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lallemantia royleana

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CHAPTER – 5

LALLEMANTIA ROYLEANA

5.1 Introduction

Lallementia royleana is commonly known as Lady’s mantle in english and Tukhm-e-

balanga in hindi (Mahmood et. al., 2013) and in urdu language it is referred to as Balangu

shirazi (Naghibi et. al., 2005). L. royleana is cultivated in North India for its highly

medicinal and mucilaginous seeds which are used widely as a remedial agent in various

diseases. L. royleana is an important medicinal plant for Iranian Jews as well. It belongs

to the family Lamiaceae or Labiateae and is known to belong to the mint family and also

considered closely related to Verbenaceae, but several recent phylogenetic studies have

shown that numerous genera classified in Verbenaceae family but instead it belongs to

Lamiaceae. The core genera of Verbenaceae are not closely related to Lamiaceae and are

more closely related to the members of Lamiales. The enlarged Lamiaceae family

contains 233 to 263 genera and 6900 to 7200 species (Kaurinovic et. al., 2010).

5.2 Description of the Plant

The plants of Lallemantia royleana are annual, biennial, or perennial, glabrous or white

hairy, herbaceous in nature. The leaves are petiolated or sessile sub-entire. The flowers

are verticillasters axillary having 6 petals, bracts ciliate or waned crenate. The pedicel is

erect, rigid, flattened. The calyx is tubular, 15-veined, straight, throat closed after anthesis

having 5 teeth which are sub-equal, posterior tooth wider than other teeth, tooth sinus

with a thickened fold. The corolla has a slender slightly exerted tube dilated at apex into

throat which is bi-lipped, where upper lip is straight, slightly concave, apex is

emarginated, with 2 longitudinal folds inside. The lower lip is spreads out as 3-lobed,

middle lobe is reniform, lateral lobes are minute and semi-circular. There are 4 stamens

present, the 2 posterior are longer having pilose filaments and anthers are divaricate

having 2 cells. The style is present at the apex having 2 clefts and subulate lobes. Nutlets

are dark brown, oblong, adaxially ribbed, and become mucilaginous when soaked in

water.

http://en.wikipedia.org/wiki/Verbenaceae

http://en.wikipedia.org/wiki/Phylogenetic

http://en.wikipedia.org/wiki/Genera

http://en.wikipedia.org/wiki/Lamiales

http://en.wikipedia.org/wiki/Species

http://zipcodezoo.com/glossary/bract.asp

http://zipcodezoo.com/glossary/pedicel.asp

http://zipcodezoo.com/glossary/calyx.asp

http://zipcodezoo.com/glossary/posterior.asp

http://zipcodezoo.com/glossary/exserted.asp

http://zipcodezoo.com/glossary/lip.asp

http://zipcodezoo.com/glossary/stamen.asp

http://zipcodezoo.com/glossary/style.asp

http://zipcodezoo.com/glossary/adaxially.asp


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Figure No. 5.1: Lallemantia royleana

Kingdom : Plantae

Division : Magnoliophyta

Class : Magnoliopsida

Order : Lamiales

Family : Lamiaceae

Genus : Lallemantia

Species : royleana


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The length of the seeds are about 1/12 inches and 1/16 inches in breadth, dark brown to

black in colour, smooth, three angled and tapering towards the umbilicus which is

marked by a tiny white spot. When moistened with water the seeds become coated with

voluminous and translucent mucilage. The odour of the seeds is mildly spicy. However,

the seeds do not impart any odour when suspended or moistened in water. The taste of

the mucilaginous seeds is mild and somewhat spicy.

5.3 Geographical Distribution

In the Indian sub-continent, the plant is found in Northern regions. It is mostly found in

the plain hills of Punjab, although, the plant does not exist in wide areas but its

cultivation is being highly encouraged. The plant is extensively found in Pakistan Forest

Institute (PFI), Pakistan, especially in the areas of Peshawar (Abbas et. al., 2012).

L. royleana is a native of Asia and found in temperate and tropical region and also in

some European regions of the world. In Western areas of Asia, It is distributed in

Afghanistan, Iran and Syria. It is also native to the Caucasian areas like Armenia and

Azerbaijan. The plant is also found in Siberian regions of Russian Federation that in

Western Siberia. In Middle Asia region, it is found in Kazakhstan, Kyrgyzstan,

Tajikistan, Turkmenistan and Uzbekistan. The plant also comes from Xinjiang, China. It

is also found in eastern part of Europe which comes under the Russian Federation. (Cao

Shu, 1994 and Abbas et. al., 2012)

5.4 Ethno-botanical Importance

Lallementia royleana seeds are ethno-botanically well-established worldwide for the

treatment of abscesses, inflammation and respiratory problems. The seeds also used in

drinks due to its sedative effects (Abbas et. al., 2012; Abdulrasool et. al., 2011and Khare,

2007).

Lallementia seeds are commonly used in the folk medicine. The studies had showed the

presence of carbohydrates, fibre, oil, protein and tannins (Naghibi et. al., 2005; Razavi

and Karazhiyan, 2009 and Razavi and Moghaddam, 2011). Other studies toward natural

remedies confirmed the fact that plant and food medicine are good source of natural and

safe healing therapies (Dogruoz et. al., 2008 and Karsha and Lakshmi, 2010).


