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INDEX – GJRMI - Volume 3, Issue 11, November 2014

MEDICINAL PLANTS RESEARCH

Bio-Chemistry

EVALUATION OF THE BIOCHEMICAL AND HEMATOLOGICAL PARAMETERS IN THE

SERUM OF ALBINO RATS FED WITH SKIMMED, WHOLE, FLAVORED AND SOYA MILK

COMMONLY CONSUMED IN NIGERIA

Essien E B, Onwuka F C, Odjoh O, Odeghe O B 389–401

Biology

PRELIMINARY SCIENTIFIC INVESTIGATION OF THE EFFECTIVENESS OF THE

MEDICINAL PLANTS PLANTAGO MAJOR AND ACHILLEA MILLEFOLIUM AGAINST THE

BACTERIA PSEUDOMONAS AERUGINOSA AND STAPHYLOCOCCUS AUREUS IN

PARTNERSHIP WITH INDIGENOUS ELDERS

Suzanne Nilson, Fidji Gendron, Jody Bellegarde, Betty McKenna, Delores Louie, Geraldine Manson, Harvey Alphonse

402–415

Natural & Life Sciences

EVALUATION OF PHENOLIC COMPOUNDS, FLAVONOIDS AND ANTIOXIDANT

PROPERTIES OF ARGANIA SPINOSA (L.) SKEELS. LEAF EXTRACTS

Saliha DJIDEL, Choubaila -Feriel CHATER, Seddik KHENNOUF, Abderrahmane BAGHIANI, Daoud HARZALLAH

416–426

INDIGENOUS MEDICINE

Ayurveda – Dravya Guna

ASSESSMENT OF ‘VIPAKA’ (METABOLISM) OF A NEW MEDICINAL PLANT IN ANIMAL

MODEL

Bidhan Mahajon, Ravi Shankar B, Remadevi R 427–434

Review Article

QUESTIONNAIRE DESIGNING AND VALIDATION IN AYURVEDIC RESEARCH

Ravi Bhat, Shivprasad Chiplunkar, Suhaskumar Shetty, Arhanth Kumar 435–444

COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R, PLANT ID – INFLORESCENCE OF CHITRAKA – PLUMBAGO ZEYLANICA L. OF THE

FAMILY PLUMBAGINACEAE PLACE – KOPPA, CHIKKAMAGALUR DISTRICT,

KARNATAKA, INDIA

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 389–401

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

EVALUATION OF THE BIOCHEMICAL AND HEMATOLOGICAL

PARAMETERS IN THE SERUM OF ALBINO RATS FED WITH

SKIMMED, WHOLE, FLAVORED AND SOYA MILK

COMMONLY CONSUMED IN NIGERIA

Essien E B1*, Onwuka F C

2, Odjoh O

3, Odeghe O B

4

1,2,3,4

Department of Biochemistry, Faculty of Science, P.M.B 5323 University of Port Harcourt, Rivers State,

Nigeria.

*Corresponding Author: E-mail: mmedara2002@yahoo.com

Received: 07/08/2014; Revised: 20/10/2014; Accepted: 30/10/2014

ABSTRACT

The effect of milk samples on haematological and biochemical parameters in the serum of twenty-

five albino rats was evaluated. Rats were subjected to feeding trial over a period of 4 weeks on diets

containing: 100g of standard rat feed and water (group A), 55g of milk sample with 45g of standard rat

feed and water for groups B (skimmed milk), C (whole milk), D (soya milk), and E (flavoured milk). At

the end of the experimental period, the highest weight gain was observed in rats fed with soya milk

(61.20%), while rats fed with skimmed milk had the least weight gain (32.40%) when compared to the

control (47.80%). Rats fed with soya milk had the highest hemoglobin concentration (13.24 ± 0.42g/dl)

and packed cell volume (39.80 ± 1.28%). The urea concentration of rats fed with soya milk was higher

(3.10 ± 0.05 mmol/l) than values obtained from the other milk samples evaluated. Results of creatinine

and bilirubin concentrations of rats in all groups were within normal values, while the values obtained

from the enzyme activities analyzed were consistent with normal reference values. Rats fed with

skimmed milk had the highest cholesterol and high density lipoprotein cholesterol concentrations as 3.70

± 0.05mmol/l and 0.91 ± 0.00mmol/l respectively, when compared to the control (3.23 ± 0.03 and

0.83 ± 0.01mmol/l respectively). A hypocholesteremic effect was observed in rats fed with whole milk,

soya milk and flavoured milk. Rats fed with flavoured milk and skimmed milk had higher concentration

of low density lipoprotein cholesterol (2.03 ± 0.61mmol/l and 2.33 ± 0.06mmol/l respectively) when

compared to the control group (1.96 ± 0.03mmol/l). The least triglyceride concentration was observed in

rats fed with soya milk (1.06 ± 0.08mmol/l) when compared to the control, while rats fed with skimmed

milk, whole milk and flavored milk had elevated triglyceride levels. Results of present investigation

demonstrate the benefits of consuming soya milk. On the other hand, the consumption of skimmed milk

with respect to weight gain is encouraged.

KEY WORDS: Milk, biochemical, hematological, enzyme activities, blood lipids.

Research Article

Cite this article:

Essien E B, Onwuka F C, Odjoh O, Odeghe O B (2014), EVALUATION OF THE BIOCHEMICAL AND

HEMATOLOGICAL PARAMETERS IN THE SERUM OF ALBINO RATS FED WITH SKIMMED,

WHOLE, FLAVORED, AND SOYA MILK COMMONLY CONSUMED IN NIGERIA,

Global J Res. Med. Plants & Indigen. Med., Volume 3(11): 389–401

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 389–401

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

INTRODUCTION

Milk is the natural secretion of the

mammary glands which plays a fundamental

role in nutrition, growth, development and

immunity of the newly born young (Woo et al.,

1995). Each species of mammals produces

milk with a unique composition designed to

meet the specific needs of the infants. For

instance, the milk of animals that grow rapidly,

such as cows which double their birth weight in

50 days is rich in protein and minerals. Milk

has also been defined as an emulsion of fat

globules in a suspension of casein micelles, all

suspended in an aqueous phase which contains

solubilized lactose, whey proteins and mineral

salts (Jensen, 1998). Milk is a highly nutritious

versatile food. People enjoy drinking milk in its

natural form and also use it to make a wide

range of food products including butter, yogurt,

cheese and ice cream. Cow’s milk and milk

products have played an important role in

human nutrition. Fresh cow milk is reported to

contain about 88% water (Kataoka et al.,

1991). During processing, the water content of

the milk is reduced, which confers desirable

qualities on the milk such as increased shelf

life, product flexibility and decreased

transportation cost (Miller et al., 1999). Milk

and milk products play an important part in a

healthy diet as they contribute to intakes of

essential nutrients and protein of a nutritionally

high quality. Milk products provide beneficial

nutrients including calcium, riboflavin, protein

and vitamin A to the diet (Block, 1985), but

whole milk products also contribute significant

amount of fats, saturated fat and cholesterol,

which have been shown to increase blood

cholesterol and subsequently pose a risk of

coronary heart disease (Kristi et al., 1994).

