6. OBSERVATIONS, RESULTS AND...

6. OBSERVATION, RESULTS & DISCUSSION 100

6. OBSERVATIONS, RESULTS AND DISCUSSION

The use of medicinal plants as raw materials in the production of new drugs is ever

increasing because of their potentials in combating the problem of drug resistance in

micro-organisms. Demand for medicinal plants is increasing in both developing and

developed countries. Research on medicinal plants is one of the leading areas of research

globally. The research into plants with alleged folkloric use as anti-inflammatory agents

should therefore be viewed as a fruitful and logical research strategy in the search for

new anti-inflammatory drugs. Sarcostemma acidum Wight. & Arn. (Syn. S. brevistigma).

Currently much interest have been paid in the searching of medicinal plants with anti-

inflammatory activity which may lead to the discovery of new therapeutic agent that is

not only used to suppress the inflammation but also used in diverse disease conditions

where the inflammation response in amplifying the disease process.

Inflammations can be uncomfortable and painful. But using natural remedies for

inflammation is much better than using any kind of chemical drugs to suppress the

symptoms. This is because, like other symptoms, such as fever, they are actually a sign of

the body healing itself of a problem, for example an infection. An area of the body is

inflamed because the immune system is sending additional "combat forces" there to try to

fight the enemies. By using artificial and chemical means to suppress an inflammation, it

is directly interfering with the body’s attempts to tackle and repair the problem which it is

facing. Such attempts to merely treat symptoms, without considering the deeper

underlying issues and attempting to achieve true and ultimate healing, is a problem not

just unique to anti-inflammatory drugs, but to chemical drugs in general.

It is commonly agreed that classical liposomes were found to be effective in forming drug

reservoir in only upper layer of the stratum corneum, for local skin therapy (Prausnitz

MR, 2004, Bouwstra JA, 2003; Cevc G, 1996a; Cevc G, 1996b; Cevc G, 2001). One of

the major advances in vesicle research was the finding a vesicle derivatives, known as an


Ethosomes (Braun-Falco O,1992). Ethosomes are noninvasive delivery carriers that

enable drugs to reach the deep skin layers and/or the systemic circulation. These are soft,

mallea-ble vesicles tailored for enhanced delivery of active agents. They are composed

mainly of phospholipids, (phosphatidylcholine, phosphatidylserine, phosphati-tidic acid),

high concentration of ethanol and water. It was found that ethosomes penetrate the skin

and al-low enhanced delivery of various compounds to the deep strata of the skin or to

the systemic circulation (Touitou E, 2002). The high concentration of ethanol makes the

ethosomes unique. The ethanol in ethosomes causes disturbance of skin lipid bilayer

organization, hence when incorporated into a vesicle membrane, it enhances the vesicle’s

ability to penetrate the stratum corneum. Also, because of their high ethanol

concentration, the lipid membrane is packed less tightly than conventional vesicles but

has equivalent stability, allowing a more malleable structure and improves drug

distribution ability in stratum corneum lipids (Merdan VM, 1998).

The plant S. acidum is an indigenous herb which was chosen for this study. The plant

belongs to the family Asclepidiaceae. The scanty availability of information on this plant

facilitates the study on it. The attempt is made to study the Pharmacognostical,

phytochemical and Anti-inflammatory activity of the plant. Attempt was made to

formulate the conventional and novel dosage and various evaluation parameters along

with the comparison of In-Vitro anti-inflammatory of the gel and ethosomes gel were

reported in present work.

6.1 PRLIMINARY INVESTIGATION

6.1.1Ethnomedicinal Claims and Folk lore uses of Plant

Herbal medicines are in great demand in both developed and the developing countries in

primary healthcare because of their great efficacy and little or no side effects. In India,

the indigenous system of medicine namely Ayurvedic, Siddha and Unani have been in

existence for several centuries. These traditional systems of medicine together with

homoeopathy and folklore medicine continue to play a significant role largely in the

health care system of the population.

During the course of investigation a well documented survey with the well designed and

approved questioner viz., informants, local name, part used, status of species, disease,

method of preparation and frequency of administration of medicine, was used to explore


the folk lore uses of the herb. It was concluded that the herb is locally known as soma,

somlata, somavalli and root and whole plant were used in the treatment of various

diseases such as asthma, swelling, fever and cold, dyspepsia, inflammatory infection,

gastric problems and as rejuvenating by the various informants in different doses and

duration along with specific method of preparation of the medicine (Table 3.1&3.2).

Further the status of the species was also recorded which conclude that the species is

endangered in almost all the survey sites, though at some cases it was found that the

species is in the verge of extinction, so there is urgent need of conservation. It has been

concluded that the selected species of Sarcosteema is very useful in the treatment of

certain kind of disease and various ex and in-situ conservation strategies may be adopted

to prevent the species from extinction.