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The mucilaginous seeds are traditionally used as curative agents against various diseases.

The poultices of moistened seeds are found to be useful in abscesses, boils and

inflammations. The seeds are also known to cure fever, common cold, act as soothing

agents for stomach warmness and intestinal troubles. The roots of the plant are known to

cure coughing. The seeds are used as an added palatable ingredient in cooling drinks

during summers mixed with brown sugar. The plant seeds are also used as sedative and

considered to be cephalic astringent, cardiac tonic and carminative (Naghibi et. al., 2005).

In Chinese medicine, L. royleana is one of the major ingredients of an ointment used in

the treatment of skin tumours. In Ivory Coast, Burkina Faso, Gabon and Tanganyika,

different parts of the plant are used for the treatment of intercostals pain, rheumatic pain

and fever. The leaf and root decoctions are used to treat pneumonia. The plant is also

used in preparation of herbal brain tonics in India (Preedy, 2011).

It is also reported that the plant extract along with the extract of Valeriana wallichii,

exhibits anti-microbial activity (Chitralekha et. al., 1964). The physico-mechanic

properties and chemical composition of L. royleana seed showed that the seeds contain

average amounts of dry matter (92.75%), ash (3.63%), crude protein (25.60%), crude fat

(18.27%), crude fiber (1.29%), NDF (30.67%) and ADF (47.80%) (Razavi et. al., 2008).

The seeds are a good source of fibre, protein and oil. The factor in its physiological fiber

behaviour is usually because of its high viscosity and gel like character in water. The

property in turn is related to the functions associated with its high molecular weight

polysaccharides (Williams and Phillips, 2010). The compounds such as Linoleic acid,

oleic acid, palmitic acid, stearic acid and beta-sitosterol are present in the seeds. The

gums of the seeds are composed of L-arabinose, D-galactose, L-rhamnose, pentosans,

proteins, uronic anhydride. It also contains all the amino acids which are present in plant

(Khare, 2008).

5.5 Determination of Extraction Yield of Seeds Extract (% Yield)

The initial weight of 30 gms of the dried seeds of Lallemantia royleana was soaked in

100 ml of methanol. The percentage yield of 2.25 percent was obtained in the methanolic

extract of the seeds of L. royleana. The percentage yield of extracts of seeds of

L. royleana in methanol is given below in Table 5.1.


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S. No. Plant name

Weight of

dried seeds

W0 (gm)

Weight of

empty

petri plate

W1 (gm)

Weight of

petri plate

with plant

extract

W2 (gm)

Percentage

yield (%)

1. Lallemantia

royleana 30 gm 46.700 gm 47.375 gm 2.25

Table No. 5.1 The percentage yield of methanolic seed extracts of L. royleana, extraction

done by soaking dried plant material in methanol and extract separation

using distilling apparatus.

5.6 Total Phenolics Estimation of Seeds Extracts

The total phenolic content was estimated spectrophotometrically using the Folin-

Ciocalteu Reagent at 765 nm. A calibration curve was drawn using Gallic Acid which

was used as standard. The level of Gallic acid in the methanolic seed extract of

Lallemantia royaleana was measured. The observed concentrations were multiplied with

dilution factor. The results were reported as Gallic Acid Equivalent, (GAE) in mg/g of

dry mass.

The gallic acid is a stable substance which is pure in nature and it is easily available. Since

this assay measures all phenolics, gallic acid is taken as standard in the study. The stability

of gallic acid standard solutions was also tested and it shows that it loses less than 5% of

their value over two weeks when refrigerated and kept tightly closed (Waterhouse, 1999

and 2009). The GAE for L. royleana methanolic seed extract was estimated to be 2.81

GAE/g. The standard calibration curve is shown in Figure 5.2.

5.7 Tannins Estimation of Seeds Extracts

The total phenolic content was also estimated spectrophotometrically at 765 nm using

Folin-Denis reagent here tannic acid was used as standard. The total phenolic content was

expressed as mg/g tannic acid equivalents per gram, (TAE) expressed in mg/g of dry

mass using the following equation based on the calibration curve.

y = 0.002x + 0.98, R² = 0.979


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The experiment was replicated thrice and average data recorded for quality assurance.

The TAE for methanolic seed extract of L. royleana was estimated to be 0.18 TAE/g. The

standard calibration curve is shown in Figure 5.3.

Figure No. 5.2: Calibration curve for gallic acid for determining the phenolic

content.

Figure No. 5.3: Calibration curve for tannic acid for determining the tannin

content.

y = 0.002x + 0.98R² = 0.979

0

0.5

1

1.5

2

2.5

0 100 200 300 400 500

Abso

rban

ce

Concentrtion of tannic acid (µg/ml)

TANNIC ACID STANDARD

Absorbance

Linear (Absorbance)

y = 0.001x + 0.147R² = 0.979

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 100 200 300 400 500

Abso

rban

ce

Concentrtion of gallic acid (µg/ml)

GALLIC ACID STANDARD

Absorbance

Linear (Absorbance)


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5.8 Phyto-chemical Analysis of Seed Extract of Lallemantia royleana

Phyto-chemical analysis involves the qualitative analysis of herbal plants extracts. The

preliminary qualitative tests have been attempted in Lallemantia royleana seeds to find

out the presence or absence of certain bio active compounds. The chemical tests were

carried out on the crude methanolic extract using standard procedures to identify the

active constituents.