Several investigations on the effect of milk

in relation to coronary heart diseases have been

carried out on both rats and man. While some

investigations reveal the benefits of milk

consumption, other studies have established a

link of the dairy product to coronary heart

disease. Still, some other researchers have

encouraged the consumption of specific milk

brands due to results obtained from their

findings. But there was no convincing evidence

that milk is harmful (Elwood et al., 2004).

Another study found no evidence that men

(aged 35–64 years) who consumed milk each

day, at a time when most milk consumed was

full fat milk, were at increased risk of death

from all causes or from coronary heart disease

(Ness et al., 2001). On the contrary, another

study has shown a high positive correlation

between milk consumption in different

countries and rates of death a few years later

from coronary heart disease (Margaret, 2002).

Milk intake is probably positively related to

blood lipids (Steinmetz et al., 1994). Although

milk has long been considered an important

factor in coronary heart disease because of the

contribution it makes to the dietary intake of

saturated fats, expert groups have advised that

milk consumption should be limited, and that

fat reduced milk should be preferred

(Nutritional Aspects of Cardiovascular Disease,

1994). This fact was further strengthened in a

report by Kritchevsky et al. (1979), in which

they pointed out that there is a factor in milk

which helps to reduce cholesterol levels in rats

and man. Although the mechanism by which

milk help to reduce cholesterol level is unclear,

they suggest that milk does not exert a

hypercholesterolemic effect. A study on eight

healthy male subjects (adults) demonstrated the

benefits of drinking skimmed milk, as

compared with whole milk (Kristi et al., 1994).

As a result of the effects of milk on human

health, some individuals now consume soya

milk in place of dairy milk products. Using soy

milk to replace foods high in animal protein

that contain saturated fat and cholesterol may

confer benefits to cardiovascular health (Sacks

et al., 2006). Comparative clinical trials have

shown that consumption of diets rich in soy

protein as opposed to those high in animal

protein significantly lowered blood total

cholesterol, low density lipoprotein, and

triglycerides, without lowering helpful high

density lipoprotein cholesterol (Anderson et al.,

1995). As a result of the previous research

investigations on the effect of milk on coronary

heart diseases examined, the present study was

carried out with a view of bringing to light the

effect of milk consumption on the

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 389–401

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

hematological and biochemical parameters in

the serum of albino rats.

MATERIALS AND METHODS

Collection and preparation of milk samples

The different types of milk used for this study

were purchased from a local market in Choba,

Port Harcourt. Dano slim milk serve as

skimmed milk, cowbell chocolate flavor milk

as flavoured milk, while peak instant full cream

milk powder as the whole milk used for this

study. Soya bean seed (Glycine maximus) were

bought from a local market, cleaned off dirt and

soaked with clean water for 12 hours. It was

thereafter hulled, washed, and ground to a

homogenous paste. To this was added water to

form a slightly liquid mixture and filtered with

cheese cloth to obtain the filtrate as milk which

was analyzed immediately.

Experimental design

Twenty-five albino rats (Wistar strain)

weighing between 182–247g were purchased

from the Animal House of the Department of

Biochemistry, University of Port Harcourt. The

animals were then divided into five groups of

five rats each designated A (control), B, C, D,

and E. Before the commencement of the dietary

regimen, the animals were fasted over night but

allowed access to water ad libitum. The

treatment protocol is as follows: group A

received 100 g of standard rat feed and water,

groups B, C, D, and E were fed 55 g of

skimmed, whole, soya, and flavoured milk

respectively with 45 g of standard rat feed.

Assay

At the end of the study period, the animals

were exposed to chloroform vapour, and about

2ml of blood sample was obtained by cardiac

puncture, which was transferred into EDTA

and heparin bottles. Blood samples were then

centrifuged at 4000 rpm for 10 minutes to

obtain serum, which was stored in the

refrigerator and analyzed three hours later. The

weights of the animals were taken before and

after the dietary regimen. The proximate

analysis was determined using the standard

method of AOAC (1984). These include the

determination of crude protein, crude fat,

moisture content, ash, crude fiber,

carbohydrates and minerals, while the

phytochemical screening of the secondary

metabolites in soya milk was by the method of

Harborne (1973). The vitamin contents were

analyzed according to the method of AOVC

(1966). The energy content was obtained by

multiplying the protein, fat and carbohydrates

by factors 4, 9 and 4 respectively.

Haematological parameters were analyzed

using microhaematocrit method and Sahli’s

haemoglobinometer as described by Ramnik,

1990. The biochemical parameters analyzed

was carried out using commercial kits from

Randox laboratories Ltd (Northern Ireland).

Total protein was determined by the Biuret

reaction described by Tietz (1990) and albumin

concentrations were estimated by method of

Doumas et al. (1971). Creatinine estimation

was done using Reflotron, a semi automated

dry chemistry analyzer, and urea was by the

method of Fawcett and Scott, (1960). Bilirubin

concentration was by the method of Jendrassik

and Grof, (1938). Serum samples were

analyzed for aspartate aminotransaminase

(AST), alanine aminotransaminase (ALT), and

alkaline phosphatase activities using

commercial kits as described by Reitman and

Frankel (1957), and Klein et al. (1960).

Determination of total cholesterol in the serum

was by the method of Trinder, (1960); high

density lipoprotein cholesterol (HDL-C) was

determined by the method of Friedewald

(1972), while the level of low density

lipoprotein cholesterol (LDL-C) was calculated

using Friedewald’s equation. Serum

triglyceride (TG) was determined using the

method of Tietz, (1990).

Statistical analysis

The data were analyzed using inferential

statistics. All values are presented as Mean ±

SEM (standard error of mean) for 5 rats in each

of the 5 groups. The significance of difference

in the means of all parameters reported was

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 389–401

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

determined using one way ANOVA by least

significant difference (LSD) comparison test.

RESULTS

Results of the study to evaluate the

proximate composition, biochemical and

hematological parameter in the serum of albino

rats fed with skimmed milk, whole milk, soya

milk and flavoured milk are presented in tables

1 to 7. The result of proximate analysis of milk

samples is presented in Table 1. The milk

samples had energy values of between

261.5kcal and 393.2kcal. Whole milk had the

highest energy value while skimmed milk had

the least energy value. The highest crude

protein obtained was recorded in skimmed milk

(28.13%), followed by whole milk (24.01%),

and soya milk (21.44%), with flavoured milk

having the least crude protein value of 12.36%.

The ash content was highest in skimmed milk

(8.70%), with the least value in flavoured milk

(2.13%). Although whole milk had the highest

fat content of 23.16% than the other milk

samples, it had the least moisture content

(0.81%), while skimmed milk had the least fat

content (2.52%). The fiber content of the milk

samples range between 0 and 36.62%, with

soya milk having the highest fiber content.