Table 3.1: Ethnomedicinal and folk lore uses of Sarcostemma acidum W. & A. from

selected study sites of India

S/No. Uses Method of preparation

1. Asthma The plant was crushed and made infusion with the water and

taken twice a day.

2. Cold The whole part of plant juice given to children to relief from

cold.

3. Inflammatory Thick Paste of plant is applied in the swelling area.

4. Rejuvenating Juice of whole plant with other herbal medicine was taken

orally regularly 1-2 month when weakness observed.

5. Dyspepsia Powder part is given twice a day with cow milk or Luke water

Status: Endangered

Conservation: In-situ and Ex-situ Conservation


Table 3.2: Results of questioner for Sarcostemma acidum from selected study sites of

Tools of

investigation

(Questioner)

Inferences

I Tribal

People

Rurul

People

Village

Farmers

Vaidhyas Hakim Ayurvedic

Doctors

Traditional

Therapist

Botanist

LN Soma Somavalli Somlata Somavalli Soma Somavalli Somlata Somavalli

PU

Whole

plant

Root

Whole

plant

Whole

plant

Whole plant

Root

Whole

plant

Root

Whole

plant

Whole

plant

Whole plant

Roots

D

Asthma,

Swelling

Cold,

Asthma

Swelling,

Gastric

problems

Rejuvenating,

Asthma

Fever,

Cold,

Asthma

Dyspepsia,

Asthma

Asthma,

Gastric

problems

Inflammatory

Infection,

Asthma

MOP

The

plant

was

crushed

and

made

infusion

with the

water.

The

whole

part of

plant

juice

given to

children

to relief

from

cold.

Thick

paste

prepared

in water

applied

to the

swelling

area

Juice of

whole plant

with other

herbal

medicine was

taken orally

Whole

part

crushed

and made

to

powdered

given

with

water

Powder

part is

given with

cow milk

or Luke

water

Powdered

part given

empty

stomach to

get relief

from gastric

problem

Thick Paste

of plant is

applied in the

swelling area

to prevent the

inflammation

FD

BD for

2-3

months

BD, when

required

TID to

the

infected

area

OD,

Regularly 1-2

month when

weakness

observed

OD when

required

OD before

sleeping

after food

for one

months

OD empty

stomach for

1-2 months

BD applied

to the

swelling area

SS E V V CE CE E E E

Status & Conservation6.1.2 Pharmacognostical Studies

Abbr. I=Informants, LN= Local Name, PU= Part Used, SS=Status of species, D=Disease, MOP=Method of preparation,

FD=Frequency of administration of medicine, OD=Once in a day, BD=twice in a day, TID=thrice in a day, E=Endangered,

V=Vulnerable, CE=Critical Endangered

India


6.1.2.1 Anatomical Studies

The plant Sarcostemma acidum was taken up for microscopic characterization and the

study revealed the presence of epidermis, hypodermis, cortex, endodermis, pericycle,

conjoint and collateral vascular bundle, medullary rays and pith.

Microscopy (Transverse section) of stem of Sarcostemma acidum reveals that it is a dicot

stem having following structure:

Epidermis : It is the outermost layer consist of a single layer of cuticle above many layer

of tangentially flattened, closely fitted parenchymatous cell are present few multicellular

hairs and stomata may also be seen.

Parenchyma cells: Cells are thin, walled, isodiametric, oval in shape with intracellular

spaces.

Hypodermis: It lies just below the epidermis & consists of 4 to 5 layered of

collenchymatous cell No intercellular space is present, but the cell contains chloroplast.

Cortex: This lies below hypodermis, made up of thin walled rounded parenchymatous

cell. It stores food and present outside the vascular bundle in 2-3 layers.

Endodermis: It is the innermost layer of the cortex and demarcates it from the stele. The

cells are more or less barrel shaped and fit closely without intracellular spaces. It contains

numerous starch grains as reserve food.

Pericycle: It is the region lying between the endodermis and the vascular bundles and is

represented by semi-lunar patches of sclerenchyma and intervening masses of

parenchyma cells.

Vascular bundles: Vascular bindles are arranged in ring and are conjoint and collateral

type. It consists of phloem, cambium and xylem.

Medullary rays: Radially elonged parenchymatous cell occur between two vascular

bundles.

Pith: Large central part, consist of parenchymatous cell with intercellular space. (Fig 3.1

& 3.2)


Figure 3.1: Transverse section of stem of Sarcostemma acidum

T = Trichome , C= cuticle, P = Parenchyma cells, H = Hypodermis

C

T

T

P

H


Figure 3.2: Transverse section of stem of Sarcostemma acidum

Co=Cortex, En = Endodermis, Pr = Pericycle, Ph = Phloem, X = Xylem, P = Pith

Co

P

X

Ph

En

Pr


6.1.2.2 Powder Microscopy

The powder is a puff white to light yellowish in color with faint odor and slightly bitter

taste having following features:

1. Parenchymatous cells are oval shaped, thin walled, isodiametric and are present in

cluster and are present in groups in union with epidermal cells.