The crude methanolic seed extract of Lallemantia royleana were evaluated

qualitatively to analyze the presence of secondary metabolites. The secondary

metabolites found in the crude methanolic extract of seeds are alkaloids,

anthraquinones, flavonoids, phlobatannins, glycosides, tannins and terpenoids.

The L. royleana crude methanolic seed extract showed positive result to the

different phyto-chemical tests indicating the presence a number of

phyto-constituents. The results of qualitative phyto-chemical analysis are given in

Table 5.2.

The presence of alkaloid was analysed in methanolic extract of seeds using Wagner’s

method. The presence of reddish brown colour precipitate indicates the presence of

alkaloids. When the methanolic seeds extract of L. royleana was evaluated using this

assay the presence of reddish brown coloured precipitate confirms the presence of

alkaloids.

The Borntrager’s test was performed for the analysis of anthraquinones in the methanolic

seed extract. The formation of rose pink colour in plant extract confirmed the presence of

anthraquinones. The methanolic seeds extract when tested using this assay confirmed the

appearance of pink colour indicating the presence of anthraquinones.

The presence of flavonoids in the crude plant extract is determined quantitatively by the

appearance of yellow colour. When the crude methanolic seeds extract of L. royleana was

evaluated using this test showed the appearance of yellow colour indicates the presence

of flavonoids.

The phlobatannins presence was evaluated qualitatively by adding 1% of aqueous

HCl in boiled crude methanoilic extract of L. royleana seeds, the presence of red colour

indicates a positive result. The crude methanolic extracts of L. royleana seed showed the

presence of red colour indicating the presence of phlobatannins.


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Table No. 5.2: Phyto-chemical analysis of seeds extract of Lallemantia royleana

The presence of glycosides in the L. royleana methanolic seeds extract evaluated using

the Fehling’s test. The brick red precipitate formation indicates the presence of

glycosides. The L. royleana methanolic seeds extract showed the presence of brick red

precipitate thus confirming the presence of glycosides.

Similarly, the presence of saponins in the plant extract evaluated using a frothing test.

The formation of froth confirmed the presence of saponins. The L. royleana seeds extract

did not show the appearance of froth indicating the absence of saponins in the crude

extract.

The L. royleana crude methanolic seed extract were also evaluated for the presence of

steroids by using the Salkwoski test. The change of colour from violet to blue indicates a

positive result. The crude methanolic seeds extract did not show the change in colour

indicating the absence of steroids in the extract.

The crude seed extract was further tested for the presence of tannins by using ferric

chloride test. The occurrence of blue black precipitate indicates the presence of tannins.

The L. royleana methanolic seeds extract showed the formation of blue black precipitate

thus confirming the presence of tannins.

S.No. Active principle Phyto-chemical Analysis Result

1. Alkaloids Wagner’s Test +

2. Anthraquinones Borntrager’s Test +

3. Flavonoids Sodium Hydroxide (NaOH) Test +

4. Phlobatanins Hydrocloric Acid (HCl) Test +

5. Glycosides Fehling’s Test +

6. Saponins Frothing Test -

7. Steroids Salkwoski Test -

8. Tannins Ferric chloride (FeCl3) Test +

9. Terpenoids Salkwoski Test +


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Similarly, Salkowski test was also performed to evaluate the presence of terpenoids in

L. royleana curde methanolic seeds extract. The formation of reddish brown colour

indicates the presence of terpenoid. The seeds extract confirmed the presence of

terpenoids as the reddish brown colour appeared in it.

5.9 Anti-oxidant Activities of Seeds Extract of Lallemantia royleana

The anti-oxidant activity of methanolic seed extract of L. royleana was determined in

vitro by using a number of assays such as super oxide scavenging activity by alkaline

DMSO method, DPPH free radical scavenging activity, nitric oxide free radical

scavenging activity, H2O2 radical scavenging activity and total anti-oxidant capacity

method.

5.9.1 Scavenging of Superoxide Radical with the Alkaline DMSO (Dimethyl

Sulfoxide) Method

The superoxide radical scavenging assay, were studied in crude methanolic seeds

extracts at different concentrations ranging from 1.95 to 1000 µg/ml and

absorbance measured at the wavelength of 560 nm. The results are given as

percentage inhibition values of the extracts. The increase in percentage showed

stronger inhibition and highest scavenging activity of the plant extract.

The percentage inhibition values of seed extract of L. royleana were found to

range between 69.78 ± 0.046 and 3.385 ± 0.385 percent, at the concentration of

1000 and 1.95 µg/ml respectively, whereas the percentage inhibition values of

standard (BHT) were found to be 63.52 ± 0.020 and 6.63 ± 0.229 percent, at the

concentration of 1000 and 1.95 µg/ml respectively. The percentage inhibition

values of L. royleana extracts along with standard (BHT) at different

concentrations are shown in Table 5.3 and 5.4 and Figure 5.4.