Flavoured milk had the highest carbohydrate

content of 56.44%, while the least carbohydrate

content was obtained from soya milk. The

vitamin and mineral contents of whole and

flavoured milk were reported as stated by the

manufacturers, while the trace elements in

skimmed milk were analyzed. The proximate

composition of soya milk was obtained by

analysis. The highest of vitamin B1 was

observed in whole milk (0.99 mg), with soya

milk obtaining the least value (0.19mg).

Skimmed and flavoured milk both had the

highest content of vitamin B2, being 1.40 mg

respectively, while soya milk had the least

vitamin B2. Although soya milk had the highest

vitamins B6 and B12 values of 5.35 mg and 3.47

mg, than the other milk samples, it obtained a

corresponding least vitamin C content (3.63

mg), with whole milk had the highest vitamin C

content. Results of macro and trace minerals

obtained revealed skimmed milk as having the

highest content of calcium (1800 mg),

phosphorus (900 mg), and potassium (1600

mg), with soya milk had the least calcium (81.5

mg), phosphorus (5.50 mg), and potassium (12

mg) contents. The magnesium content was

higher in soya milk (192.69 mg), and least in

whole milk (85 mg). The iron content range

between 0.17 and 15.30 mg, with the highest

value obtained from soya milk and whole milk

having the least value. The zinc content of

whole milk was higher than the other milk

samples analysed. Results of phytochemical

screening of soya milk indicate the presence of

glycosides, steroids, terpenoids, and reducing

sugars (Table 2).

The initial and final body weights of rats

fed with milk samples are presented in Table 3.

An increase in body weight was observed in

each group at the end of the dietary regimen.

However, there was no significant difference in

the initial and final body weights of the control

and treatment group. Rats fed with soya milk

(61.20%) gained the highest weight when

compared with the control group (47.80%),

while the least weight gained was observed in

rat groups fed with skimmed milk (32.40%).

The hematological investigation on rats fed

the milk samples is presented in Table 4. Group

D rats fed soya milk had the highest

hemoglobin concentration (13.24 ± 0.42g/dl)

and packed cell volume of 39.80 ± 1.28%,

while the least values was obtained from the

control group (10.04 ± 0.98 g/dl and

30.00 ± 2.94%) respectively. No significant

difference was observed in the hemoglobin

concentration of groups B, C and E rats (fed

with skimmed, whole, and flavoured milk

respectively) when compared with the control,

while a significant difference was observed in

the packed cell volume of groups B and E rats

when compared with the control group.

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 389–401

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Table 1. Proximate analysis of milk samples and soya milk analyzed

Parameter Skimmed milk whole milk Flavoured milk Soya milk

Energy (kcal) 261.5 393.2 339.2 339.2

Protein (%) 28.13 ± 0.15 24.01 ± 0.03 12.36 ± 0.64 21.44 ± 0.29

Ash (%) 8.70 ± 0.01 5.44 ± 0.03 2.13 ± 0.13 6.50 ± 0.02

Fats (%) 2.52 ± 0.08 23.16 ± 0.23 7.11 ± 0.20 21.90 ± 0.42

Moisture (%) 3.96 ± 0.03 0.81 ± 0.01 21.52 ± 0.57 1.20 ± 0.01

Fiber (%) 0.00 0.00 0.00 36.62 ± 0.03

Carbohydrate (%) 31.57 ± 0.82 22.21 ± 0.03 56.44 ± 2.23 12.34 ± 0.17

Vitamin A (IU) 2500* 2700 3750 23.21 ± 0.02

Vitamin B1(mg) NI 0.99 0.90 0.19 ± 0.02

Vitamin B2 (mg) 1.40 1.10 1.40 0.15 ± 0.03

Vitamin B3 (mg) NI 0.60 11.00 0.98 ± 0.08

Vitamin B6 (mg) NI 0.90 1.50 5.35 ± 0.04

Vitamin B12(mg) 0.003 0.0024 0.0045 3.47 ± 0.07

Vitamin C (mg) NI 90 30 3.63 ± 0.03

Vitamin E (mg) NI 0.60 4.00 0.34 ± 0.05

Calcium (mg) 1800 930 380 81.5 ± 0.06

Phosphorus (mg) 900 750 312 5.50 ± 0.02

Magnesium (mg) 120 85 121 192.69 ± 0.07

Potassium (mg) 1600 1200 523 12.00 ± 0.06

Sodium (mg) 19.87 340 106 2.59 ± 0.11

Iron (mg) NI 0.17 13.50 15.30 ± 0.06

Zinc (mg) 0.08 31 3.8 0.20 ± 0.03

Copper (mg) 0.12 0.02 0.1 0.20 ± 0.03

Selenium (µg) NI 10 17.5 7.15 ± 0.02mg

Manganese (mg) 0.04 0.02 0.10 0.01 ± 0.02 *Enriched, NI= Not indicated

Table 2. Phytochemical Screening Of Soya Milk

Secondary metabolites Relative abundance

Alkaloids −

Flavonoids −

Glycosides ++

Saponins ND

Steroids ++

Terpenioids +++

Carbohydrates ND

Reducing sugar ++

Resin ND

Tannins −

Proteins ND

Oils ND

Acid compounds ND Key: − = Absent; + = Low in concentration; ++ = Moderate in concentration; +++ = High in concentration;

ND = Not determined

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 389–401

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Table 3. Mean body weights of rats (grams) fed the milk samples

Groups Initial body weight Final body weight Weight gained (%)

Control 218.20 ± 12.31b 266.00 ± 14.50

b 47.80

Skimmed milk 208.00 ± 9.43b 240.40 ± 12.25

b 32.40

Whole milk 206.60 ± 6.79b 242.20 ± 7.45

b 35.60

Soya milk 209.80 ± 10.05b 271.00 ± 10.95

b 61.20

Flavoured milk 209.60 ± 8.61b 245.60 ± 9.26

b 36.00

Values are mean ± SEM (n=5/group). bP>0.05. One way ANOVA by least significant difference comparison (LSD) test

Table 4. Hematological parameters of rats fed with milk samples

Group Hemoglobin (g/dl) Packed cell volume (%)

Control 10.04 ± 0.98b 30.00 ± 2.94

b

Skimmed milk 11.22 ± 0.44b 33.60 ± 1.20

a

Whole milk 10.47 ± 0.77b 31.50 ± 2.32

b

Soya milk 13.24 ± 0.42a 39.80 ± 1.28

a

Flavoured milk 11.50 ± 0.16b 34.80 ± 0.37

a

Values are mean ± SEM (n=5 rats/group). Values in the same row carrying different superscripts are significantly

different (P<0.05).