2. A cortex cell consists of round shaped parenchyma.

3. The abundant starch grains which are composed with two, three or more

components, individual granules are polyhedral and fairly small. A hilum is

visible in some of the granules.

4. Many multicellular trichomes are visible. They are numerous in number, 3-4

celled.

5. The fibrous cells which are generally found in groups vary in shape but are

usually narrow and elongated with blunt end.

6. Tracheids are also visible in cluster, 2-3 celled and compact in nature.

6.1.3 Physico-Chemical Analysis

The dried parts of S. acidum were subjected to standard procedure for the determination

of various physicochemical parameters- ash values (total ash, acid insoluble ash and

water soluble ash) were determined, Swelling index, Moisture content (M.C.), Foreign

organic matter (F.O.M.) was determined.(Table.3.3)

6.2 EXTRACTION

The dried powder of plant was extracted with various solvents i.e., water, ethanolic,

chloroform, petroleum ether and ethyl acetate. The solvents were removed by distillation

under reduced pressure and the resulting semisolid mass was vacuum dried using rotary

flash evaporator to obtain the extract. The percentage yields of various extract was

presented in Table 3.4.

6.2.1 Phytochemical Screening

The various extracts obtained were subjected to preliminary phytochemical screening.

The extraction was carried out with water, ethanol, chloroform, ethyl acetate and

petroleum ether the extract were screened for the presence of various medicinally active

constituents. Aqueous extract shows presence of alkaloids, carbohydrates, glycosides,


tannins, protein, amino acids, steroids. Ethanolic extract shows presence of alkaloids,

carbohydrates, glycosides, tannins, protein, amino acids, steroids. Chloroform extract

shows presence of carbohydrates, tannins while petroleum ether extract shows presence

of alkaloids, carbohydrates, glycosides, protein and amino acids, steroids.

Table 3.3: Physico-chemical analysis of S. acidum

Parameters

(% w/w)

Results

R1 R2 R3 X X+SD X+SEM

Total ash (TA) 7.0532 7.1958 7.0972 7.1154 7.11+0.05 7.11+0.03

Water soluble ash (WSA) 4.9674 4.8260 4.9062 4.8998 4.89+0.05 4.89+0.03

Acid insoluble ash (AIA) 1.8856 1.8074 1.7988 1.8306 1.83+0.00 1.83+0.00

Moisture content (MC) 6.0 5.79 5.90 5.89666 5.896+0.07 5.89+0.04

Swelling index (SI) 0.21 0.17 0.23 0.203333 0.20+0.02 0.20+0.01

Foreign organic matters

(FOM) 0.90 0.87 0.89 0.88666 0.88+0.10 0.88+0.06

Abbr.: R1=Reading 1, R2=Reading 2, R3=Reading 3, X=Mean, SD=Standard Deviation,

SEM=Standard Mean Error

Table 3.4: Extractive value of different extract of S. acidum

S/No. Type of Extract % Yield (w/w) Color of Extract

1. Aqueous extract 2.0947 Light Green

2. Ethanolic extract 12.7123 Dark Green

3. Chloroflam extract 10.6321 Light Green

4. Pet. Ether extract 9.7915 Brownish Green

5. Ethyl acetate extract 14.6511 Dark Green


Since, the major active constituents are present in ethanolic extract and ethyl acetate

extract therefore; the ethanolic and ethyl acetate extract was taken for further

investigation. The results are shown in Table 3.5.

6.3 PHARMACOLOGICAL SCREENING

In-vitro Anti-inflammatory activity of Extracts of S. acidum

During inflammation, lysosomal hydrolytic enzymes are released into the sites which

cause damages of the surrounding organelles and tissues with attendance of variety of

disorders (Sadique et al., 1989). Various methods were employed to screen and study

drugs, chemicals, herbal preparations that exhibit anti-inflammatory properties or

potentials. These techniques include uncoupling of oxidative phosphorylation (ATP

biogenesis linked to respiration), inhibition of denaturation of protein, erythrocyte

membrane stabilization, lysosomal membrane stabilization, fibrinolytic assays and

platelet aggregation (Kalyanpur et al., 1968; Lee and Thong, 1970; Swingle, 1974;

Kumar and Sadique, 1987; Pal and Chaudhuri, 1992; Oyedapo et al., 1999). In the

present study, stabilization of erythrocyte membranes exposed to both heat and hypotonic

induced lyses was employed due to its simplicity and reproducibility. The ethanolic and

ethyl acetate extract of the stem of Sarcostemma acidum were studied for in vitro anti-

inflammatory activity by HRBC membrane stabilization method. Four different

concentration of extract 1mg/ml, 2 mg/ml, 4 mg/ml and 6 mg/ml were used. Among

which EE extract at concentration 6 mg/ml showed 51.4 % protection of HRBC in

hypotonic solution and EAE extract at concentration 6 mg/ml showed 68 % protection of

HRBC in hypotonic solution. All the results were compared with standard indomethacin

which showed 69.6 % protection at concentration 2.5 mg/ml (Table 3.6 & 3.7, Fig.3.3)

.The activity may be due to the presence of one or more phytochemical constituents

present in the extract. The result obtained have been supported by Photomicrographical

pictures of the HRBC (Fig.3.4).