The methanolic extract of L. royleana scavenges super oxide radical and thus

inhibits formazan formation. In Table 5.3 it is illustrated that increase scavenging

of superoxide radicals in dose dependent manner due to the scavenging ability of

the L. royleana methanolic crude extract. The IC50 value of L. royleana was found

to be 308.67 ± 7.12 μg/ml, whereas the IC50 value of BHT was 792.49 ± 1.16

μg/ml.


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5.9.2 Nitric Oxide Free Radical Scavenging Activity

The seeds of L. royleana methanolic crude extracts were evaluated using the nitric

oxide free radical scavenging activity. The standard used in the study was

butylated hydroxytoluene (BHT). The methanolic extract of Lallemantia royleana

seeds showed significant scavenging activity, and the percentage inhibition ranges

between 72.65 ± 0.268 and 2.831 ± 0.401 percent, at the concentration of 1000

µg/ml and 1.95 µg/ml respectively, whereas the percentage inhibition values of

BHT were found to be 56.44 ± 0.113 and 1.90 ± 0.380 percent, at the

concentration of 1000 µg/ml and 1.95 µg/ml respectively. The nitric oxide radical

scavenging activity values of the methanolic seed extracts along with standard

(BHT) shown in Table 5.5 and 5.6 and Figure 5.5. The high percentage inhibition

indicates high scavenging activity of the seed extract. The IC50 value of

L. royleana was found to be 99.93 ± 5.70 μg/ml, whereas the IC50 value of BHT

was found to be 364.60 ± 3.51 μg/ml.

5.9.3 Scavenging of Radical with the H2O2 (Hydrogen peroxide) Method

The hydrogen peroxide is a quit weak oxidizing agent. It can cause inactivation of

some enzymes directly, by oxidation of the thiol (-SH) groups. It can easily cross

cell membrane rapidly. Once reached inside the cell, H2O2 can possibly reacts with

Fe2+

and possibly Cu2+

to form hydroxyl radical. The formation of hydroxyl radical

is the initial step of the formation of many toxic effects (Miller et. al., 1993). It is

therefore very important and necessary for the cells to control the production of

hydrogen peroxide which was built up in vivo. The scavenging of H2O2 attributes to

their phenolic content which donate electrons to H2O2, thus was neutralizing it to

water (Halliwell and Gutteridge, 1985). The ability of the extract to effectively

scavenge hydrogen peroxide, determined according to the method done by Ruch

et. al., (1989), where they were compared with BHT. The L. royleana extracts were

capable of scavenging hydrogen peroxide in a concentration dependent manner.

The methanolic seed extracts exhibited 61.30 ± 0.234 and 3.44 ± 0.376 percent

inhibition, at the concentration of 1000 μg/ml and 1.95 µg/ml respectively, by

hydrogen peroxide anti-oxidant method. On the other hand, using the above

concentration butylated hydroxy toluene exhibited 77.03 ± 0.128 and 4.14 ± 0.128


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percent inhibition respectively, by hydrogen peroxide scavenging activity. The

percentage inhibition values of methanolic seed extracts and standard (BHT) have

shown in Table 5.7 and 5.8 and Figure 5.6. The IC50 value of L. royleana was

found to be 576.50 ± 0.00 μg/ml, whereas the IC50 value of BHT was found to be

26.16 ± 0.351 μg/ml.

5.9.4 Anti-oxidant Activity by DPPH (2, 2 – Diphenyl – 1- Picryl Hydrazyl)

Radical Scavenging Assay

The DPPH radical scavenging assay showed the ability of the extracts and the

standard to scavenge DPPH free radicals. The DPPH radical exists naturally in

deep violet colour but when reacts with anti-oxidant it turn into a yellow coloured

diphenyl picryl hydrazine. The degree of discoloration indicates the

radical-scavenging potential of the anti-oxidant (Tirzitis and Bartosz, 2010).

The results are shown as percentage inhibition values of the extracts at different

concentrations ranging from 1.95 to 1000 µg/ml. The crude methanolic extracts of

L. royleana seed gave percent inhibition of 68.72 ± 0.236 and 0.57 ± 0.197 when

tested using 1000 and 1.95 µg/ml respectively, of the plant extract which was

comparative with BHT, used as standard having percent inhibition of 73.03 ±

0.128 and 12.59 ± 0.128, at the concentration of 1000 µg/ml and 1.95 µg/ml

respectively. The DPPH radical scavenging activity values of the methanolic

extracts along with standard (BHT) have shown in Table 5.9 and 5.10 and Figure

5.7. The high percentage inhibition indicates high scavenging activity of the plant

extract. The IC50 value of L. royleana was found to be 140.53 ± 4.22 μg/ml.

whereas the IC50 value of BHT was found to be 43.40 ± 1.307 μg/ml.