The results obtained for the biochemical

parameters considered in this study is presented

in Table 5. The control rats had the highest

serum total protein concentration being 67.40 ±

0.86g/l, while animals fed soya milk had the

least total protein concentration of 44.73 ±

4.78g/l. The least albumin concentration was

obtained by rats fed with soymilk (27.13 ±

2.70g/l), followed by rats fed with flavoured

milk (31.13 ± 2.54g/l) and whole milk (31.70 ±

3.09g/l) respectively. The control group had the

highest albumin concentration as 40.20 ±

0.49g/l, followed by rats fed with skimmed

milk (39.40 ± 0.00g/l). Although no significant

difference was observed in serum creatinine,

urea and bilirubin concentration of rats in each

groups, the creatinine concentration ranged

from 57.33 ± 0.66 to 60.66 ± 0.33µmol/l. Rats

fed with flavoured milk had the least creatinine

concentration, with the highest concentration

observed in rats fed with soya milk. Rats fed

with soya milk had the highest urea

concentration of 3.10 ± 0.05 when compared

with the control rats (2.90 ± 0.10), with the

least value obtained by rats fed with flavoured

milk (2.80 ± 0.05mmol/l). A slight significant

increase in urea concentration was observed in

rats fed with skimmed milk and whole milk

when compared to the control group, while rats

fed flavoured milk had slight reduction as

compared to the control group. The direct

bilirubin concentrations of rats ranged between

3.26 ± 0.14 µmol/l (soya milk) and 4.00 ±

0.57µmol/l (whole milk). The control rats

obtained the highest total bilirubin

concentration being 7.53 ± 0.37µmol/l,

followed by rats fed with soya milk (7.26 ±

0.21µmol/l), while rats fed with whole milk

had the least total bilirubin concentration (6.96

± 0.32µmol/l).

The enzyme activities of animals fed the

various experimental diets and the control is

depicted in Table 6. From the results obtained,

no significant difference (P>0.05) was

observed in the activities of aspartate

aminotransaminase (AST), alanine

aminotransminase (ALT) and alkaline

phosphatase when compared with the values

obtained from the control rats.

The effect of the various milk samples on

serum lipid profile of rats is shown in Table 7.

From the results obtained, the highest

cholesterol level was obtained by rats fed

skimmed milk being 3.70 ± 0.05mmol/l, while

rats fed whole milk had the least cholesterol

value (1.76 ± 0.08 mmol/l). A significant

reduction in the cholesterol level of rats fed

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 389–401

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

with whole milk, soya milk and flavoured milk

was observed when compared with the control

group (3.23 ± 0.03mmol/l). The high density

lipoprotein cholesterol levels of rats had values

of between 0.47 ± 0.03 and 0.91 ± 0.00mmol/l.

Although no significant difference in the high

density lipoprotein cholesterol levels of both

the control and treatment groups was observed,

rats fed with skimmed milk had the highest

high density lipoprotein cholesterol value with

the least value obtained by rats fed with whole

milk. The low density lipoprotein cholesterol of

the rats had values between 0.77 ± 0.08 and

2.33 ± 0.06 mmol/l. The highest value was

obtained from rats fed with skimmed milk, with

rats fed with whole milk having the least value.

The triglyceride concentration in the test and

non-test groups had values between 1.06 ± 0.08

and 2.43 ± 0.12mmol/l. The highest triglyceride

value was obtained by rats fed whole milk,

while rats fed with soya milk had the least

value. A significant increase in triglyceride

concentration of rats fed with skimmed and

whole milk was observed when compared with

the control group (1.73 ± 0.03mmol/l), with

rats fed with soya and flavoured milk showing

a significant decrease.

Table 5. Serum concentrations of the biochemical parameters analyzed.

Parameter Control

Skimmed

milk

Whole milk Soya milk Flavoured

milk

Total protein (g/l) 67.40 ± 0 .86b 65.40 ± 0.29

b 52.83 ± 5.13

a 44.73 ± 4.78

a 55.23 ± 4.76

a

Albumin (g/l) 40.20 ± 0.49b 39.40 ± 0.00

b 31.70 ± 3.09

a 27.13 ± 2.70

a 31.13 ± 2.54

a

Creatinine (µmol/l) 58.00 ± 2.00b 59.33 ± 1.76

b 59.33 ± 0.66

b 60.66 ± 0.33

b 57.33 ± 0.66

b

Urea (mmol/l) 2.90 ± 0.10b 2.96 ± 0.08

b 2.96 ± 0.03

b 3.10 ± 0.05

b 2.80 ± 0.05

b

Direct bilirubin

(µmol/l)

3.66 ± 0.24b 3.93 ± 0.06

b 4.00 ± 0.57

b 3.26 ± 0.14

b 3.70 ± 0.35

b

Total bilirubin

(µmol/l)

7.53 ± 0.37b 6.70 ± 0.20

b 6.96 ± 0.32

b 7.26 ± 0.21

b 6.73 ± 0.83

b

Values are mean ± SEM (n=5). Values in the same row carrying different superscripts are significantly different

(P<0.05).

Table 6. Serum activities of enzymes studied (U/L)

Parameter

Control Skimmed

milk

Whole milk Soya milk Flavoured

milk

Aspartate

aminotransaminase

5.90 ± 0.05b 5.66 ± 0.33

b 6.00 ± 0.57

b 6.03 ± 0.03

b 5.86 ± 0.13

b

Alanine

aminotransaminase

6.10 ± 0.05b 6.00 ± 0.57

b 6.00 ± 0.00

b 5.30 ± 0.40

b 5.33 ± 0.33

b

Alkaline phosphatase 17.60 ± 0.30b 18.03 ± 0.03

b 17.33 ± 1.45

b 15.26 ± 0.37

b 16.00 ± 1.05

b

Values are Mean ± SEM (n=5). Values in the same row carrying the same superscripts are not significantly different

(P>0.05).

Table 7. Serum Lipid Profile of rats fed the various milk samples (mmol/l)

Parameter Control Skimmed

milk

Whole milk Soya milk Flavoured

milk

Cholesterol 3.23 ± 0.03b 3.70 ± 0.05

b 1.76 ± 0.08

a 2.10 ± 0.15

b 3.13 ± 0.78

b

High density lipoprotein 0.83 ± 0.01b 0.91 ± 0.00

b 0.47 ± 0.03

b 0.58 ± 0.04

b 0.64 ± 0.17

b

Low density lipoprotein 1.96 ± 0.03b 2.33 ± 0.06

b 0.77 ± 0.08

a 1.26 ± 0.08

b 2.03 ± 0.61

b

Triglycerides 1.73 ± 0.03a 2.26 ± 0.08

a 2.43 ± 0.12

a 1.06 ± 0.08

a 1.60 ± 0.05

a

Values are Mean ± SEM (n=5). Values in the same row carrying different superscripts are significantly different

(P<0.05).

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DISCUSSION

The results of proximate composition of the

milk samples as depicted in Table 1 shows a

slight variation in the energy values of whole

milk, flavoured milk and soya milk. This

variation was due to the energy contents

obtained from the carbohydrate, crude protein

and fat contents of the milk samples. The

highest energy value was obtained from whole

milk, while skimmed milk had the least energy

value. The consumption of skimmed milk is

therefore recommended for those who wish to

reduce their calorie intake.

