavica L.
(Contd …… Fig. 3.11)
91
Calotropis gigantea (L.) R.Br.
Gymnema sylvestre (Retz.) R.Br.
(Contd …… Fig. 3.11)
92
Holostemma ada-kodien Schult.
Pergularia daemia (Forssk.) Chiov.
(Contd …… Fig. 3.11)
93
Tylophora indica (Burm.f.) Merr.
Wattakaka volubilis (L.f.) Stapf.
94
Fig. 3.12 HPTLC profile of methanolic stem extracts
Asclepias curassavica L.
Calotropis gigantea (L.) R.Br.
(Contd …… Fig. 3.12)
95
Gymnema sylvestre (Retz.) R.Br.
Holostemma ada-kodien Schult.
(Contd …… Fig. 3.12)
96
Pergularia daemia (Forssk.) Chiov.
Tylophora indica (Burm.f.) Merr.
(Contd …… Fig. 3.12)
97
Wattakaka volubilis (L.f.) Stapf.
Fig. 3.13 HPTLC chromatogram of β-sitosterol (standard)
98
Fig. 3.14 Overlapping chromatographic comparison of leaf and stem extracts and β-sitosterol standard
Stem extract
1 Asclepias curassavica L. 2 Calotropis gigantea (L.) R.Br. 3 Gymnema sylvestre (Retz.) R.Br. 4 Holostemma ada-kodien Schult. 5 Pergularia daemia (Forssk.) Chiov. 6 Tylophora indica (Burm.f.) Merr. 7 Wattakaka volubilis (L.f.) Stapf.
Leaf extract 8 Asclepias curassavica L. 9 Calotropis gigantea (L.) R.Br. 10 Gymnema sylvestre (Retz.) R.Br. 11 Holostemma ada-kodien Schult. 12 Pergularia daemia (Forssk.) Chiov. 13 Tylophora indica (Burm.f.) Merr. 14 Wattakaka volubilis (L.f.) Stapf.
15 β-sitosterol standard
3.2.6.5 Detection of lupeol in the stem and leaf extracts using HPTLC
The chromatogram was developed using all the 7 leaf and 7 stem
extracts and the standard pure compound lupeol on a single plate using
the solvent system Toluene: Ethyl acetate: Formic acid (50:30:4).
Densitometric scanning was done at λ max 230nm where it was showing
99
maximum absorption. Lupeol showed a peak with Rf 0.80 at λ max
230nm. In the chemopofile of the leaf extracts lupeol was found to be
present in all the plants except Asclepias curassavica L. In the stem
extracts also a peak with Rf 0.80 was seen in all the plants except
Asclepias curassavica L. The results are presented in Fig. 3.15 (1-7), Fig.
3.16 (1-7) and Fig. 3.17. Clear superimpossibility at the Rf of 0.80 in all
the 6 leaf and 6 stem extract’s chromatogram and the standard lupeol
confirmed its presence in Calotropis gigantea (L.) R.Br., Gymnema
sylvestre (Retz.) R.Br., Holostemma ada-kodien Schult., Pergularia
daemia (Forssk.) Chiov., Tylophora indica (Burm.f.) Merr. and
Wattakaka volubilis (L.f.) Stapf. leaf and stem. The superimpossed graph
is shown in Fig. 3.18.
Fig. 3.15 HPTLC profile of methanolic leaf extracts
Asclepias curassavica L.
(Contd …… Fig. 3.15)
100
Calotropis gigantea (L.) R.Br.
Gymnema sylvestre (Retz.) R.Br.
(Contd …… Fig. 3.15)
101
Holostemma ada-kodien Schult.
Pergularia daemia (Forssk.) Chiov.
(Contd …… Fig. 3.15)
102
Tylophora indica (Burm.f.) Merr.
Wattakaka volubilis (L.f.) Stapf.
103
Fig. 3.16 HPTLC profile of methanolic stem extracts
Asclepias curassavica L.
Calotropis gigantea (L.) R.Br.
(Contd …… Fig. 3.16)
104
Gymnema sylvestre (Retz.) R.Br.
Holostemma ada-kodien Schult.
(Contd …… Fig. 3.16)
105
Pergularia daemia (Forssk.) Chiov.
Tylophora indica (Burm.f.) Merr.
(Contd …… Fig. 3.16)
106
Wattakaka volubilis (L.f.) Stapf.
Fig. 3.17 HPTLC chromatogram of lupeol (standard)
107
Fig. 3.18 Overlapping chromatographic comparison of leaf and stem extracts and lupeol standard
Leaf extract
1 Asclepias curassavica L. 2 Calotropis gigantea (L.) R.Br. 3 Gymnema sylvestre (Retz.) R.Br. 4 Holostemma ada-kodien Schult. 5 Pergularia daemia (Forssk.) Chiov. 6 Tylophora indica (Burm.f.) Merr. 7 Wattakaka volubilis (L.f.) Stapf.
Stem extract
8 Asclepias curassavica L. 9 Calotropis gigantea (L.) R.Br. 10 Gymnema sylvestre (Retz.) R.Br. 11 Holostemma ada-kodien Schult. 12 Pergularia daemia (Forssk.) Chiov. 13 Tylophora indica (Burm.f.) Merr. 14 Wattakaka volubilis (L.f.) Stapf.
15 Lupeol standard
3.2.7 HPLC analysis of amino acids
High Performance Liquid Chromatography (HPLC) separation and
quantification of amino acids were done. The percentage of amino acids
and the amount in gm/16g-N2 were represented in Table 3.11.
108
Table 3.11 Amino acid composition
Test Amount of amino acid (g/16g-N2) Sl. No
Amino Acid A.C. C.G. G.S. H.A. P.D. T.I. W.V.
1 Aspartic acid 44.5 49.1 13.0 6.4 8.6 30.9 7.7
2 Threonine 4.1 6.1 6.0 2.7 3.6 3.0 2.9
3 Serine 9.9 8.9 5.7 2.6 3.7 5.9 3.1
4 Glutamic acid - - 13.6 7.3 10.6 - 8.3
5 Proline - - 6.4 - 3.3 - -
6 Glycine 3.2 3.5 3.2 1.8 2.7 2.4 2.4
7 Alanine 0.6 0.7 4.1 2.1 3.7 0.4 3.4
8 Cysteine - - - - - - -
9 Valine - - 7.4 3.7 5.1 - 4.3
10 Methionine - - - - - - -
11 Isoleucine 4.5 4.8 5.9 2.9 3.9 3.9 3.5
12 Leucine 9.3 10.3 10.3 5.0 8.0 7.5 7.1
13 Tyrosine 2.1 2.4 2.6 1.3 1.9 1.7 1.8
14 Phenylalanine 5.8 6.4 6.7 3.6 4.9 4.9 4.3
15 Histidine 3.0 3.6 4.8 2.4 2.6 2.9 2.6
16 Lysine - - - - - - -
17 Arginine - - - - - - -
18 Tryptophan 2.6 3.0 5.5 2.6 2.3 3.5 4.0
- Absent
A.C. Asclepias curassavica L. C.G. Calotropis gigantea (L.) R.Br. G.S. Gymnema sylvestre (Retz.) R.Br. H.A. Holostemma ada-kodien Schult. P.D. Pergularia daemia (Forssk.) Chiov. T.I. Tylophora indica (Burm.f.) Merr. W.V. Wattakaka volubilis (L.f.) Stapf.