5.9.5 Total Anti-oxidant Capacity by Phosphomolybdenum Method

The total anti-oxidant capacity of the methanolic crude plant extracts and BHT

were determined by using the method of phosphomolybdenum. The higher

absorbance value indicates the greater anti-oxidant activity. The total anti-oxidant

capacity of plant extracts were measured spectrophotometrically at 695 nm using

phosphomolybdenum method, which is based on the reduction of Mo (IV) to Mo

(V) by the test sample and the formation of green phosphate/Mo (V) compounds


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(Abbasi et. al., 2010). A high absorbance value of the sample indicates its strong

anti-oxidant activity. The total anti-oxidant capacity may be contributed due to

their chemical composition and phenolic acid content.

The percentage inhibition values of the L. royleana crude methanolic seed

extracts were found to range between 66.66 ± 2.309 and 1.33 ± 2.309 percent, at

the concentration of 1000 µg/ml and 1.95 µg/ml respectively, while the

percentage inhibition values of BHT were found to range between 77.12 ± 0.322

and 20.10 ± 0.207 percent, at the concentration of 1000 µg/ml and 1.95 µg/ml

respectively. The values of the methanolic seed extracts along with standard

(BHT) of total anti-oxidant capacity by phosphomolybdenum method were shown

in Table 5.11 and 5.12 and Figure 5.8. The high percentage inhibition indicates

high scavenging activity of the plant extract. IC50 value of L. royleana was found

to be 187.46 ± 0.55 μg/ml. whereas the IC50 value of BHT was found to be 124.25

± 3.04 μg/ml.

S.No. Plant conc.

(μg/ml)

Lallemantia royleana

(Absorbance)

Percent Inhibition

(%)

1. 1000 0.377 ± 0.000 69.78 ± 0.046

2. 500 0.255 ± 0.000 55.35 ± 0.10

3. 250 0.221 ± 0.001 48.41 ± 0.233

4. 125 0.183 ± 0.001 37.92 ± 0.517

5. 62.5 0.148 ± 0.001 22.97 ± 0.520

6. 31.25 0.142 ± 0.000 19.90 ± 0.324

7. 15.62 0.138 ± 0.000 17.78 ± 0.343

8. 7.812 0.131 ± 0.000 13.20 ± 0.380

9. 3.906 0.123 ± 0.001 7.31 ± 0.753

10. 1.95 0.118 ± 0.001 3.38 ± 0.385

Table No. 5.3: The scavenging effect of methanolic seeds extract of L. royleana

by Alkaline DMSO method. The different concentrations of

extracts used from 1000 to 1.95 µg/ml. The data represent the

percentage alkaline DMSO inhibition. Values are expressed as

mean ± SD (n=3).


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Table No. 5.4: The scavenging effect of BHT by Alkaline DMSO method. The

different concentrations of extracts used from 1000 to 1.95 µg/ml. The

data represent the percentage alkaline DMSO inhibition. Values are

expressed as mean ± SD (n=3).

Table No. 5.5: The nitric oxide radical scavenging activity of methanollic seeds

extract of L. royleana. The different concentrations of extracts used

from 1000 to 1.95 µg/ml. The data represent the percentage nitric

oxide inhibition. Values are expressed as mean ± SD (n=3).

S. No. Plant conc.

(μg/ml)

Butylated

Hydroxytoluene

(Absorbance)

Percent Inhibition

(%)

1. 1000 1.041 ± 0.000 63.52 ± 0.020

2. 500 0.549 ± 0.000 30.82 ± 0.072

3. 250 0.532 ± 0.000 28.61 ± 0.077

4. 125 0.526 ± 0.001 27.75 ± 0.137

5. 62.5 0.488 ± 0.001 22.13 ± 0.159

6. 31.25 0.479 ± 0.001 20.66 ± 0.165

7. 15.625 0.461 ± 0.001 17.62 ± 0.206

8. 7.8125 0.435 ± 0.000 12.71 ± 0.115

9. 3.906 0.422 ± 0.001 9.95 ± 0.213

10. 1.95 0.407 ± 0.001 6.63 ± 0.229

S.No. Plant conc.

(μg/ml)


(Absorbance)

Percent Inhibition

(%)

1. 1000 0.366 ± 0.001 72.65 ± 0.268

2. 500 0.342 ± 0.002 67.61 ± 0.269

3. 250 0.317 ± 0.000 51.94 ± 0.331

4. 125 0.280 ± 0.001 47.16 ± 0.338

5. 62.5 0.231 ± 0.000 43.71 ± 0.343

6. 31.25 0.212 ± 0.001 38.49 ± 0.059

7. 15.625 0.199 ± 0.001 25.48 ± 0.300

8. 7.8125 0.181 ± 0.001 15.66 ± 0.245

9. 3.906 0.122 ± 0.001 9.20 ± 0.548

10. 1.95 0.103 ± 0.001 2.83 ± 0.401


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Table No. 5.6: The nitric oxide radical scavenging activity of standard. The different

concentrations of standards used were 1000 to 1.95 µg/ml. The standard

from was butylated hydroxytoluene. The data represent the percentage

nitric oxide inhibition. Values are expressed as mean ± SD (n=3).

Table No. 5.7: The hydrogen peroxide radical scavenging activity of methanollic seeds

extract of L. royleana. The different concentrations of extracts used

from 1000 to 1.95 µg/ml. The data represent the percentage hydrogen

peroxide inhibition. Values are expressed as mean ± SD (n=3).

