RESULTS - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/25924/16/16_chapter3.pdf · and Kakatundi in Hindi. It is distributed throughout Kerala. Erect herb. Leaves lanceolate,

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

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(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


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


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.


69

Pergularia daemia (Forssk.) Chiov.

Tylophora indica (Burm.f.) Merr.


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


72

Fig. 3.6 HPTLC profile of phenols in the stem extracts




73




74




75


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


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




79




80




81


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


83

Fig. 3.8 HPTLC profile of flavonoids in the stem extracts




84




85




86


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



91




92




93



94

Fig. 3.12 HPTLC profile of methanolic stem extracts




95




96




97


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



100




101




102



103

Fig. 3.16 HPTLC profile of methanolic stem extracts




104




105




106


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


Stem extract


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


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


(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


(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



Methanol 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



Methanol extract



(+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



Methanol extract



(+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)


Methanol extract



(+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)


Methanol extract



(+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



Methanol extract



(+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



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



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



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


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



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


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


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.


Absorbance at 516nm


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..


Absorbance at 516nm


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.


Absorbance at 516nm


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.


Absorbance at 516nm


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


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


A4


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


A9


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


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


pendulous apical oval irregular concavities

simple triangular

long

Calotropis gigantea ( L.) R.Br.

pendulous apical oval irregular concavities

complex elongated

long


erect basal globular convoluted complex elongated

medium stout


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


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


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


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


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


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.


153

Plate 3.12 Scanning electron photomicrographs of Gymnema sylvestre (Retz.) R.Br.


Plate 3.13 Scanning electron photomicrographs of Holostemma ada-kodien Schult.


154

Plate 3.14 Scanning electron photomicrographs of Pergularia daemia (Forssk.) Chiov.


Plate 3.15 Scanning electron photomicrographs of Tylophora indica (Burm.f.) Merr.


155

Plate 3.16 Scanning electron photomicrographs of Wattakaka volubilis (L.f.) Stapf.


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