Results of crude protein revealed that the

consumption of skimmed milk, whole milk and

soya milk are good dietary sources of protein. It

is reported that an adult would have to drink

about two liters of milk to satisfy the

recommended daily allowance for protein (60–

70g) (Pamplona-Roger, 2004).

The ash content of a food sample is a

reflection of its mineral element composition.

Skimmed milk was shown to have the highest

ash content, followed by soya milk, with the

least ash content obtained from flavoured milk.

The higher ash content in the skimmed milk

analysed revealed a rich composition of

mineral elements, especially the macro

elements.

One cup of 236 ml whole milk contains

approximately 629kJ (150kcal) and 8 grams of

fat (5 grams of which are saturated) as

compared with 356kJ (85kcal) and 0.4g fat in

one cup of skimmed milk (United States

Department of Agriculture, 1976). The highest

crude fat was obtained from whole milk when

compared with the fat contents of the other

milk samples while skimmed milk had the least

crude fat content. The least crude fat obtained

from skimmed milk is a reflection of its

reduced fat (calorie) content during the

production process. Hence, adults who wish to

reduce their calorie intake from milk products

should be encouraged to consume skimmed

milk. The moisture content of the milk samples

was highest in flavoured milk, and this tends to

decrease its keeping property. Whole milk had

the least moisture content which may indicate

that it would keep longer than other samples.

Of the milk samples analysed, only soya

milk had a fiber content which could be

attributed to the fact that it is obtained from

plant source, as opposed to processed milk

from animal source. Soya milk proved to be an

excellent source of dietary fiber, hence its

consumption should be highly recommended.

The carbohydrate content of soya milk was

low, as compared with the carbohydrate

contents in the processed milk samples.

Flavoured milk was shown to contain the

highest carbohydrate content.

Vitamin content analyses as presented in

Table 1 showed skimmed milk, whole milk,

and flavoured milk to be good sources of

vitamin A as opposed to the content in soya

milk. Thus processed dairy milk powder

provides the daily recommended intake of this

vitamin being 600–900 mg for adults, and 300–

400 mg for children (Daily Reference Intakes,

2001).

Results shows the milk samples to be a

poor source of vitamin B1 (thiamin), as they do

not provide the daily recommended need of

0.9–1.2 mg/day (for adults) and 0.5–0.6 mg/day

for children (Daily Reference Intakes, 1998).

The consumption of approximately 28 grams of

skimmed and flavoured milk by adults and

children can provide the daily intake of vitamin

B2 (riboflavin), being 0.5–0.6 mg for children

and 0.9–1.3 mg for adults (Daily Reference

Intakes, 1998). These milk samples show them

to be poor sources of vitamin B3

(Nicotinamide) as they do not provide the daily

requirement for this vitamin. Soya milk proved

to be good sources of vitamins B6 (pyridoxine)

and B12 (cobalamine), when compared to the

contents derived from the processed milk

samples. The Daily Reference Intake of vitamin

C by adults is 45–90 mg and 15–25 mg for

children. Result of proximate composition of

the whole milk powder used for this study is

shown to provide the daily need of this vitamin

by both adults and children. The milk samples

also proved to be poor sources of vitamin E

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 389–401

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

since they do not provide the required daily

intake for both children and adults. The mineral

elements constitute an important group of

nutrients required by the body for optimal

functions (WHO, 1996). They are divided into

macro minerals (sodium, potassium,

magnesium, calcium and phosphorus) and trace

elements (iron, zinc, copper and manganese).

The sodium content of the milk samples

prove to be poor sources of sodium since they

do not provide the daily reference intake of

1.0–1.2g (for children), and 1.2–1.5g for adults

(DRI, 2001). The estimated safe and adequate

daily dietary intake for potassium is 550–4575

mg in children, and 1875–5625 mg in adults

(Daily Reference Intakes, 2001). Result shows

that the milk samples analyzed do not

contribute to the daily requirement of this

element. Skimmed milk had the highest

calcium and phosphorus content with soya milk

had the least calcium and phosphorus content.

This indicates that consumption of skimmed

and whole milk can provide the daily need for

calcium and phosphorus in both children and

adults. In this present study, soya milk was

shown to contain the highest magnesium

content over the dairy milk products analyzed.

Soya milk consumption contributes to the

recommended daily allowance of magnesium in

children, being 70–170mg/day, but not for

adults who require about 270–400mg/day

(Food and Nutrition Board, 1989).

The milk samples analyzed were found to

contain 0.17–15.30mg of iron per 100 gram. Of

these, soya milk had the highest iron content

than the processed milk samples. Consumption

of soya milk as dietary source of iron should be

encouraged. The milk samples had zinc

contents of between 0.20 and 31 mg per 100

gram. The recommended daily allowance of

this element is 10 mg for children and 12–

15 mg for adults (FNB, 1989). Result of

analysis shows that the consumption of whole

milk provides a remarkable contribution of this

element in both adults and children, due to its

high zinc content. The copper content of the

milk samples was found to be below the

recommended daily intake. Although soya milk

had the highest copper intake, these milk

samples should not be consumed by adults or

children deficient of this element as sources of

dietary copper. The estimated safe and

adequate daily intake of manganese is 1–2mg

in children, and 2–5 mg in adults (FNB, 1989).

Results of proximate composition of

manganese range between 0.01 and 0.10 mg

per 100 gram. This shows that the milk samples

to be poor sources of this element.

The results of phytochemical screening of

soya milk as shown in Table 2 indicates the

presence of moderate concentration of

glycosides, steroids, reducing sugars, and high

concentration of terpenoids. Alkaloids,

flavonoids and tannins were found to be absent

in soya milk.

Results obtained from rat feeding studies

shows increase in body weights of rats in all

groups (Table 3). Rats fed soya milk showed a

considerable weight gain when compared with

rats fed dairy milk samples. The least weight

gain was observed in rats fed skimmed milk,

followed by rats fed whole milk. As a result,

adults who wish to control their weight, with a

significant reduction of their calorie intake

should be encouraged to consume skimmed

milk in preference to whole and flavoured milk.

Skimmed milk ability to cause the least weight

gain is due to its low calorie content (Table 1)

when compared with those of whole milk, soya

milk and flavoured milk.

From the results of hematological

investigations (Table 4), it was observed that

consumption of skimmed milk, whole milk and

flavoured milk by rats resulted in a significant

decrease in hemoglobin concentration and

packed cell volume. The hemoglobin and

packed cell volume concentrations are basic

values revealing the degree of anemia.

Although the hemoglobin concentration and

packed cell volume of rats fed soya milk were

slightly lower than the reference values, these

values were significantly higher than the values

obtained for rats fed the other milk samples.

Soya milk has been reported to be a rich source

of iron (Murray-Kolb, et al., 2003).

Consumption of soya milk as a source of

dietary iron is therefore encouraged.

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The results of biochemical estimations are

presented in Table 5. Total protein is the sum

of albumin (60%) and globulins in the serum.