S.No. Plant conc.

(μg/ml)

Butylated Hydroxytoluene

(Absorbance)

Percent Inhibition

(%)

1. 1000 0.011 ± 0.000 56.44 ± 0.113

2. 500 0.121 ± 0.001 53.63 ± 0.4997

3. 250 0.138 ± 0.000 46.87 ± 0.111

4. 125 0.144 ± 0.001 44.82 ± 0.332

5. 62.5 0.151 ± 0.001 42.14 ± 0.604

6. 31.25 0.176 ± 0.001 32.56 ± 0.641

7. 15.625 0.203 ± 0.001 22.22 ± 0.681

8. 7.8125 0.212 ± 0.001 18.77 ± 0.352

9. 3.906 0.222 ± 0.001 14.94 ± 0.358

10. 1.95 0.257 ± 0.001 1.90 ± 0.380

S.No. Plant conc.

(μg/ml)


(Absorbance)

Percent

Inhibition

(%)

1. 1000 0.101 ± 0.001 61.30 ± 0.234

2. 500 0.134 ± 0.002 48.53 ± 0.670

3 250 0.154 ± 0.001 40.74 ± 0.365

4. 125 0.165 ± 0.001 36.78 ± 0.547

5. 62.5 0.177 ± 0.001 32.05 ± 0.842

6. 31.25 0.186 ± 0.001 28.35 ± 0.108

7. 15.625 0.197 ± 0.001 24.52 ± 0.093

8. 7.8125 0.212 ± 0.001 18.77 ± 0.694

9. 3.906 0.221 ± 0.002 15.07 ± 1.058

10. 1.95 0.252 ± 0.001 3.44 ± 0.376


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Table No. 5.8: The hydrogen peroxide radical scavenging activity of standard.

The different concentrations of standard used from 1000 to 1.95 µg/ml.

The standard used was butylated hydroxytoluene. The data represent the

percentage hydrogen peroxide inhibition. Values are expressed as mean

± SD (n=3).

Table No. 5.9: The DPPH radical scavenging activity of methanollic seeds extract of

L. royleana.The different concentrations of extracts used from 1000 to

1.95 µg/ml. Values are expressed as mean ± SD (n=3).

S.No. Plant conc.

(μg/ml)

Butylated hydroxytoluene

(Absorbance)

Percent

Inhibition (%)

1. 1000 0.103 ± 0.005 77.03 ± 0.128

2. 500 0.119 ± 0.000 73.48 ± 0.128

3. 250 0.136 ± 0.000 69.70 ± 0.128

4. 125 0.167 ± 0.001 62.88 ± 0.222

5. 62.5 0.200 ± 0.001 55.55 ± 0.222

6. 31.25 0.217 ± 0.001 51.77 ± 0.222

7. 15.625 0.241 ± 0.000 46.37 ± 0.128

8. 7.8125 0.313 ± 0.001 30.37 ± 0.339

9. 3.906 0.380 ± 0.001 15.55 ± 0.222

10. 1.95 0.431 ± 0.000 4.14 ± 0.128

S.No. Plant conc. (μg/ml) Lallemantia royleana

(Absorbance)

Percent

Inhibition

(%)

1. 1000 0.091 ± 0.001 68.72 ± 0.236

2. 500 0.118 ± 0.000 59.33 ± 0.327

3. 250 0.128 ± 0.000 55.78 ± 0.250

4. 125 0.147 ± 0.000 49.25 ± 0.264

5. 62.5 0.166 ± 0.001 42.95 ± 0.539

6. 31.25 0.187 ± 0.000 35.62 ± 0.296

7. 15.625 0.208 ± 0.000 28.40 ± 0.124

8. 7.8125 0.227± 0.002 21.87 ± 0.464

9. 3.906 0.268 ± 0.001 7.90 ± 0.660

10. 1.95 0.289 ± 0.000 0.57 ± 0.197


138

Table No. 5.10: The DPPH radical scavenging activity standard. The different

concentrations of extracts used from 1000 to 1.95 µg/ml. The standard

used was butylated hydroxytoluene. Values are expressed as mean ± SD

(n=3).

Table No. 5.11: Total Anti-oxidant Capacity of methanollic seeds extract of L. royleana.

The different concentrations of extracts used from 1000 to 1.95 µg/ml.

Values are expressed as mean ± SD (n=3).