Amino acids like aspartic acid, threonine, serine, glycine, alanine,
isoleucine, leucine, tyrosine, phenyl alanine, histidine and tryptophan
were present in all the seven plants. Similarly cysteine, methionine, lysine
and arganine were absent in all the seven plants. Glutamic acid was
present only in Gymnema sylvestre (Retz.) R.Br., Holostemma ada-
109
kodien Schult., Pergularia daemia (Forssk.) Chiov. and Wattakaka
volubilis (L.f.) Stapf. Proline was present only in Gymnema sylvestre
(Retz.) R.Br. and Pergularia daemia (Forssk.) Chiov. Valine was present
in all the plants except Asclepias curassavica L., Calotropis gigantea (L.)
R.Br. and Tylophora indica (Burm.f.) Merr. Out of the eighteen amino acids
tested among the seven plants, fourteen amino acids were present in
Gymnema sylvestre (Retz.) R.Br. and Pergularia daemia (Forssk.) Chiov.
Aspartic acid was seen in large quantity in Asclepias curassavica L.,
Calotropis gigantea (L.) R.Br. and Tylophora indica (Burm.f.) Merr.
Among the eight essential amino acids, threonine, isoleucine,
leucine, phenyl alanine and tryptophan were present in all the seven
plants in comparatively larger percentage. Two essential amino acids
methionine and lysine were altogether absent in all the seven plants.
Histidine and arganine are two amino acids essential for growing children
of which arganine was absent in all the plants and histidine was present in
comparatively larger quantities in all the plants (Table 3.11). The highest
percentage of essential amino acid was observed in Gymnema sylvestre
(Retz.) R.Br. followed by Calotropis gigantea (L.) R.Br. and Pergularia
daemia (Forssk.) Chiov. (Fig. 3.19). The highest percentage of non-
essential amino acid was observed in Calotropis gigantea (L.) R.Br.
followed by Asclepias curassavica L. and Gymnema sylvestre (Retz.)
R.Br. (Fig. 3.20).
110
Fig. 3.19 Essential amino acids in percentage
111
Fig. 3.20 Non- essential amino acids in percentage
112
3.2.8 Estimation of phytoconstituents
The total polyphenolic compounds and flavonoids were estimated
quantitatively in all the leaves of the seven plants under study and it was
found that the quantity varies agewise, seasonally and habitatwise.
Table 3.12 Comparison of total polyphenolic compounds in mature and young plants.
Plant name
Total polyphenolic content (%) mature plant Mean ± SD
Total polyphenolic content (%) young plant Mean ± SD
Asclepias curassavica L. 0.758 ± 0.03 0.730 ± 0.05
Calotropis gigantea (L.) R.Br. 0.13 ± 0.02 0.09 ± 0.03
Gymnema sylvestre (Retz.)R.Br. 0.931 ± 0.12 0.892 ± 0.15
Holostemma ada-kodien Schult. 0.9575 ± 0.08 0.926 ± 0.12
Pergularia daemia (Forssk.) Chiov. 1.524 ± 0.14 1.49 ± 0.13
Tylophora indica (Burm.f.) Merr. 1.03 ± 0.12 1.01 ± 0.09
Wattakaka volubilis (L.f.) Stapf. 0.44 ± 0.02 0.39 ± 0.04
Among the mature plants the highest total polyphenolic content was
recorded in Pergularia daemia (Forssk.) Chiov. (1.524 ± 0.14%)
followed by Tylophora indica (Burm.f.) Merr. (1.03 ± 0.12 %). The
lowest total polyphenolic content was found in Calotropis gigantea (L.)
R.Br. (0.13 ± 0.02 %) (Table 3.12; Fig 3.21). In the case of young plants
also the highest quantity was observed in Pergularia daemia (Forssk.)
Chiov. and the lowest quantity in Calotropis gigantea (L.) R.Br. But the
quantity of the content was lower in young plants when compared to the
mature plants.
113
Table 3.13 Comparison of total polyphenolic compounds during the dry and rainy season
Plant name
Total polyphenolic content (%) dry season Mean ± SD
Total polyphenolic content (%) rainy season Mean ± SD
Asclepias curassavica L. 0.758 ± 0.03 0.536 ± 0.04 Calotropis gigantea (L.) R.Br. 0.13 ± 0.02 0.06 ± 0.02 Gymnema sylvestre (Retz.)R.Br. 0.931 ± 0.12 0.743 ± 0.14 Holostemma ada-kodien Schult. 0.9575 ± 0.08 0.715 ± 0.01 Pergularia daemia (Forssk.) Chiov. 1.524 ± 0.14 1.03 ± 0.07 Tylophora indica (Burm.f.) Merr. 1.03 ± 0.12 0.83 ± 0.10 Wattakaka volubilis (L.f.) Stapf. 0.44 ± 0.02 0.21 ± 0.03
The highest quantity of polyphenol was observed in Pergularia
daemia (Forssk.) Chiov. and the lowest quantity in Calotropis gigantea
(L.) R.Br. both in the dry as well as in the rainy season. The gradation of
content variation was the same among the plants in both the seasons. But
the quantity of the content showed a marked reduction during the rainy
season (Table 3.13; Fig. 3.22)
Table 3.14 Comparison of total polyphenolic compounds in wild and cultivated plants.
Plant name
Total polyphenolic content (%) wild plant Mean ± SD
Total polyphenolic content (%)
cultivated Plant Mean ± SD
Asclepias curassavica L. 0.758 ± 0.03 0.612 ± 0.01 Calotropis gigantea (L.) R.Br. 0.13 ± 0.02 0.10 ± 0.05 Gymnema sylvestre (Retz.)R.Br. 0.931 ± 0.12 0.85 ± 0.16 Holostemma ada-kodien Schult. 0.9575 ± 0.08 0.903 ± 0.07 Pergularia daemia (Forssk.) Chiov. 1.524 ± 0.14 1.44 ± 0.08 Tylophora indica (Burm.f.) Merr. 1.03 ± 0.12 0.98 ± 0.08 Wattakaka volubilis (L.f.) Stapf. 0.44 ± 0.02 0.39 ± 0.14
114
The highest quantity of polyphenol was observed in Pergularia
daemia (Forssk.) Chiov. and the lowest quantity in Calotropis gigantea
(L.) R.Br. both in the wild as well as in the cultivated plants. The gradation
of content variation was the same among the plants in both the seasons.
But the quantity of the content showed a marked reduction in the cultivated
plants. (Table 3.14; Fig. 3.23)
Table 3.15 Comparison of total flavonoids in mature and young plants.