The study also provides a strong evidence for the use of the leaves S. acidum in folkloric

treatment as anti-inflammatory agent. The plant therefore could be regarded as a natural

source of membrane stabilizers and was capable of providing an alternative remedy for

the management and treatment of inflammatory related disorders and diseases.


Table 3.5: Preliminary phytochemical screening of different extract of Sarcostigmma

acidum

S/No. Constituents Test AE EE CE PE EA

1. Alkaloids Mayer’s test - + - - -

Dragendroff’ test + + - + +

Hager’s test - - - + -

Wagner’s test - - - - -

2. Carbohydrates Molisch’s test + + + + +

Fehling’s test + + - - +

3. Glycosides Brontrager’s test - + - - -

Legal’s test + + - + +

4. Fixed oil and fats Spot test - - - - -

Soap formation test - - - - -

5. Tannins FeCl3 - + - - -

Vanillin hydrochloride + + + - +

Alkaline reagent - + - - -

6. Protein and amino

acid

Million’s test + + - + +

Ninhydrin test + - - +

Biuret test - - - + -

7. Flavanoids With NaOH - - - - -

Shinoda test - - - - -

With H2SO4 - - - - -

8. Steroids and

triterpenoids

Libermann’s

Burchard test

- - - - -

Salkowski’s test + + - + +

9. Mucilage and gum With 90% alcohol - - - - -

10. Waxes With alc. KOH - - - - -

AE: Aqueous Extract; EE: Ethanolic Extract; CE: Chloroform Extract; PE: Petroleum ether

Extract, EA: Ethyl acetate Extract (+ Present, - Absent)


Figure 3.3: In-vitro Anti-inflammatory activity of Extracts of S. acidum

Table 3.6: In-Vitro anti-inflammatory activity of Ethanolic extract of S. acidum by

membrane stabilization method

Treatment Con(mg/ml) Absorbance(560nm) % of Inhibition

Control - 0.250±0.29 -

Ethanolic extract 1.00

2.00

4.00

6.00

0.208±0.25 a

0.182±0.22a

0.147±0.28b

0.123 ±0.42c

19.8

31.2

43.2

51.4

Indomethacin (Standard drug) 2.50 0.070±0.18b 69.6

Values are expressed as X (Mean) +SEM, n=3. (One way ANOVA followed by Student t-

test). Statistically significance of aP < 0.05, bP<0.01, cP<0.001 and dNS in comparison to

respective control.

Antiinflammetory action of S. acidum

0

10

20

30

40

50

60

70

80

0 2 4 6 8Con. mg/ml

% In

hib

itio

n o

f H

em

oly

sis

Ethyl acetate

extract

Ethanolic

extract

In -vitro Anti-inflammatory activity of S. acidum


Table 3.7: In-Vitro anti-inflammatory activity of Ethyl acetate extract of S. acidum

by membrane stabilization method

Treatment Con(mg/ml) Absorbance(560nm) % of Inhibition

Control - 0.250±0.29 -

Ethyl acetate extract 1.00

2.00

4.00

6.00

0.175±0.12a

0.143±0.23a

0.115±0.44c

0.081±0.39b

30.0

42.8

54.0

67.6

Indomethacin (Standard drug) 2.50 0.070±0.18b 69.6

Values are expressed as X (Mean) +SEM, n=3. (One way ANOVA followed by Student t-

test). Statistically significance of aP < 0.05, bP<0.01, cP<0.001 and dNS in comparison to

respective control.

Figure 3.5: Percentage Inhibition of haemolysis of Ethyl acetate extract


HRBC in Isotonic Solution Control HRBC in Hypertonic Solution

(Lysis of Hypertonic induced HRBC membrane)

RBC in Hypertonic solution with Plant extract (6mg/ml)

(Protection of Hypertonic induced HRBC membrane lysis)

Figure 3.4: HRBC Membrane in isotonic and hypertonic solution


6.4 FORMULATION CHARACTERIZATION AND EVALUATION OF

…..SUITABLE TOPICAL THERAPEUTIC SYSTEM

6.4.1 Evaluation of Gel Formulation:

During the trial, the excipients concentrations of carbapol and Sodium CMC were

gradually increased and then decreased as several problems like homogeneity,

spreadibility and viscosity were encountered. These problems occurred in some of the

batches (F1, F2, F6 and F7) of polymer based gel containing S. acidum. Hence, these

batches were discarded and remaining batches (F3, F4 and F5) were considered for

further study.