S.No. Plant conc. (μg/ml) Butylated Hydroxytoluene

(Absorbance)

Percent Inhibition

(%)

1. 1000 0.121 ± 0.000 73.03 ± 0.128

2. 500 0.149 ± 0.000 66.81 ± 0.128

3. 250 0.194 ± 0.001 56.88 ± 0.222

4. 125 0.201 ± 0.000 55.25 ± 0.128

5. 62.5 0.213 ± 0.002 52.59 ± 0.462

6. 31.25 0.232 ± 0.000 48.44 ± 0.000

7. 15.625 0.248 ± 0.001 44.88 ± 0.222

8. 7.8125 0.287 ± 0.000 36.14 ± 0.128

9. 3.906 0.331 ± 0.000 26.29 ± 0.128

10. 1.95 0.393 ± 0.000 12.59 ± 0.128

S.No. Plant conc. (μg/ml) Lallemantia royleana

(Absorbance)

Percent Inhibition

(%)

1. 1000 0.008 ± 0.000 66.66 ± 2.309

2. 500 0.010 ± 0.000 57.33 ± 2.309

3. 250 0.011 ± 0.000 53.33 ± 2.309

4. 125 0.013 ± 0.000 46.66 ± 2.309

5. 62.5 0.015 ± 0.000 40.00 ± 0.000

6. 31.25 0.017 ± 0.000 30.66 ± 2.309

7. 15.625 0.019 ± 0.000 24.00 ± 0.000

8. 7.8125 0.020 ± 0.000 17.33 ± 2.309

9. 3.906 0.022 ± 0.000 9.33 ± 0.369

10. 1.95 0.024 ± 0.000 1.33 ± 2.309


139

Table No. 5.12: Total Anti-oxidant Capacity of standard. The different concentrations of

extracts used from 1000 to 1.95 µg/ml. The standard used was butylated

hydroxytoluene. Values are expressed as mean ± SD (n=3).

Figure No. 5.4: Graphical representation of percent inhibition of methanolic extract

of the seeds of Lallementia royleana (LR) and butylated hydroxy

toluene (BHT) as standard by using Alkaline DMSO method.

0

10

20

30

40

50

60

70

80

1000 500 250 125 62.5 31.25 15.625 7.81 3.9 1.95

Percen

tage I

nh

ibit

ion

(%

)

Plant Concentration (µg/ml)

LR

BHT

S.No. Plant conc. (μg/ml) Butylated HydroxyTotuene

(Absorbance)

Percent Inhibition

(%)

1. 1000 0.099 ± 0.001 77.12 ± 0.322

2. 500 0.143 ± 0.001 66.92 ± 0.290

3. 250 0.176 ± 0.001 59.40 ± 0.365

4. 125 0.217 ± 0.001 50.03 ± 0.175

5. 62.5 0.234 ± 0.000 46.05 ± 0.061

6. 31.25 0.269 ± 0.000 38.06 ± 0.082

7. 15.625 0.300 ± 0.000 30.92 ± 0.091

8. 7.812 0.319 ± 0.001 26.47 ± 0.233

9. 3.906 0.337 ± 0.001 22.25 ± 0.347

10. 1.95 0.347 ± 0.001 20.10 ± 0.207


140

Figure No. 5.6: Graphical representation of percent inhibition of methanolic extract of

the seeds of Lallementia royleana (LR) and Butylated hydroxy toluene

(BHT) as standard by using hydrogen peroxide scavenging method.

0

10

20

30

40

50

60

70

80

90

1000 500 250 125 62.5 31.25 15.625 7.81 3.9 1.95

Per

cen

tage

Inh

ibit

ion

(%)


LR

BHT

Figure No. 5.5: Graphical representation of percent inhibition of methanolic extract of the

seeds of Lallementia royleana (LR) and Butylated hydroxy toluene (BHT)

as standard by using nitric oxide radical scavenging activity.

0

10

20

30

40

50

60

70

80

1000 500 250 125 62.5 31.25 15.625 7.81 3.9 1.95

Pe

rce

nta

ge In

hib

itio

n (%

)


LR

BHT


141


seeds of Lallementia royleana (LR) and Butylated hydroxy toluene (BHT) as

standard by using DPPH radical scavenging activity.


seeds of Lallementia royleana (LR) and Butylated hydroxy toluene

(BHT) as standard by using total anti-oxidant capacity.

0

10

20

30

40

50

60

70

80

1000 500 250 125 62.5 31.25 15.625 7.81 3.9 1.95

Pe

rce

nta

ge In

hib

itio

n (%

)


LR

BHT

0

10

20

30

40

50

60

70

80

90

1000 500 250 125 62.5 31.25 15.625 7.81 3.9 1.95

Percen

tag

e I

nh

ibit

ion

(%

)


LR

BHT


142

5.10 IC50 value of different anti-oxidant activity

The IC50 values of the methanolic extracts were calculated based on the results of

different anti-oxidant assay were conducted such as DPPH, Alkaline DMSO, Nitric oxide

scavenging assay, total anti-oxidant assay and hydrogen peroxide method. The results are

given below in Table 5.13.

Table No.5.13: Comparative chart of IC50 value of different anti-oxidant activity

of methanolic seeds extract of L. royleana and standard. The

standard used was butylated hydroxytoluene (BHT). Unit for

IC50 for all the activities are µg/ml. Data are expressed as mean

± SD (n=3).

5.11 Thin Layer Chromatography

Thin layer chromatography (TLC) is a sophisticated method and a type of planar

chromatography used in the present study to identify the components in the Lallemantia

royleana methanolic seed extract such as alkaloids, phenols, flavonoids, etc. The

separation depends on the relative affinity of the compounds towards stationary and

mobile phase. The compounds travel under the influence of mobile phase (driven by

capillary action) over to the surface of the stationary phase. The compound with higher

affinity travels slowly in stationary phase while others travel faster. All the silica gel

glass plates contained a reference spot along with the L.royleana methanolic seed extract.