Plant name
Total flavonoid content (%)
Mature plant Mean ± SD
Total flavonoid content (%) Young plant Mean ± SD
Asclepias curassavica L. 2.73 ± 0.18 2.61 ± 0.09 Calotropis gigantea (L.) R.Br. 1.28 ± 0.06 0.93 ± 0.08 Gymnema sylvestre (Retz.) R.Br. 2.19 ± 0.13 2.08 ± 0.16 Holostemma ada-kodien Schult. 2.94 ± 0.16 2.78 ± 0.13 Pergularia daemia (Forssk.) Chiov. 4.41 ± 1.02 4.21 ± 0.92 Tylophora indica (Burm.f.) Merr. 3.21 ± 1.03 3.09 ± 0.96 Wattakaka volubilis (L.f.) Stapf. 2.42 ± 0.05 2.39 ± 0.06
Among the mature plants the highest flavonoid content was
recorded in Pergularia daemia (Forssk.) Chiov. (4.41 ± 1.02%) followed
by Tylophora indica (Burm.f.) Merr., Holostemma ada-kodien Schult.,
Asclepias curassavica L., Wattakaka volubilis (L.f.) Stapf., Gymnema
sylvestre (Retz.) R.Br. and the lowest flavonoid content was found in
Calotropis gigantea (L.) R.Br. (1.28 ± 0.06%) (Table 3.15; Fig.3.24). In
the case of young plants also the highest quantity was observed in
Pergularia daemia (Forssk.) Chiov. and the lowest quantity in Calotropis
gigantea (L.) R.Br., but the quantity of the content was lower in young
plants when compared to the mature plants.
115
Table 3.16 Comparison of total flavonoids in dry and rainy season.
Plant name
Total flavonoid content (%) dry season Mean ± SD
Total flavonoid content (%) rainy season Mean ± SD
Asclepias curassavica L. 2.73 ± 0.18 1.08 ± 0.18 Calotropis gigantea (L.) R.Br. 1.28 ± 0.06 0.92 ± 0.13 Gymnema sylvestre (Retz.) R.Br. 2.19 ± 0.13 1.84 ± 0.09 Holostemma ada-kodien Schult. 2.94 ± 0.16 1.83 ± 0.19 Pergularia daemia (Forssk.) Chiov. 4.41 ± 1.02 3.02 ± 0.94 Tylophora indica (Burm.f.) Merr. 3.21 ± 1.03 2.71 ± 1.01 Wattakaka volubilis (L.f.) Stapf. 2.42 ± 0.05 1.98 ± 0.05
The highest quantity of flavonoid was observed in Pergularia daemia
(Forssk.) Chiov. and the lowest quantity in Calotropis gigantea (L.) R.Br. both
in the plants collected during dry and rainy season. But the quantity of the
content showed a marked reduction in the plants collected during the rainy
season (Table 3.16; Fig. 3.25).
Table 3.17 Comparison of total flavonoids in wild and cultivated plants.
Plant name
Total flavonoid content (%) wild plant Mean ± SD
Total flavonoid content (%)
cultivated plant Mean ± SD
Asclepias curassavica L. 2.73 ± 0.18 2.21± 0.13 Calotropis gigantea (L.) R.Br. 1.28 ± 0.06 0.97 ± 0.02 Gymnema sylvestre (Retz.) R.Br. 2.19 ± 0.13 2.02 ± 0.05 Holostemma ada-kodien Schult. 2.94 ± 0.16 2.82 ± 0.09 Pergularia daemia (Forssk.) Chiov. 4.41 ± 1.02 3.98 ± 0.18 Tylophora indica (Burm.f.) Merr. 3.21 ± 1.03 2.98 ± 1.03 Wattakaka volubilis (L.f.) Stapf. 2.42 ± 0.05 2.08 ± 0.02
The highest quantity of flavonoid was observed in Pergularia
daemia (Forssk.) Chiov. and the lowest quantity in Calotropis gigantea
(L.) R.Br. both in the wild as well as in the cultivated plants. But the
quantity of the content showed a marked reduction in the cultivated plants
(Table 3.17; Fig. 3.26).
116
Fig. 3.21 Comparison of total polyphenolic compounds in mature and young plants.
Fig. 3.22 Comparison of total polyphenolic compounds in dry season and
rainy season.
117
Fig. 3.23 Comparison of total polyphenolic compounds in wild and cultivated lants.
Fig. 3.24 Comparison of total flavonoids in mature and young plants.
118
Fig. 3.25 Comparison of total flavonoids in dry and rainy season.
Fig. 3.26 Comparison of total flavonoids in wild and cultivated plants.
3.2.8.1 Statistical analysis
The range and mean along with standard deviation of total
polyphenol and flavonoid content were analysed seasonwise, agewise and
habitatwise. The values were subjected to statistical analysis using t test.
The P value was taken at 5% and 1% level of significance.
119
120
121
122
123
124
125
3.3 Biological activity
From the qualitative studies and HPTLC results, it was very clear
that secondary metabolites were more in the leaf extracts. So the
biological activities of only the leaf extracts were done.
3.3.1 Antibacterial activity
Table 3.24 Antibacterial activity of Asclepias curassavica L. leaf extract
Zone of inhibition (mm)
Culture Water extract
Methanol extract
Hydro alcoholic extract
Ethanolic extract Flavonoid Gentamycin
(+ve control)
Gram positive bacteria Staphylococcus aureus 11 ± 1.16 21 ± 2.01 Nil 14 ± 1.04 20 ± 1.02 21 ± 1.04 Bacillus subtilis Nil 16 ± 1.02 Nil 13 ± 1.04 Nil 26 ± 1.41 Gram negative bacteria Klebsiella pneumoniae Nil Nil Nil Nil 11 ± 1.01 20 ± 1.02 Salmonella typhymurium Nil Nil Nil 11 ± 1.01 Nil 20 ± 1.16
Escherichia coli Nil 12 ± 1.02 Nil 12 ± 1.02 12 ± 1.01 28 ± 1.81
The gram negative bacteria were weakly sensitive while the gram
positive bacteria were highly sensitive to Asclepias curassavica L. leaf
extract. The methanolic extract of the leaf and the flavonoids isolated
from the leaves were showing a zone of inhibition almost equal to that of
standard gentamycin (Table 3.24; Plate 3.3).
Table 3.25 Antibacterial activity of Calotropis gigantea (L.) R.Br. leaf extract
Zone of inhibition (mm)
Culture Water extract
Methanol extract
Hydro alcoholic extract
Ethanolic extract Flavonoid Gentamyci
(+ve control)
Gram positive bacteria Staphylococcus aureus Nil 19 ± 1.18 12 ± 1.16 15 ± 1.09 16 ± 1.81 21 ± 1.04 Bacillus subtilis Nil 14 ± 1.02 Nil 16 ± 1.08 12 ± 1.01 26 ± 1.41 Gram negative bacteria Klebsiella pneumoniae Nil Nil Nil Nil 11 ± 1.04 20 ± 1.02 Salmonella typhymurium Nil 16 ± 1.04 Nil Nil Nil 20 ± 1.16 Escherichia coli Nil Nil Nil 11 ± 1.02 Nil 28 ± 1.81
126
All the organisms were resistant to the water extract of Calotropis
gigantea (L.) R.Br. but sensitive to the methanol and ethanol extract and
the flavonoids of Calotropis gigantea (L.) R.Br. (Table 3.25; Plate 3.4).