The result showed that the developed herbal gel was greenish in color, translucent in

appearance and showed good homogeneity with absence of lumps. Formulation F5 had

good values of spreadability, viscosity, pH, drug content and during the accelerated

stability studies the appearance was clear and no significant variation in spreadability, pH

and drug content was observed. Hence Hydroalcohalic and Ethosomal gel was formulated

from hydrogel F5 formulation and its physiochemical study was found to be good.

(Fig.3.6, 3.7 & Table.3.8)

Table 3.8: Physical evaluation of all formulations

Batch Color Appearance Spreadibility

(gm.cm/sec)

Consistency

(60 mm)

Viscosity

(cps) Ph

Drug

content

(%)

F3 Greenish Homogeneous 19.75 5 22410 7.00 99.81



FEAE Greenish Homogeneous 21.45 6 22393 7.00 99.97

FA Greenish Homogeneous 23.96 8 17053 7.00 99.80

FAEAE Greenish Homogeneous 24.08 8 17862 7.00 99.92

FE Greenish Homogeneous 24.21 8 16915 7.00 99.94

FEEAE Greenish Homogeneous 24.42 8 16995 7.00 99.95


Hydrogel formulation F-5

Hydrogel formulation FEAE

Figure 3.6: Microscopic photograph’s of Hydrogel Formulation containing S.

acidum plant extract


Ethosomal gel formulation FE

Ethosomal gel formulation FE EAE

Figure 3.7: Microscopic photograph’s of Ethosomal Gel Formulation

containing S. acidum plant extract


Fig

ure

3.8

: F

TIR

sp

ectr

a o

f S

. aci

du

m e

xtr

act

s (e

thyl

ace

tate

+ e

han

oli

c ex

tract

)


Fig

ure

3.9

: F

TIR

sp

ectr

a o

f H

yd

rogel

base


Figure FTIR spectra of Gel base

Fig

ure

3.1

0 :

FT

IR s

pec

tra o

f H

yd

rogel

base

+ S

. aci

du

m E

xtr

act

s


Fig

ure

3.1

1 :

FT

IR s

pec

tra o

f E

thoso

mal

gel

base


Figure FTIR spectra of Ethosomal gel base

Fig

ure

3.1

2 :

FT

IR s

pec

tra o

f E

thoso

mal

gel

base

+ S

. aci

du

m E

xtr

act

s


Compatibility studies

Fourier transformed infrared (FTIR) spectra technique have been used here to study the

physical and chemical interaction between extracts and excipients used in formulation.

From the figure 3.8-3.12 ,it has been observed that there is no changes in these main

peaks in IR spectra of mixture of S.acidum extract, Soya Phaphodilcholine and polymers

used in formulation which shown there were no physical and chemical interaction. The

peaks obtained in the spectra’s of formulation correlated with the peaks of S. acidum

extract. This indicates that the extract was compatible with the formulation.

6.4.2 Evaluation of Ethosome suspension

Image analysis of ethosomes by optical microscope

For the initial vesicle characterization of ethosome suspension were examined by

compound microscope. The result revealed that formulation without added SLS and

Tween 80 shown aggregation process among the structure (Fig.3.14). Formulation in

which SLS and Tween 80 added shown spherical shaped vesicles like structure without

aggregation process.(Fig.3.13, a & b) Hence, Formulation containing SLS and Tween 80

in ehanolic extract (FE3, FE4,FE7,FE8) and ethyl acetate extract (FE3EAE, FE4EAE,

FE7 EAE,FE8EAE) were considered for further study and remaining formulation batches

were discarded.

Vesicular shape and surface morphology by TEM

The vesicular shape and surface morphology of ethosomes of formulation FE8EAE

examined by Transmission Electron Microscope (TEM). The TEM image showed that

ethosomes were spherical shaped. (fig.3.15)

Determination of Entrapment efficiency of ethosomes suspension:

The entrapment efficiency of various ethosomes formulations are presented in Table.3.9.

The entrapment efficiency of formulation containing ethanolic extract FE8 was found to

be highest (75.91%) while FE3 formulation showed least entrapment efficiency (66.66

%). The entrapment efficiency of formulation containing EAE was observed to be highest

for FE8,EAE formulation (74.54%) and least for FE3,EAE (58.87%). The EE of formulation

containing EAE and EE were comparable. However it has been observed the formulation

containing phospholipid (3 gm) with ethanol has maximum entrapment efficiency.


(a) Ethosome suspension FE8

(b) Ethosome suspension FE8,EAE

Figure 3.13: Microphotographs of Ethosome suspensions containing

S. acidum plant extract


Ethosome suspension, FE6,EAE

Figure 3.14: Microphotographs of Ethosome suspensions containing S.

acidum plant extract (without added SLS and Tween 80)

Figure 3.15: Transmission electron microphotograph Visualization of ethosomes by

transmission electron microscopy.