The gallic acid and tannic acid were used as reference. The silica gel glass plates were

S.No. Test Performed IC50 value for

Seeds Extract

Butylated

Hydroxytoluene

1. Alkaline DMSO Method 308.67 ± 7.12 792.49 ± 1.16

2. DPPH Method 140.53 ± 4.22 43.40 ± 1.307

3. H2O2 Method 576.50 ± 0.00 26.166 ± 0.351

4. Nitric Oxide Method 99.93 ± 5.70 364.60 ± 3.510

5. Total Anti-oxidant Capacity

Method 187.46 ± 0.55 124.25 ± 3.04


143

developed in an iodine chamber in the presence of iodine fumes. The yellow to purplish

pink colour spots or rockets were observed. The retention factor (Rf) of the unknown

coumpound is compared with Rf of the known compound (gallic acid and tannic acid). Rf

is the retention factor, indicating how far the compound has travelled on the silica gel

plates.

The thin layer chromatogram was prepared by using 2 µg ml-1

of L. royleana methanolic

seed extract and standard compounds on silica gel plates. The Rf values of the extract and

standard were observed, calculated and compared. It was found that the chromatogram

had been showing bands at the similar distances as that of the band of tannic acid and

gallic acid and their Rf values calculated were similar.

The Rf value of L. royleana methanolic seed extract was observed to be 0.80. The Rf

value of gallic acid and tannic acid are 0.88 and 0.91.Thus, it indicates there are

phenolics present in the L. royleana methanolic seed extract. Apart from these, other

bands were also seen indicating the presence of other compounds as well. The results are

shown in Figure 5.9.

5.12 GC-MS Analysis of Lallemantia royleana Methanolic Extract

The crude methanolic seeds extract of L. royleana was characterized using GC-MS

analysis to evaluate the compounds present in it. The results of GC-MS analysis showed

that at least 21 compounds are present in methanolic extract of L. royleana. The

compounds which were identified through mass spectrometry are attached with GC. The

mass spectra of these compounds were matched with those found in the NIST05 and

WILEY 8 spectral database. The fragmentation of major compound was found in 9, 12,

15-Octadecatrienoic acid, (Z, Z, Z)-74.54% (retention time: 15.442 min), n-Hexadecanoic

acid 13.77% (retention time: 14.206 min) and 1, 2, 3-propanetriol (retention time:

4.943min) 1.27%. The active principles along with their retention time, area, area percent

and compound name in the methanolic extract of L. royleana are given in Table 5.14.

The chromatogram of GC-MS is given in Figure 5.10.


144

Figure No. 5.9: Thin layer chromatography plate showing resolution of

methanolic crude extracts. Tannic acid (T.A.), Gallic acid

(G.A.) and L. royleana (L.R.)

Figure No. 5.10: Chromatogram of Lallemantia royleana methanolic seeds extract

L.R. G.A. T.A.


145

Peak R. Time Area Area% Name

1 4.165 5843004 0.76 1-Butane, 4-Isothiocyanato-

2 4.943 9758315 1.27 1,2,3-Propanetriol

3 5.960 1649384 0.22 Undecane

4 8.611 748205 0.10 Cyclohexasiloxane, dodecamethyl-

5 10.592 571484 0.07 trans-Z-.alpha.-Bisabolene epoxide

6 10.810 1418485 0.19 Phenol, 3,5-bis(1,1-dimethylethyl)-

7 11.409 1615616 0.21 3',5'-Dimethoxyacetophenone

8 12.804 1759571 0.23 Tetradecanoic acid

9 13.140 541339 0.07 Isopropyl Myristate

10 13.492 4015724 0.52 1,2-Benzenedicarboxylic acid, Bis(2-

Methylpropyl) Ester

11 14.206 105549348 13.77 n-Hexadecanoic acid

12 14.670 2020914 0.26 Eicosanoic acid

13 14.961 29815108 3.89 9,12-Octadecadienoic acid, methyl ester

14 15.442 571182242 74.54 9,12,15-Octadecatrienoic acid, (Z,Z,Z)-

15 18.373 4291323 0.56 Tetratriacontane

16 20.404 3003773 0.39

Naphthalene, 1,2,3,5,6,7,8,8a-

octahydro-1,8a-dimethyl-7-(1-

methylethenyl)-, [1R-(21

17 21.998 1373739 0.18 9,19-Cyclolanostan-3-ol, acetate,

(3.beta.)-

18 24.940 1565530 0.20 7-(Trimethylsilyloxy)-3-[4-

(Trimethylsilyloxy)Phenyl]-4H-1-

19 26.679 1099323 0.14 7-(Trimethylsilyloxy)-3-[4-

(Trimethylsilyloxy)Phenyl]-4H-1-

20 27.009 1546408 0.20 2-Butanone, 4-(2,2-Dimethyl-6-

Methylenecyclohexylidene)-

21 27.698 6488647 0.85 Tetrahydroabietic acid

Table No. 5.14: The table shows peak results of L. royleana methanolic seed extract


146

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