Albumin is synthesized by the liver using

dietary protein. A decrease in serum total

protein and albumin concentration was

observed in rats fed soya milk. This decrease

may be due to the soy protein not digested and

absorbed properly.

The creatinine concentration of rats in all

groups reveals normal and healthy values.

Although no significant difference (P>0.05) in

the creatinine concentration of rats in all groups

was observed, rats fed with soya milk had the

highest creatinine concentration. The effect of

the milk samples on serum creatinine did

indicate any harmful benefits on milk

consumption.

Rats fed with soya milk had the highest

urea concentration when compared to the

control group. The urea concentration of rats

fed with skimmed milk, whole milk, and

flavoured milk were slightly lower than the

value obtained from rats fed soya milk.

The bilirubin concentration of rats in each

group was with normal clinical values (Table

5). Bilirubin is formed by the breakdown of

hemoglobin in the liver, bone marrow and

spleen. An increase in plasma bilirubin results

in jaundice. Although no significant difference

was observed (P>0.05) in the bilirubin

concentration of both the test and non-test

groups, the highest direct bilirubin

concentration was observed in rats fed with

whole milk, with the control group having the

highest total bilirubin concentration. The effect

of the milk samples analysed on serum

bilirubin did not indicate the presence of

jaundice.

Enzyme assay is usually conducted to

determine the health condition of tissues

especially the liver and heart. High activities of

these enzymes in the blood are an indication of

tissue damage. No significant difference

(P>0.05) was observed in aspartate

aminotransaminase, alanine aminotransaminase

and alkaline phosphatase activities of rats in all

groups. The results indicates a slight lower

aspartate aminotransaminase activity in rats fed

skimmed and flavoured milk when compared

with the control, while rats fed soya milk and

flavoured milk had lower alanine

aminotransaminase activities as compared with

the control group. The alkaline phosphatase

activity of rats fed with skimmed milk was

higher than the control, while rats fed with

whole milk, soya milk and flavoured milk had

lower alkaline phosphatase activities when

compared with the control. The effect of the

milk samples on enzyme activities of rats in

each group revealed healthy concentrations

when with normal clinical values.

The result of lipid profile analyses is shown

in Table 7. Rats fed with skimmed milk had

the highest serum cholesterol and high density

lipoprotein cholesterol, which were within

normal clinical values. Contrary to the expected

elevated level of serum cholesterol

concentration in rats fed whole milk, a

hypocholesteremic effect was observed. Rats

fed with soya milk and flavoured milk also had

reduced cholesterol concentration when

compared with the control group. Soya milk

has been shown to decrease serum total

cholesterol level in rats (Anderson et al., 1995;

Zhan and Ho, 2005). Consumption of whole

milk, soya milk and flavoured milk did not lead

to increased concentration of high density

lipoprotein cholesterol in this study, instead, a

reduction was observed. Comparison of the

cholesterol and high density cholesterol

concentrations with normal clinical values,

revealed healthy levels in rats fed with

skimmed milk, while rats in the other groups

had lower concentrations.

Rats fed with skimmed and flavoured milk

had a higher low density lipoprotein cholesterol

concentration when compared with control,

which is between normal reference values. It is

important to note that soya milk and whole

milk consumption by rats led to a significant

lowering of serum low density lipoprotein

cholesterol.

An elevated concentration of triglycerides

was observed in rats fed skimmed and whole

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 389–401

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milk when compared with the control, with

whole milk having the highest triglyceride

concentration. The least concentration was

observed in rats fed with soya milk, which

indicate that soya milk had beneficial effect on

triglyceride. Result shows that consumption of

skimmed milk, whole milk and flavoured milk

had positive effect on serum triglyceride level.

CONCLUSION

Although the highest weight gained was

observed in rats fed with soya milk, results of

lipid profile analysis revealed the health benefit

of consuming the non-dairy product. A

hypocholesteremic effect was observed in rats

fed whole milk, soya milk and flavoured milk.

The benefit of skimmed milk on weight gain

was also observed. Thus, the consumption of

skimmed milk by adults who wish to control

their weight should be encouraged, since

consumption of this milk led to the least weight

gain by rats. The effect of these milk samples

did not reveal potential harm on human health.

Consumption of soya milk over the processed

milk samples evaluated should be encouraged.

In conclusion, the hypothesis that consumption

of whole milk leads to coronary heart diseases

due to its saturated fat content was not

confirmed by this study.

RECOMMENDATION

This relatively short-term study indicates

that soya milk appears to have beneficial

advantages over the processed milk samples

studied. A longer term effect of these milk

samples, with further investigation on

flavoured milk should be evaluated.

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Source of Support: NIL Conflict of Interest: None Declared

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Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

PRELIMINARY SCIENTIFIC INVESTIGATION OF THE EFFECTIVENESS

OF THE MEDICINAL PLANTS PLANTAGO MAJOR AND

ACHILLEA MILLEFOLIUM AGAINST THE BACTERIA

PSEUDOMONAS AERUGINOSA AND STAPHYLOCOCCUS AUREUS IN

PARTNERSHIP WITH INDIGENOUS ELDERS.

Suzanne Nilson1, Fidji Gendron

2*, Jody Bellegarde

3, Betty McKenna

4, Delores Louie

5,

Geraldine Manson6, Harvey Alphonse

7

1,5,6,7Biology Department, Vancouver Island University, 900 Fifth Street, Nanaimo, British Columbia, V9R 5S5

Canada 2,3,4

First Nations University of Canada, 1 First Nations Way, Regina, Saskatchewan, S4S 7K2 Canada

*Corresponding Author: Email: fgendron@fnuniv.ca; Telephone: 306-790-5950 ext 3335; Fax: 306-790-

5994

Received: 25/09/2014; Revised: 07/11/2014; Accepted: 10/11/2014

ABSTRACT This preliminary investigation was undertaken in partnership with Indigenous elders to

investigate the antibacterial effectiveness of common Plantain (Plantago major L.) and Yarrow

(Achillea millefolium L.) against the skin pathogens Pseudomonas aeruginosa and Staphylococcus

aureus. Plants were selected, prepared and antibacterial chemicals were tested from plants harvested

according to elders‟ guidance. Spectrophotometry, Kirby Bauer disc diffusion testing, standard

bacterial population counts, and determination of concentrations of the plant antibacterial chemicals,

alkaloids and saponins, were conducted. The spectrophotometry method provided results that were

ineffective at determining viable bacterial biomass. Kirby Bauer disc diffusion testing and standard

bacterial population counts showed that both plants were more consistently effective against the

gram positive bacterium, S. aureus, versus the gram negative, P. aeruginosa. Although not

significant, alkaloid concentration in P. major was higher at the 7:00 p.m. picking time compared to

the 11:30 a.m. picking time, which agreed with the elder‟s Indigenous science knowledge. Saponin

concentration in P. major, on the other hand, showed similar results for the 11:30 a.m. and 7:00 p.m.

picking times. In addition to determining antibacterial effectiveness against common skin pathogens,

the use of local plant species for medicinal preparations also contributes to the discussion of possible

alternatives to antibiotic preparations for topical healing of bacterial skin infections.