Table 3.26 Antibacterial activity of Gymnema sylvestre (Retz.) R.Br. leaf extract
Zone of inhibition (mm)
Culture Water extract
Methanol extract
Hydro alcoholic extract
Ethanolic extract Flavonoid Gentamycin
(+ve control)
Gram positive bacteria
Staphylococcus aureus 14 ± 1.21 17 ± 1.32 Nil 14 ± 1.01 14 ± 1.83 21 ± 1.04
Bacillus subtilis Nil Nil 15 ± 1.21 12 ± 1.02 13 ± 1.72 26 ± 1.41
Gram negative bacteria
Klebsiella pneumoniae Nil Nil Nil Nil Nil 20 ± 1.02
Salmonella typhymurium Nil Nil Nil Nil Nil 20 ± 1.16
Escherichia coli Nil Nil Nil Nil Nil 28 ± 1.81
The gram negative bacteria were resistant to the different extracts.
The gram positive bacteria were sensitive to the extracts but the zone of
inhibition was less when compared to standard gentamycin except for the
methanolic extract. Water extract was having some activity against
Staphylococcus aureus (Table 3.26; Plate 3.5).
Table 3.27 Antibacterial activity of Holostemma ada-kodien Schult. leaf extract
Zone of inhibition (mm)
Culture Water extract
Methanol extract
Hydro alcoholic extract
Ethanolic extract Flavonoid Gentamycin
(+ve control)
Gram positive bacteria
Staphylococcus aureus Nil 16 ± 1.31 15 ± 1.61 18 ± 1.82 20 ± 1.27 21 ± 1.04
Bacillus subtilis Nil 14 ± 1.01 12 ± 1.52 14 ± 1.61 13 ± 1.01 26 ± 1.41
Gram negative bacteria
Klebsiella pneumoniae Nil Nil Nil Nil 14 ± 1.31 20 ± 1.02
Salmonella typhymurium Nil 23 ± 1.81 13 ± 1.02 Nil 10 ± 1.01 20 ± 1.16
Escherichia coli Nil Nil 11 ± 1.01 Nil Nil 28 ± 1.81
127
The methanolic, ethanolic and hydroalcoholic extracts showed
antibacterial activity against gram positive bacteria Staphylococcus aureus
(MTCC 3160) and Bacillus subtilis (MTCC 3053). The methanolic extract
was showing greater activity than the antibiotic Gentamycin against the
gram negative bacteria Salmonella typhymurium (MTCC 98) (Table 3.27;
Plate 3.6). The secondary metabolites present in the plant could be
responsible for some of the observed antimicrobial activity. All the tested
organisms were resistant to the water extract. No antibacterial activity
was observed in negative controls.
Table 3.28 Antibacterial activity of Pergularia daemia (Forssk.) Chiov. leaf extract Zone of inhibition (mm)
Culture Water extract
Methanol extract
Hydro alcoholic extract
Ethanolic extract Flavonoid Gentamycin
(+ve control)
Gram positive bacteria Staphylococcus aureus Nil 21 ± 1.62 12 ± 1.23 15 ± 1.21 16 ± 1.46 21 ± 1.04 Bacillus subtilis Nil 16 ± 1.01 Nil 16 ± 1.32 12 ± 1.01 26 ± 1.41 Gram negative bacteria Klebsiella pneumoniae Nil 11 ± 1.10 Nil 11 ± 1.02 12 ± 1.02 20 ± 1.02 Salmonella typhymurium Nil Nil Nil Nil Nil 20 ± 1.16 Escherichia coli Nil Nil Nil Nil Nil 28 ± 1.81
All the tested bacterial strains were resistant to the water extract and
sensitive to all other extracts. The zone of inhibition was equivalent to
that of the standard gentamycin drug for Salmonella typhymurium
(MTCC 98) in the methanolic extract (Table 3.28; Plate 3.7).
Table 3.29 Antibacterial activity of Tylophora indica (Burm.f.) Merr. leaf extract Zone of inhibition (mm)
Culture Water extract
Methanol extract
Hydro alcoholic extract
Ethanolic extract Flavonoid Gentamycin
(+ve control)
Gram positive bacteria Staphylococcus aureus Nil 17 ± 1.41 18 ± 1.23 21 ± 1.31 18 ± 1.43 21 ± 1.04 Bacillus subtilis Nil 15 ± 1.07 17 ± 1.02 19 ± 1.02 14 ± 1.05 26 ± 1.41
Gram negative bacteria Klebsiella pneumoniae Nil 11 ± 1.04 Nil Nil 13 ± 1.01 20 ± 1.02 Salmonella typhymurium Nil Nil Nil Nil Nil 20 ± 1.16 Escherichia coli Nil 12 ± 1.03 Nil 12 ± 1.01 Nil 28 ± 1.81
128
Methanolic, ethanolic and hydro alcoholic extracts showed
antibacterial activity against Staphylococcus aureus (MTCC 3160),
Bacillus subtilis (MTCC 3053) - gram positive bacteria and Klebsiella
pneumonia (MTCC 3384) and Escherichia coli (MTCC 727) - gram
negative bacteria (Table 3.29; Plate 3.8)). Salmonella typhymurium
(MTCC 98) was resistant to the leaf extract.
Table 3.30 Antibacterial activity of Wattakaka volubilis (L.f.) Stapf. leaf extract
Zone of inhibition (mm)
Culture Water extract
Methanol extract
Hydro alcoholic extract
Ethanolic extract Flavonoid Gentamycin
(+ve control)
Gram positive bacteria Staphylococcus aureus Nil Nil 16 ± 1.21 11 ± 1.10 14 ± 1.01 21 ± 1.04
Bacillus subtilis Nil Nil Nil 15 ± 1.01 12 ± 1.02 26 ± 1.41 Gram negative bacteria Klebsiella pneumoniae Nil Nil Nil Nil Nil 20 ± 1.02
Salmonella typhymurium Nil Nil Nil Nil Nil 20 ± 1.16
Escherichia coli Nil Nil Nil Nil Nil 28 ± 1.81
All the gram negative bacteria were resistant to all the extracts.
Gram positive bacteria were weakly sensitive to the hydro alcoholic,
ethanolic extracts and flavonoids. Hydro alcoholic extract was showing a
zone of inhibition of 16 mm in Staphylococcus aureus (MTCC 3160)
(Table 3.30, Plate 3.9).