Table 3.9: Entrapment efficiency of ethosomes suspension

S/No. Ethosomes Qt Qs %EE

1. FE3,EAE 2.5 1.0281 58.87

2. FE4,EAE 2.5 0.8831 64.67

3. FE7,EAE 2.5 0.7416 70.30

4. FE8,EAE 2.5 0.6364 74.54

5. FE3 2.5 0.8326 66.66

6. FE4 2.5 0.7916 68.33

7. FE7 2.5 0.6921 72.31

8. FE8 2.5 0.6021 75.91

EE =entrapment efficiency, Qt = amount of extract added, Qs = amount detected in the

supernatant.

6.4.3 In-vitro drug release study

Percentage drug release of hydrogel containing EE & EAE extract was observed to be-

9.89 & 11.25% (at 30 min.) and 47.63% & 48.63% (at 240 min.) respectively where

hydroalcohalic gel containing EE & EAE extract was observed to be- 13.62% & 15.43%

(at 30 min.) and 60.81% & 62.75%(at 240 min.) respectively and Ethosomal gel

containing EE & EAE extract was observed to be 26.86% and 28.31% (at 30 min.) and

77.98% & 78.42% (at 240 min.) respectively. It was observed that addition of ethanol in

formulation increase the release by increasing permeation properties of gel. The

ethosomal gel containing EAE extract (formulation FEEAE) showed maximum drug

release as compared to others formulation. (Table.3.11, Fig.3.17, 3.18)

Standard calibration curve of S. acidum plant extract for its active constituent

Standard calibraction curve of S. acidum extract was determined by plotting absorbace vs

concentraction at 255 nm and it follow the beer’s law. The results are shown in

Table.3.10 and Fig.3.16


Figure 3.16: Standard calibration curve of S. acidum at 255 nm.

y=0.27x-, r2=0.997

Table 3.10: Standard calibration curve of S. acidum at 255 nm

S.No Concentration (µg/ml) Absorbance

1.

2.

3.

4.

5.

6.

Blank

0.2

0.4

0.6

0.8

1.0

0.000

0.049

0.112

0.174

0.213

0.271


Table 3.11: Percentage Drug release of Formulated Hydrogel, Hydroalcohalica Gel

containing EE and EAE extract

Time

Interval

(Min)

% Drug release of formulations

F7 FEAE FA FAEAE FE FEEAE

15

30

45

60

90

120

180

240

05.12

09.89

18.91

26.41

35.62

46.43

46.85

47.63

05.41

11.25

19.68

25.23

38.32

46.72

47.81

48.12

08.42

13.62

20.81

28.23

45.58

50.24

56.12

60.81

10.23

15.43

22.21

29.32

41.12

49.47

57.32

62.75

15.21

26.86

31.81

42.18

55.43

60.25

74.28

77.98

15.43

28.31

32.12

43.53

54.01

61.89

73.21

78.42

(F7= Hydrogel containing ethnolic extract, FFEAE =Hydrogel containing ethyl acetate extract,

FA =Hydroalcohalic gel containing ethanolic extract, FAEAE =Hydroalcohalic gel containing ethyl

acetate extract, FE= Ethosomal gel containing ethanolic extract, FEEAE = Ethosomal gel

containing ethyl acetate extract.)


Figure 3.17: Release profile of Hydrogel, Hydroalcohalic gel and Ethosome

gel containing EAE extract

Figure 3.18: Release profile of Hydrogel, Hydroalcohalic gel and Ethosome

gel containing EE extract

Time (min)

Per

cen

tage

dru

g r

elea

se

Per

cen

tage

dru

g r

elea

se

Time (min)


Drug release kinetic modeling

On comparison of kinetic modeling and release profile data it was evident that ethosomal

gel FEEAE was found to release the drug in accordance to Higuchi kinetics, the regression

coefficient was not found to be exactly near to 1, which could be due to influence of

some other factors.(fig. 3.19- 3.21)

Figure 3.19: Zero order kinetics of formulatio FEEAE through fabricated

diffusion cell

Figure 3.20: First order kinetics of formulatio FEEAE through fabricated

diffusion cell

Zero order kinetic

y = 0.3077x + 16.326

R2 = 0.8748

0

10

20

30

40

50

60

70

80

90

100

0 50 100 150 200 250 300

time in min

% d

rug

rele

ase

% Drug Release

Linear (% Drug

Release)

First order kinetic of Etosome gel of EAE

y = 0.005x + 1.018

R2 = 0.4592

0

0.5

1

1.5

2

2.5

0 100 200 300

time (min)

log

of

% d

rug

rele

ase

log %dru release

Linear (log %dru

release)


Figure 3.21: Release of formulatio FEEAE through fabricated diffusion cell

data fitted to Higuchi model kinetics

6.5 IN-VITRO ANTI-INFLAMMATORY STUDY OF OPTIMIZED

FORMULATION

During inflammation, lysosomal hydrolytic enzymes are released into the sites which

cause damages of the surrounding organelles and tissues with attendance variety of

disorders. Various methods were employed to screen and study of drugs, chemicals,

herbal preparations that exhibit anti-inflammatory properties or potentials. These

techniques include uncoupling of oxidative phosphorylation (ATP biogenesis linked to

respiration), inhibition of denaturation of protein, erythrocyte membrane stabilization,

lysosomal membrane stabilization, fibrinolytic assays and platelet aggregation. In the

present study, HRBC membrane stabilization method was employed due to its simplicity

and reproducibility.