KEYWORDS: alkaloids, antimicrobial, saponins, traditional medicine in Northern America,

antibiotics, Plantago major L. and Achillea millefolium L.

Research Article

Cite this article:

Suzanne Nilson, Fidji Gendron, Jody Bellegarde, Betty McKenna, Delores Louie, Geraldine

Manson, Harvey Alphonse (2014), PRELIMINARY SCIENTIFIC INVESTIGATION OF THE

EFFECTIVENESS OF THE MEDICINAL PLANTS PLANTAGO MAJOR AND ACHILLEA

MILLEFOLIUM AGAINST THE BACTERIA PSEUDOMONAS AERUGINOSA AND

STAPHYLOCOCCUS AUREUS IN PARTNERSHIP WITH INDIGENOUS ELDERS,

Global J Res. Med. Plants & Indigen. Med., Volume 3(11): 402–415

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 402–415

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

INTRODUCTION

The development of resistant bacteria from

prolonged exposure to antibacterial agents,

such as antibiotics, and harmful effects

resulting from the toxicity of antibiotic usage is

an increasing public health challenge.

Considering these concerns, bioactive chemical

agents in plants might be one helpful solution,

requiring further investigation. Plant medicines

are widely known and continue to make an

important contribution to health care for many

Indigenous people (Holetz et al., 2002; Ferreira

et al., 2012; Alkholy et al., 2013; Ferreira et

al., 2013), however less than 10% of higher

plant species have been investigated for

biological activity, such as antibacterial

effectiveness (Fabricant and Farnsworth, 2001).

Two plants historically used by local

Indigenous people of British Columbia and

Saskatchewan in treating skin and wound

infection are the common Plantain (Plantago

major L.) and Yarrow (Achillea millefolium

L.). This study works in partnership with First

Nations elders to bring Indigenous science

knowledge together with Western science

knowledge and further the investigation of

local plants used as medicines to treat skin &

wound infections caused by the bacteria P.

aeruginosa and S. aureus.

Plantago major is a perennial species that

grows from a short, thick taproot. It has broad

oval dark green basal leaves with green to

white small flowers that are borne in a dense

spike. This commonly used medicinal plant is

an introduced species that grows in disturbed

places such as roadsides, trails, and urban areas

(Vance et al., 1999). Plantain leaves have been

used as a wound healing remedy for centuries

in almost all parts of the world and have also

been used in the treatment of a number of

diseases apart from wound healing (Samuelsen,

2000). For example, this plant is known as

nature‟s “Band-Aid” and is invaluable as a first

aid remedy for cuts, scrapes, bee stings, and

burns (Keane, 2009). Indigenous elders from

British Columbia and Saskatchewan often use

P. major in the treatment of skin

wounds/infection. In British Columbia, plantain

is called “Frog‟s Leaves” by elder Geraldine

Manson. Also known as “Frog‟s Pants”, the

following is a story told by elder Betty

McKenna from Saskatchewan: “Plantain is

called frog‟s pants because of the Woman‟s

medicine wheel. On this medicine wheel,

woman is facing north, the fish is facing south

and the turtle and the frog are facing the right

and left sides, respectively. All these living

organisms have their eggs when they are born,

so they share the same healing ways. Plantain is

called the Frog‟s Pants because it is believed

that the frog came, hopped away and left its

pants, which are the plantain‟s leaves. Women

take the frog‟s pants, chew the leaves and apply

them as a compress on the skin to cure certain

diseases. As people were living close to the

land, plantain was especially useful for soil-

borne diseases such as rashes, sty and pink

eyes. The compress is also good at drawing the

infection out. Chewing plantain is an important

step as it is believed that the medicinal

properties of plantain are released when

combined with saliva. It is important to the

woman who is chewing the leaves not to have

fillings or gold teeth as these materials change

the medicines. Although it is a cure for

everyone, it is traditionally the women who

would chew it because they were the medicine

people in their family. Women would chew

several plantain leaves and spit them out in a

container to give them to people who would

then bring the container home for future uses.

Roots were also used once they were boiled”

(B. McKenna, personal communication, 2011).

Elders Geraldine Manson and Delores Louie of

British Columbia agree with elder Betty‟s

shared knowledge, which aligns similarly with

their own knowledge.

Achillea millefolium possesses white flower

heads that are densely packed in a round topped

terminal cluster. Its woolly leaves are divided

into many segments that grow from a branched

rhizome. Achillea millefolium is one of the

most abundant white flowers growing across

the Canadian prairie and British Columbia. In

North America, Indigenous people use it for

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healing wounds (Chandler et al., 1982). The

traditional knowledge keeper Harvey Alphonse

from British Columbia advised on the use of A.

millefolium as traditional medicine to treat skin

infection while reducing inflammation. Elders

in Saskatchewan call this plant species

porridge-on-a-stick and share that “a tea made

using the entire top of the plant helps support

the immune system and can be used for chest

infections. It can also be boiled in water and

used as a rinse to make your hair shiny and get

rid of dandruff” (Yuzicapi et al., 2013).

Antibacterial properties associated with

many plants are attributed to the biologically

active compounds identified as alkaloids and

saponins. Alkaloids are a large family of

nitrogen-containing secondary metabolites

whose main function is to defend against

predators (Taiz and Zeiger, 2002). Saponins are

glycosides with soap-like properties that act as

feeding deterrents against herbivores (Taiz and

Zeiger, 2002). Alkaloids and saponins are both

found in P. major (Cowan 1999; Mojab et al.,

2003; Cordeiro et al., 2006) and A. millefolium

(Chandler et al., 1982; Khan and Gilani, 2011),

and have shown marked antibacterial activities

against gram positive bacteria (Avato et al.,

2006; Khan et al., 2012). Saponins are known

to be particularly effective against gram

positive bacteria (such as S. aureus) compared

to gram negative bacteria (such as P.

aeruginosa) (Pistelli et al., 2002, Avato et al.,

2006, Soetan et al., 2006).

Previous studies have also indicated that

environmental conditions associated with

different geographical locations may influence

levels of biologically active plant compounds

(Lagalante and Montgomery, 2003). Similarly,

Indigenous science knowledge also informs

that picking the plant leaves at specific times of

the day may provide more or fewer benefits

relative to the effectiveness of the plant

medicine against bacterial wound infections

(elder Geraldine Manson, personal

communication, 2011).

This preliminary study has employed

selected standard methods including

spectrophotometry, Kirby Bauer disc diffusion

testing, and bacterial population counts to

investigate the effectiveness of local plant

medicines. The plants selected for study, P.

major and A. millefolium, are used by

Indigenous people‟ in British Columbia and

Saskatchewan against wound infection and will

be used for study on the known bacterial skin

pathogens P. aeruginosa and S. aureus. The

investigation also includes the Indigenous

science knowledge and advisement of local

elders by determining the antibacterial

effectiveness of plant medicine treatments

intended to parallel advised usage by the elders,

and determines the levels of alkaloid and

saponin concentrations at the advised picking

times of 11:30 a.m. and 7:00 p.m.