129
Plate 3.3 Screening for antibacterial activity as Asclepias curassavica L. leaf extract
A and B - Bacillus subtilis E and F - Staphylococcus aureus
C and D - Klebsiella pneumoniae G and H - Salmonella typhymurium
I and J - Escherichia coli HA – Hydroalcoholic extract, EE – Ethanolic extract, WE-Water extract, ME-Methanolic extract, E-Pure ethanol (negativ e control), M-Pure methanol (negative control), F-Flavonoid, G-Gentamycin.
130
Plate 3.4 Screening for antibacterial activity of Calotropis gigantea (L.) R.Br. leaf extract
A and B - Bacillus subtilis E and F - Klebsiel la pneumoniae
C and D - Staphylococcus aureus G and H - Salmonella typhymurium
I and J - Escherichia coli HA – Hydroalcoholic extract, EE – Ethanolic extract, WE-Water extract, ME-Methanolic extract, E-Pure ethanol (negative control), M-Pure methanol (negative control), F-Flavonoid , G-Gentamycin .
131
Plate 3.5 Screening for antibacterial activity of Gymnema sylvestre (Retz.) R.Br. leaf extract
A and B - Bacillus subtilis E and F - Staphylococcus aureus
C and D - Klebsiella pneumoniae G and H - Salmonella typhymurium
I and J - Escherichia col i
HA – Hydroalcoholic ex tract, EE – Ethanolic extract, WE-Water extract, ME-Methanolic extract, E-Pure ethanol (negative control) , M-Pure methanol (negative control), F-Flavonoid, G-Gentamycin .
132
Plate 3.6 Screening for antibacterial activity of Holostemma ada-kodien Schult. leaf extract
A and B - Bacillus subtilis E and F - Staphylococcus aureus
C and D - Klebsiella pneumoniae G and H - Salmonella typhymurium
I and J - Escherichia coli
HA – Hydroalcoholic extract, EE – Ethanolic extract, WE-Water extract, ME-Methanolic extract, E-Pure ethanol (negative control), M-Pure methanol (negative control), F-Flavonoid, G-Gentamycin .
133
Plate 3.7 Screening for antibacterial activity of Pergularia daemia (Forssk.) Chiov. leaf extract
A and B - Klebsiella pneumoniae E and F - Bacillus subtilis
C and D - Staphylococcus aureus G and H - Salmonella typhymurium
I and J - Escherichia coli
HA – Hydroalcoholic extract, EE – Ethanolic extract, WE-Water extract, ME-Methanolic extract, E-Pure ethanol (negative control) , M-Pure methanol (negative control), F-Flavonoid, G-Gentamycin.
134
Plate 3.8 Screening for antibacterial activity of Tylophora indica (Burm.f.) Merr. leaf extract
A and B - Bacillus subtilis E and F - Staphylococcus aureus
C and D - Klebsiella pneumoniae G and H - Escherichia coli
I and J - Salmonella typhymurium
HA – Hydroalcoholic extract, EE – Ethanolic extract, WE-Water extract, ME-Methanolic extract, E-Pure ethanol (negative control), M-Pure methanol (negative control), F-Flavonoid, G-Gentamycin.
135
Plate 3.9 Screening for antibacterial activity of Wattakaka volubilis (L.f.) Stapf. leaf extract
A and B - Bacillus subtilis E and F - Klebsiel la pneumoniae
C and D - Staphylococcus aureus G and H - Salmonella typhymurium
I and J - Escherichia coli
HA – Hydroalcoholic extract, EE – Ethanolic extract, WE-Water extract, ME-Methanolic extract, E-Pure ethanol (negative control), M-Pure methanol (negative control), F-Flavonoid, G-Gentamycin .
136
Table 3.31 Minimum inhibitory concentrations (MIC) of flavonoids on Staphylococcus aureus
No: Name of the plant MIC mg/ml
1. Asclepias curassavica L. 20 mg/ml
2. Calotropis gigantea (L.) R.Br. 30 mg/ml
3. Gymnema sylvestre (Retz.) R.Br. 80 mg/ml
4. Holostemma ada-kodien Schult. 30 mg/ml
5. Pergularia daemia (Forssk.) Chiov. 60 mg/ml
6. Tylophora indica (Burm.f.) Merr. 20 mg/ml
7. Wattakaka volubilis (L.f.) Stapf. 100 mg/ml
3.3.2 Antioxidant activity
Highest DPPH free radical scavenging activity was found in
Pergularia daemia (Forssk.) Chiov. followed by Holostemma ada-kodien
Schult., Asclepias curassavica L., Wattakaka volubilis (L.f.) Stapf.,
Tylophora indica (Burm.f.) Merr., Gymnema sylvestre (Retz.) R.Br. and
the lowest activity was found in Calotropis gigantea (L.) R.Br.
Table 3.32 Anti oxidant activity of Asclepias curassavica L.
Concentration (µg/ml)
Absorbance at 516nm
Log conc. Percentage inhibition %
Control .832 - - 132.5 0.237 2.4652 28.065 156.8 0.324 2.5815 40.168 182.2 0.488 2.7542 58.202
Fig. 3.27 Anti oxidant activity of Asclepias curassavica L.
137
Table 3.33 Anti oxidant activity of Calotropis gigantea (L.) R.Br.
Concentration(µg/ml)
Absorbance at 516nm
Log conc. Percentage inhibition %
Control .832 - - 291.9 0.609 2.4652 27.121 381.5 0.523 2.5815 38.128 567.8 0.481 2.7542 57.630
Fig. 3.28 Anti oxidant activity of Calotropis gigantea (L.) R.Br.
Table 3.34 Anti oxidant activity of Gymnema sylvestre (Retz.) R.Br.
Concentration ( µg/ml)
Absorbance at 516nm
Log conc. Percentage inhibition %
Control .832 - - 154.025 0.517 2.1875 37.8605 308.05 .271 2.4886 67.4278 770.125 .146 2.8866 82.4519
Fig. 3.29 Anti oxidant activity of Gymnema sylvestre (Retz.) R.Br.
Table 3.35 Anti oxidant activity of Holostemma ada-kodien Schult.
Concentration(µg/ml)
Absorbance at 516nm
Log conc. Percentage inhibition %
Control .832 - - 112.8 0.580 2.1875 31.046 120.6 0.547 2.4886 35.019 160.2 0.422 2.8866 50.234
Fig. 3.30 Anti oxidant activity of Holostemma ada-kodien Schult.
138
Table 3.36 Anti oxidant activity of Pergularia daemia (Forssk.) Chiov..
Concentration(µg/ml)
Absorbance at 516nm
Log conc. Percentage inhibition %
Control .839 - - 137.7 0.457 2.1389 46.331 142.3 0.441 2.1532 47.887 159.0 0.387 2.2014 54.307
Fig. 3.31 Anti oxidant activity of Pergularia daemia (Forssk.) Chiov.
Table 3.37 Anti oxidant activity of Tylophora indica (Burm.f.) Merr.
Concentration(µg/ml)
Absorbance at 516nm
Log conc. Percentage inhibition %
Control .839 - - 144.1 0.369 2.1587 43.965 175.1 .0.413 2.2433 49.136 208.1 0.445 2.3183 53.002
Fig. 3.32 Anti oxidant activity of Tylophora indica (Burm.f.) Merr.