Percentage inhibition of HRBC haemolysis in hypotonic solution of hydrogel containing

EE & EAE extract was observed to be- 18% & 31.3% (at 30 min.) and 46.4% & 58.1%

(at 240 min) respectively where as percentage inhibition of HRBC haemolysis in

hydroalcohalic gel containing EE & EAE was observed to be- 20% & 35% (at 30 min.)

and 52% & 67% (at 240 min.) respectively and for ethosomal gel containing EE & EAE

extract was observed to be-37% & 46% respectively and 53% & 75% (at 240 min.)

respectively. All the results were compared with standard voveran emulgel.

Higuchi model of Ethosome gel containing EAE

y = 5.5415x - 2.2218

R2 = 0.9759

0

10

20

30

40

50

60

70

80

90

0 5 10 15 20

under root of time(min)

% d

rug

rele

ase

% drug release drug

Linear (% drug release

drug)


The gel exhibited membrane stabilization effect by inhibiting hypotonicity induced lyses

of erythrocyte membrane. The activity may be due to the presence of one or more

phytochemical constituents present in the formulation. The result also showed that ethyl

acetate extract of S. acidum has better anti-inflammatory action as compared to ethanolic

extract. The ethosomal gel containing EAE extract, formulation FEEAE showed maximum

percentage inhibition of HRBC haemolysis in hypotonic solution as compared to others

formulation.(Table.3.12)

Table 3.12: The In-vitro anti-inflammatory activity of formulated gel by Red blood

Cell Membrane Stabilization method

Time

Interval

(Min)

% Inhibition of haemolysis of Formulations

F7 FEAE FA FAEAE FE FEEAE Voveran Emulgel

(Standard Gel)

15

30

45

60

90

120

180

240

-

18

30.8

35.2

41.3

45.6

46.1

46.4

15.1

31.3

40.8

45.4

51.3

55.8

57.9

58.1

16

20

31

38

44

47

50

52

30

35

42

49

54

59

60

67

26

37

39

43.6

48.2

52.6

53.1

53.9

35

46

49

55

59

67

70

75

31

45

48

56

60

70

74

78





6.6 IN-VIVO ANTI-INFLAMMATORY STUDY OF OPTIMIZED

FORMULATION

Percentage inhibition of edema by hydrogel containing EE & EAE extract in rat’s left

hind paw was observed to be- 13.87% & 15.32% (at 1 hr.) and 20.72% & 24.16% (at 2

hr.) respectively where as in case of hydroalcohalic gel containing EE & EAE was


observed to be- 23.19% & 25.0% (at 1 hr.) and 35.00% & 37.23% (at 4 hr.) respectively

and for ethosomal gel containing EE & EAE extract inhibition was observed to be-

28.10% & 32.13% 9 (at 1 hr.) respectively and 39.10% & 42.03% (at 4 hr. min.)

respectively. All the results were compared with standard voveran emulgel gel.

The result revealed that formulations containing ethyl acetate extract of S. acidum has

better anti-inflammatory action as compared to ethanolic extract. The ethosomal gel

containing EAE extract, FEEAE show highest percentage inhibition of edema as compared

to others formulation (Table.3.13, 3.14 & fig.3.22).

Table 3.13: Mean Paw edema Volume of the albino rats

Hind Paw (Mean+SEM)

Values are expressed as X (Mean) +SEM, n=3. (One way ANOVA followed by Student t-test).

Statistically significance of *P < 0.05, **P<0.01, in comparison to control.





Formulation Mean Paw edema Volume

1 hr 2hr 3hr 4hr

Control 1.74+0.03 1.8+0.05 1.74+0.05 1.68+0.58

F7 1.61+0.03* 1.52+0.03** 1.46+0.04* 1.41+0.20*

FEAE 1.60+0.04* 1.50+0.13* 1.49+0.50* 1.39+0.05*

FA 1.61+0.04* 1.52+0.03* 1.32+0.60* 1.28+0.03*

FAEAE 1.58+0.90** 1.42+0.07* 1.39+0.03* 1.22+0.60*

EE 1.46+0.05* 1.28+0.02* 1.24+0.02* 1.16+0.04*

FEEAE 1.22+0.03* 1.12+0.03** 1.10+0.03* 0.97+0.04*

Standard Drug

(Voveran Emulgel) 1.2±0.05 0.84±0.04 0.78±0.03 0.74±0.024


Figure 3.22: Percentage inhibition of edema

Table 3.14: Mean Percentage inhibition of edema

% Inhibition (Mean+SEM)