Table 3.38 Anti oxidant activity of Wattakaka volubilis (L.f.) Stapf.
Concentration(µg/ml)
Absorbance at 516nm
Log conc. Percentage inhibition %
Control .832 - - 131.3 0.258 2.4652 30.224 159.7 0.349 2.5815 41.255 195.5 0.476 2.7542 56.836
Fig. 3.33 Anti oxidant activity of Wattakaka volubilis (L.f.) Stapf.
139
Fig. 3.34 Antioxidant activity of the seven plants under study
A.C. Asclepias curassavica L. C.G. Calotropis gigantea (L.) R.Br. G.S. Gymnema sylvestre (Retz.) R.Br. H.A. Holostemma ada-kodien Schult. P.D. Pergularia daemia (Forssk.) Chiov. T.I. Tylophora indica (Burm.f.) Merr. W.V. Wattakaka volubilis (L.f.) Stapf.
Table 3.39 Antioxidant activity of the seven plants under study
Plant name IC50 value (µg/ml )
Asclepias curassavica L. 170.600
Calotropis gigantea (L.) R.Br. 486.400
Gymnema sylvestre (Retz.) R.Br. 199.526
Holostemma ada-kodien Schult. 158.490
Pergularia daemia (Forssk.) Chiov. 147.700
Tylophora indica (Burm.f.) Merr. 181.970
Wattakaka volubilis (L.f.) Stapf. 179.640
Table 3.39 shows the comparative data of DPPH free radical
scavenging activity, as determined by the IC50 values of the different leaf
extracts. IC50 value is inversely related to the activity (Fig 3.34).
140
The highest DPPH free radical scavenging activity was observed in
Pergularia daemia (Forssk.) Chiov. followed by Holostemma ada-kodien
Schult., Asclepias curassavica L., Wattakaka volubilis (L.f.) Stapf.,
Tylophora indica (Burm.f.) Merr. and Gymnema sylvestre (Retz.) R.Br..
The lowest activity was found in Calotropis gigantea (L.) R.Br. (Table
3.39; Fig. 3.34).
3.3.2.1 Statistical analysis
The relationship between the phytochemical content and the anti
oxidant activity was analysed using the statistical tool, correlation
coefficient.
Fig. 3.35 Correlation coefficient (R2) of antioxidant capacity and total polyphenol content
141
Table 3.40 Model summary of correlation between antioxidant capacity and total polyphenol content
Model Summary b
Change Statistics
Model R R
Square
Adjusted R
Square
Std. Eoor of the
Estimate R
Square Change
F Change df 1 df2 Sig. F
Change
1 .810a .656 .587 .0989233 .656 9.524 1 5 .027 a. Predictors: ( Constant ), TRIND b. Dependent Variable: REDEP
Table 3.41 ANOVA of antioxidant capacity and total polyphenol content ANOVAb
Model Sum of Squares df Mean
Square F Sig.
1 Regression Residual
Total
.093
.049
.142
1 5 6
.093
.010 9.524 .027a
Table 3.42 Coefficients of independent variables of the regression analysis done between antioxidant capacity and total polyphenol content
Coefficentsa
Unstandardized Coefficients
Standardized Coefficients
95% Confidence Interval for B
Model B Std.
Error Beta t Sig
Lower Boound
Upper Bound
1 (Constant ) TRIND
.158
.398 .126 .129
.810
1.249 3.086
.267
.027 -.167 .067
.482
.730
a. Dependent Variable: REDEP
Fig. 3.35 shows the relationship between antioxidant activity and
total polyphenol content. In order to make a normal distribution the
square root of the variables on the X axis and the reciprocal percentage of
the variables on the Y axis were taken. The transformed variables give a
linear relationship with correlation coefficient (R2) value 0.6557 which
shows that these two variables were correlated.
142
Fig. 3.36 Correlation coefficient (R2) of antioxidant capacity and flavonoid content
Table 3.43 Model summary of correlation between antioxidant capacity
and flavonoid content
Model Summary
Change Statistics
Model R R
Square
Adjusted R
Square
Std. Eoor of the
Estimate R
Square Change
F Change df 1 df2 Sig. F
Change
1 .913a .834 .800 .0687624 .834 25.060 1 5 .004
a. Predictors ( Constant ), TRIND
Table 3.44 ANOVA of antioxidant capacity and flavonoid content
ANOVAb
Model Sum of Squares df Mean
Square F Sig.
1 Regression Residual
Total
.118
.024
.142
1 5 6
.118
.005 25.060 .004a
a. Predictos: (Constant), TRIND b. Dependent Variable: REDEP
143
Table 3.45 Coefficients of independent variables of the regression analysis done between antioxidant capacity and total polyphenol content
Coefficentsa
Unstandardized Coefficients
Standardized Coefficients Model
B Std. Error Beta t Sig
1 (Constant ) TRIND
-.223 .439
.153
.088 .913
-1.461 5.006
.204
.004
a. Dependent Variable: REDEP
Fig. 3.36 shows the relationship between antioxidant activity and
flavonoid content. In order to make a normal distribution the square root
of the variables on the X axis and the reciprocal percentage of the
variables on the Y axis were taken. Correlation coefficient (R2) value
0.8337 shows that these two variables are highly correlated.
3.4 Cluster analysis
Cluster analysis is a statistical procedure for the grouping of similar
objects using multivariate data about the different objects. The graphical
representation of clustering is called dendrogram. In the present
investigation, the seven plants species were grouped based on the
distribution of different quantities of amino acids, phenols and flavonoids
among them.
Dendrogram was prepared for the distribution. Here hierarchical
clustering was done. The clusters were themselves grouped into
bigger clusters. The process was repeated at different levels to form a
tree of clusters until all the plants combined together to form a single
group.
144
3.4.1 Clustering of the plants based on amino acid distribution
Table 3.46 Agglomeration schedule for cluster analysis of amino acid
Agglomeration Schedule
Cluster Combined Stage Cluster First Appears Stage
Cluster 1 Cluster 2 Coefficients
Cluster 1 Cluster 2
Next Stage
1 4 7 6.450 0 0 3 2 1 2 20.610 0 0 5 3 4 5 41.473 1 0 4 4 3 4 150.193 0 3 6 5 1 6 335.979 2 0 6 6 1 3 2433.569 5 4 0
Fig. 3.37 Dendrogram based on the distribution of amino acids
145
Table 3.47 ANOVA of amino acid variation among the plants ANOVA
Sum of Squares df Mean
Square F Sig.