Group Percentage inhibition of edema

1 hr 2hr 3hr 4hr

Control - - - -

F 13.87+0.01 15.90+0.72 18.43+0.12 20.72+0.16

FEAE 15.32+0.04 17.12+0.19 21.90+0.13 24.16+0.17

FA 23.19+0.40 28.18+0.13 30.97+0.43 35.00+0.12

FAEAE 25.50+0.10 31.03+0.10 36.09+0.12 37.23+0.04

EE 28.10+0.41 33.40+0.19 35.07+0.03 39.10+0.23

FEEAE 32.13+0.16 38.52+0.70 41.78+0.24 42.03+0.97

Standard Drug

(Voveran Emulgel) 41.02+0.16 49.00+0.03 51.10+0.05 55.95+0.90

(F7= Hydrogel containing ethnolic extract, FFEAE =Hydrogel containing ethyl acetate extract, FA

=Hydroalcohalic gel containing ethanolic extract, FAEAE =Hydroalcohalic gel containing ethyl



Per

cen

tage

inhib

itio

n o

f ed

ema

Time (hr.)


6.7 Skin Irritation Study

The skin irritation test was conducted for a period of seven days and the results are

tabulated in Table 3.15. The results indicated that the control preparation, test gel, FEEAE

and marketed products did not cause any skin reaction. It can be assured that ethyl acetate

extract of S. acidum and the excipients did not cause any skin irritation and can be used in

the gel formulation.

Table 3.15: Skin irritation study

Treatment Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7

Control A A A A A A A

FEEAE A A A A A A A

Voveran emulgel A A A A A A A

A – No reaction, B – Slight patchy erythema, C –Slight but confluent or moderate but patchy

erythema, D – Moderate erythema, E – Severe erythema with or without edema.

6.8 Stability Study

The formulated gels were subjected to stability studies. No color fading was observed for

all prepared gels. The pH of all formulations remained unchanged and was found to be

within the range of 6.2-7.2. The viscosity and spreadability of all gels remained unaltered

and found to be within the range. The drug content was found to be in the limit 90% -

103% for all gel formulation. (Table.3.16)


Table 3.16: Accelerated Stability study of formulated gel

Batch Color Appearance Spreadibility

(gm.cm/sec)

Consistency

(60 Sec)

Viscosity

(cps) Ph

Drug

content (%)

F3 Greenish

black Homogeneous 14.60

5 22230 6.84 99.77

F4 Dark

Greenish Homogeneous 18.50 6 24010 6.93 98.20


FEAE Greenish Homogeneous 20.47 6 17828 6.91 99.96

FA Greenish Homogeneous 23.00 9 16042 6.95 98.95

FAEAE Greenish Homogeneous 23.12 9 16837 6.92 99.03

FE Greenish Homogeneous 23.43 9 16042 7.01 99.82

FEEAE Greenish Homogeneous 23.54 9 16042 6.99 99.86

(F7= Hydrogel containing ethnolic extract, FFEAE =Hydrogel containing ethyl acetate extract, FA

=Hydroalcohalic gel containing ethanolic extract, FAEAE =Hydroalcohalic gel containing ethyl




Table 3.2: Results of questioner for Sarcostemma acidum from selected study sites of India

Tools of

investigation

(Questioner)

Inferences

I Tribal

People

Rurul

People

Village

Farmers Vaidhyas Hakim

Ayurvedic

Doctors

Traditional

Therapist Botanist

LN Soma Somavalli Somlata Somavalli Soma Somavalli Somlata Somavalli

PU Whole plant

Root Whole plant Whole plant

Whole plant

Root

Whole plant

Root

Whole plant

Whole plant

Whole plant

Roots

D Asthma,

Swelling

Cold,

Asthma

Swelling,

Gastric

problems

Rejuvenating,

Asthma

Fever, Cold,

Asthma

Dyspepsia,

Asthma

Asthma,

Gastric problems

Inflammatory

Infection,

Asthma

MOP

The plant

was crushed

and made

infusion

with the

water.

The whole

part of plant

juice given to

children to

relief from

cold.

Thick paste

prepared in

water

applied to

the swelling

area

Juice of whole

plant with other

herbal medicine

was taken orally

Whole part

crushed and

made to

powdered

given with

water

Powder part is

given with cow

milk or Luke

water

Powdered part

given empty

stomach to get

relief from

gastric problem

Thick Paste of

plant is applied in

the swelling area

to prevent the

inflammation

FD BD for 2-3

months

BD, when

required

TID to the

infected area

OD, Regularly

1-2 month when

weakness

observed

OD when

required

OD before

sleeping after

food for one

months

OD empty

stomach for 1-2

months

BD applied to the

swelling area

SS E V V CE CE E E E

Abbr. I=Informants, LN= Local Name, PU= Part Used, SS=Status of species, D=Disease, MOP=Method of preparation, FD=Frequency of administration of medicine,

OD=Once in a day, BD=twice in a day, TID=thrice in a day, E=Endangered, V=Vulnerable, CE=Critical Endangered

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