Between Groups 1819.081 1 1819.081 44.649 .001 Within Groups 203.707 5 40.741
A1
Total 2022.789 6 Between Groups .617 1 .617 .261 .631 Within Groups 11.840 5 2.368
A2
Total 12.457 6 Between Groups 34.074 1 34.074 11.986 .018 Within Groups 14.214 5 2.843
A3
Total 48.289 6 Between Groups 169.719 1 169.719 36.126 .002 Within Groups 23.490 5 4.698
A4
Total 193.209 6 Between Groups 10.081 1 10.081 1.779 .240 Within Groups 28.328 5 5.666
A5
Total 38.409 6 Between Groups .443 1 .443 1.323 .302 Within Groups 1.674 5 .335
A6
Total 2.117 6 Between Groups 13.043 1 13.043 28.426 .003 Within Groups 2.294 5 .459
A7
Total 15.337 6 Between Groups 45.027 1 45.027 28.543 .003 Within Groups 7.888 5 1.578
A9
Total 52.914 6 Between Groups .210 1 .210 .191 .680 Within Groups 5.490 5 1.098
A11
Total 5.700 6 Between Groups 3.522 1 3.522 .953 .374 Within Groups 18.487 5 3.697
A12
Total 22.009 6 Between Groups .048 1 .048 .215 .662 Within Groups 1.107 5 .221
A13
Total 1.154 6 Between Groups 1.167 1 1.167 .908 .384 Within Groups 6.428 5 1.286
A14
Total 7.594 6 Between Groups .008 1 .008 .009 .928 Within Groups 4.167 5 .833
A15
Total 4.174 6 Between Groups .550 1 .550 .401 .554 Within Groups 6.867 5 1.373
A18
Total 7.417 6
146
Holostemma ada-kodien Schult. and Pergularia daemia (Forssk.)
Chiov. are very close based on the amino acids distribution. Similarly
Asclepias curassavica L. and Calotropis gigantea (L.) R.Br. stand closer.
Wattakaka volubilis (L.f.) Stapf. and Gymnema sylvestre (Retz.) R.Br. form
the next closest members. Tylophora indica (Burm.f.) Merr. stand closer to
the group containing Asclepias curassavica L. and Calotropis gigantea (L.)
R.Br. The group containing Holostemma ada-kodien Schult. and Pergularia
daemia (Forssk.) Chiov. is much closer to the group containing Wattakaka
volubilis (L.f.) Stapf. and Gymnema sylvestre (Retz.) R.Br. and this group
stands distinct from the other group of three plant members namely Asclepias
curassavica L., Calotropis gigantea (L.)R.Br. and Tylophora indica (Burm.f.)
Merr. As shown in the dendrogram, hierarchical clustering divides the seven
plants into two clusters. Cluster one containing Holostemma ada-kodien
Schult., Wattakaka volubilis (L.f.) Stapf., Pergularia daemia (Forssk.) Chiov.
and Gymnema sylvestre (Retz.) R.Br. Cluster two contains Asclepias
curassavica L., Calotropis gigantea (L.) R.Br. and Tylophora indica
(Burm.f.) Merr. The length of the horizontal lines connecting the plants
represents the percentage of dissimilarity among the plants. As the length
decreases the plants are more closer. The long horizontal line separating the
two clusters clearly shows the dissimilarity between the clusters (Fig. 3.37).
3.4.2 Clustering of the plants based on the distribution of phenols Table 3.48 Agglomeration schedule for cluster analysis of phenol
Agglomeration Schedule Cluster Combined Stage Cluster First Appears Stage
Cluster 1 Cluster 2 Coefficients
Cluster 1 Cluster 2 Next Stage
1 1 7 .000 0 0 6 2 3 4 4.000 0 0 4 3 2 6 11.000 0 0 5 4 3 5 19.000 2 0 5 5 2 3 30.400 3 4 6 6 1 2 48.571 1 5 0
147
Fig. 3.38 Dendrogram based on the distribution of phenols
The dendrogram in Fig. 3.38 exhibits the grouping of the seven
plant species under study with respect to phenols. Asclepias curassavica
L. and Wattakaka volubilis (L.f.) Stapf. stand closer and form the first
cluster. Gymnema sylvestre (Retz.) R.Br. and Holostemma ada-kodien
Schult. are more similar and together with Pergularia daemia (Forssk.)
Chiov. form the second cluster. Calotropis gigantea (L.) R.Br. and
Tylophora indica (Burm.f.) Merr. form cluster three.
3.4.3 Clustering of the plants based on the distribution of flavonoids
Table 3.49 Agglomeration schedule for cluster analysis of flavonoids
Agglomeration Schedule
Cluster Combined Stage Cluster First Appears Stage
Cluster 1 Cluster 2
Coefficients
Cluster 1 Cluster 2 Next Stage
1 2 7 4.500 0 0 3 2 3 6 9.500 0 0 5 3 1 2 15.000 0 1 6 4 4 5 22.000 0 0 5 5 3 4 30.000 2 4 6 6 1 3 38.286 3 5 0
148
Fig. 3.39 Dendrogram based on the distribution of flavonoids
In the distribution of flavonoids, Calotropis gigantea (L.) R.Br. and
Wattakaka volubilis (L.f.) Stapf. are the closest and together with
Asclepias curassavica L. form cluster one. Gymnema sylvestre (Retz.)
R.Br. and Tylophora indica (Burm.f.) Merr. are closer and form the
second cluster. Holostemma ada-kodien Schult. and Pergularia daemia
(Forssk.) Chiov. show similarity and form cluster three (Fig. 3.39).
3.5 Study of pollinial morphology
Pollinia has a central corpusculum and a pair of caudicles by which
the pollinial sacs are attached to the corpusculum. The size, shape and the
nature of pollinial sac, its position, structure of caudicle are the important
features for the analysis of phylogenetic study.
149
Table 3.50 Qualitative characters of pollinia
Name of the plant
Stature of
pollinia
Caudicle attachment
to the pollinia
Shape of pollinial
sac
Surface sculpturing of pollinial
sac
Nature of corpusculam
Nature of
caudicle
Asclepias curassavica L.
pendulous apical oval irregular concavities
simple triangular
long
Calotropis gigantea ( L.) R.Br.
pendulous apical oval irregular concavities
complex elongated
long
Gymnema sylvestre (Retz.) R.Br.
erect basal globular convoluted complex elongated
medium stout
Holostemma ada-kodien Schult.
pendulous apical elongated irregular concavities
complex elongated
long
Pergularia daemia ( Forssk.) Chiov.
pendulous apical oval convoluted simple triangular
short
Tylophora indica (Burm.f.)Merr.
horizontal apical globular convoluted simple triangular
medium stout
Wattakaka volubilis (L.f.) Stapf.
erect basal oval irregular concavities
complex elongated
short
Pollinia are found to be pendulous in Asclepias curassavica L.,
Calotropis gigantea (L.) R.Br., Holostemma ada-kodien Schult., and
Pergularia daemia (Forssk.) Chiov. They are horizontal in Tylophora indica
(Burm.f.) Merr. erect in Wattakaka volubilis (L.f.) Stapf. and suberect in
Gymnema sylvestre (Retz.) R.Br. Caudicle is attached to the apex in all the
species except Gymnema sylvestre (Retz.) R.Br. and Wattakaka volubilis
(L.f.) Stapf. Pollinial sac is oval in Asclepias curassavica L., Calotropis
gigantea (L.) R.Br., Pergularia daemia (Forssk.) Chiov. and Wattakaka
volubilis (L.f.) Stapf., elongated in Holostemma ada-kodien Schult., globular
in Tylophora indica (Burm.f.) Merr. and subglobular in Gymnema sylvestre
(Retz.) R.Br. Pollinial sac sculpturing is similar in Asclepias curassavica L.,
Calotropis gigantea (L.) R.Br., Holostemma ada-kodien Schult. and
150
Wattakaka volubilis (L.f.) Stapf. with irregular concavities, some markings
corresponding to the pollen grains inside can be seen and convoluted or
having some infoldings in Gymnema sylvestre (Retz.) R.Br., Pergularia
daemia (Forssk.) Chiov. and Tylophora indica (Burm.f.) Merr. Corpusculum
is with a simple structure in Asclepias curassavica L., Pergularia daemia
(Forssk.) Chiov. and Tylophora indica (Burm.f.) Merr., but it is complex in
Calotropis gigantea (L.) R.Br., Gymnema sylvestre (Retz.) R.Br.,
Holostemma ada-kodien Schult. and Wattakaka volubilis (L.f.) Stapf.
Caudicle is elongated and slender in Asclepias curassavica L., Calotropis
gigantea (L.) R.Br., Holostemma ada-kodien Schult., very short in
Pergularia daemia (Forssk.) Chiov. and medium sized stout in Gymnema
sylvestre (Retz.) R.Br. and Tylophora indica (Burm.f.) Merr.
Table 3.51 Quatitative characters of pollinia
Pollinial sac Corpusculam Caudicle Name of the
plant Length mm
Breadth mm
Length mm
Breadth mm
Length mm
Breadth mm
Ratio of length of
pollinia by length of caudicle
Asclepias curassavica L.
2.056 ± 0.05
0.796 ±0.02
0.889 ± 0.03
0.527 ± 0.02
1.075 ± 0.02
0.186 ± 0.02 1.913
Calotropis gigantea ( L.) R.Br.
2.976 ± 0.06
1.426 ± 0.05
1.302 ± 0.06
0.517 ± 0.03
0.982 ± 0.03
0.114 ± 0.01 3.031
Gymnema sylvestre (Retz.) R.Br.
0.069 ± 0.01
0.026 ± 0.01
0.039 ± 0.01
0.013 ± 0.01
0.026 ± 0.01
0.013 ± 0.01 2.650
Holostemma ada-kodien Schult.
0.714 ± 0.02
0.117 ± 0.02
0.229 ± 0.02
0.065 ± 0.01
0.294 ± 0.03
0.051 ± 0.01 2.429
Pergularia daemia (Forssk.) Chiov.
0.569 ± 0.03
0.317 ± 0.02
0.229 ± 0.02
0.145 ± 0.01
0.140 ± 0.01
0.028 ± 0.01 4.064
Tylophora indica (Burm.f.)Merr.
0.182 ± 0.01
0.147 ± 0.03
0.113 ± 0.02
0.065 ± 0.01
0.069 ± 0.02
0.026 ± 0.01 2.640
Wattakaka volubilis ( L.f.) Stapf.
0.407 ± 0.03
0.121 ± 0.02
0.069 ± 0.01
0.030 ± 0.02
0.186 ± 0.03
0.043 ± 0.02 5.900
151
It was found that within the seven members, the largest pollinial sac
was found in Calotropis gigantea (L.) R.Br. (Table 3.51; Plate 3.11) and
the smallest in Gymnema sylvestre (Retz.) R.Br. (Table 3.51; Plate 3.12).
The largest corpusculum is also seen in Calotropis gigantea (L.) R.Br.
(Table 3.51; Plate 3.11) and the smallest in Gymnema sylvestre (Retz.)
R.Br. (Table 3.51; Plate 3.12). Asclepias curassavica L. possesses the
longest caudicle (Table 3.51; Plate 3.10) while the shortest is seen in
Gymnema sylvestre (Retz.) R.Br. (Table 3.51; Plate 3.12).
Pollinial wall is smooth without any ornamentation. The impressions
of enclosed pollen grains are visible through the translucent wall.
Asclepias curassavica L. (Plate 3.10), Calotropis gigantea (L.) R.Br.
(Plate 3.11) and Holostemma ada-kodien Schult. (Plate 3.13) are having
elongated slender caudicle oriented in vertical position. Pergularia
daemia (Forssk.) Chiov. is having short caudicle (Plate 3.14) kept in
vertical position. Gymnema sylvestre (Retz.) R.Br. (Plate 3.12),
Tylophora indica (Burm.f.)Merr. (Plate 3.15) and Wattakaka volubilis
(L.f.) Stapf. (Plate 3.16) are having caudicles oriented in horizontal
position. In Gymnema sylvestre (Retz.) R.Br. (Plate 3.12) and
Wattakaka volubilis (L.f.) Stapf. (Plate 3.16) the attachment of caudicle
to pollinia is basal keeping the pollinia erect. In all others the
attachment is basal. The surface of the pollinial sac is highly convoluted
or having some infoldings in Gymnema sylvestre (Retz.) R.Br., (Plate
3.12), Pergularia daemia (Forssk.) Chiov. (Plate 3.16) and Tylophora
indica (Burm.f.)Merr. (Plate 3.15). In Asclepias curassavica L. (Plate
3.10), Calotropis gigantea (L.) R.Br. (Plate 3.11) and Holostemma ada-
kodien Schult. (Plate 3.13) instead of infoldings some markings
corresponding to the pollen grains inside can be seen.
152
Plate 3.10 Scanning electron photomicrographs of Asclepias curassavica L.
A - Pollinia entire view, B - Pollinial sac, C - Caudicle, D – corpusculum
Plate 3.11 Scanning electron photomicrographs of Calotropis gigantea ( L.) R.Br.
A - Pollinia entire view, B - Pollinial sac, C - Caudicle, D – corpusculum
153
Plate 3.12 Scanning electron photomicrographs of Gymnema sylvestre (Retz.) R.Br.
A - Pollinia entire view, B - Pollinial sac, C - Caudicle, D – corpusculum
Plate 3.13 Scanning electron photomicrographs of Holostemma ada-kodien Schult.
A - Pollinia entire view, B - Pollinial sac, C - Caudicle, D – corpusculum
154
Plate 3.14 Scanning electron photomicrographs of Pergularia daemia (Forssk.) Chiov.
A - Pollinia entire view, B - Pollinial sac, C - Caudicle, D – corpusculum
Plate 3.15 Scanning electron photomicrographs of Tylophora indica (Burm.f.) Merr.
A - Pollinia entire view, B - Pollinial sac, C - Caudicle, D – corpusculum
155
Plate 3.16 Scanning electron photomicrographs of Wattakaka volubilis (L.f.) Stapf.
A - Pollinia entire view, B - Pollinial sac, C - Caudicle, D – corpusculum
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