MATERIALS AND METHODS

Plant material

Plantago major and Achillea millefolium

were identified by S.N. and F.G. and collected

with elders following traditional protocols. In

British Columbia, whole plants were picked

along the Nanaimo River in the town of Cedar

during June 2012. Plantago major was picked

at different times during the day (11:30 a.m.

and 7:00 p.m.) because the elder informed the

research team that the late picking time is the

most recommended for antibacterial

effectiveness in wounds. In Saskatchewan,

whole plants were collected from the vicinity of

Moose Jaw in July 2011. Plant material was

washed in a 10% bleach solution (Kinney et al.,

1987) and dried at 40o

C (Thakhiew et al.,

2014) until constant weight was observed and

ground to powder. The powered plant material

was used for the Soxhlet extraction procedures.

Ground plant material was also exchanged

between laboratories in British Columbia and

Saskatchewan. Spectrophotometry, Kirby

Bauer disc diffusion testing and bacterial

population counts occurred in British

Columbia, while alkaloid and saponin

determinations were conducted in

Saskatchewan.

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Spectrophotometry

In accordance with communication with the

traditional knowledge keeper H. Alphonse and

the elder D. Louie, P. major and A. millefolium

were dried, weighed, and placed into sterile

beakers. P. major was weighed at 0.5 g, 5.0 g

and 10.0 g while A. millefolium was weighed at

0.08 g, 5.38 g and 10.76 g. To conduct a

combined treatment to test for synergistic

effects using a P. major / A. millefolium

combination, each plant was weighed at 0.08 g,

5.38 g and 10.76 g and placed into beakers

(triplicate). The weighed plant matter was then

soaked in a 10% bleach solution for 15

minutes, followed by rinsing twice with

distilled water (Kinney et al., 1987).

Elder D. Louie advised us that chewing P.

major is an important step for preparation of

the plant medicine. The chewing process may

help with the release of plant chemical

components. Therefore, according to the elder‟s

advisement, the different weights of plant

matter for the P. major trials and the combined

P. major / A. millefolium trials were placed into

individual sterile mortars and saliva (from same

individual) was added depending on weight.

Based on the elder‟s knowledge, 1.0, 3.0 and

6.0 ml of saliva were added to the 0.5, 5.0 and

10.0 g of P. major, respectively. For the

combination study, 6.0 ml of saliva was added

to the P. major / A. millefolium for each of the

sample weights investigated. The plant / saliva

mixtures were pressed twenty times each using

a sterile pestle to conduct a procedure to

parallel chewing practices, as recommended by

the elder‟s local Indigenous science knowledge.

The plant / saliva mixtures were then

aseptically transferred into different sterile

beakers, and 40 ml of sterile Trypticase Soy

Broth (TSB) were added to each beaker. Each

of the three weights of A. millefolium was

covered with tin foil and steeped using 62.5 ml

of TSB for one hour in keeping with the local

Indigenous science knowledge of H. Alphonse.

Following this procedure, 5 ml of each of the

plant/broth solutions were pipetted into

spectrophotometry test tubes (triplicates), and

each test tube was inoculated with 100 µl of

bacteria P. aeruginosa (ATCC 10145) or S.

aureus (ATCC 25923). Controls were also

developed in triplicates. The control for the P.

major and P. major / A. millefolium treatments

consisted of broth with 3 ml of saliva. The

control for the A. millefolium treatments

paralleled the teachings of H. Alphonse and

consisted of TSB broth with no saliva.

Immediately following inoculation with

bacterial cultures, initial absorbance readings

were conducted. The test tubes were then

incubated for 18 hours at 37° C, followed by

the taking of an absorbance reading using a

Spectronic 20 (Milton Roy Company) and the

recording of the difference between the two

readings. Absorbance readings were taken at an

optical density of 600 nm for both P.

aeruginosa (Davies et al., 1993; Kim et al.,

2012) and S. aureus (Nychas et al., 1990).

Soxhlet extraction for Kirby Bauer disc

diffusion testing

To conduct Soxhlet extractions, 79 g of

dried plant material were used to fill Soxhlet

thimbles and 150 ml of methanol (ACS

Laboratory grade) were used to conduct

extraction procedures. The final extracts were

then roto-evaporated at 30o C, at 235 RPM,

until thick in consistency, but not yet solidified.

The extracts were then transferred to sterile

glass vials and maintained in dark conditions

by wrapping in tin foil.

From the freshly prepared plant extract,

final extract solutions of 500 mg/ml, 50 mg/ml,

5 mg/ml and 0.5 mg/ml (10% sterile dimethyl

sulfoxide (DMSO)) were filtered using 0.45

microliter filter syringes. Sterile filter discs (6

mm) were then saturated with 40 µL of each

plant extract solution, placed into sterile,

covered petri dishes and stored at room

temperature in the dark and overnight to

remove excess methanol (Mistry et al., 2010).

Following this time interval, the filter discs (6

mm) were applied to agar plates (in triplicates)

previously swabbed with bacterial cultures of

P. aeruginosa (ATCC 10145) and S. aureus

(ATCC 25923), using McFarland Standard

procedures for conducting the Kirby Bauer disc

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diffusion test (Kelly et al., 1999). The selected

bacterial populations were cultured at a density

adjusted to 0.5 of McFarland scale for uniform

swabbing of bacteria onto the surface of the

agar plates (Kelly et al., 1999). In addition to

plant extract treatments, positive controls using

the antibiotics Ciprofloxacin and Gentimicin

were tested for effectiveness. Ciprofloxacin is

a fluoroquinolone that acts against both gram

positive (e.g. S. aureus) and gram negative

bacteria (e.g. P. aeruginosa) (Agrawal et al.,

2007). Gentimicin is an aminoglycoside that

acts best against gram negative bacteria.

Negative controls included methanol and

DMSO saturated filter discs.

Bacterial population counts

Following incubation of test tubes

containing plant treatments for

spectrophotometry procedures, serial dilutions

were conducted on randomly selected test tube

solutions. Procedures for bacterial population

counts were then conducted and recorded as log

of colony forming unit per ml (cfu ml-1

)

(Harley and Prescott, 2002; Nilson and Holley,

2012; Hazan et al., 2012) following a 24 hour

incubation period at 37o C. For each bacterial

species, controls with broth and saliva (P.

major) and broth only (A. milifolium) were also

prepared.

Soxhlet extraction for determination of the

plant biological compounds

Chemicals used throughout these

procedures were of analytical grade (hexanes,

HCL, methanol, acetone (from Fisher, ON,

Canada), 95% ethanol and NH4OH (from

Sigma, ON, Canada), KOH (from Occidental

Chemical Corporation, TX, USA), petroleum

ether (from BDH, ON, Canada), and

chloroform and CH2Cl2 (from EMD, ON,

Canada). The extraction was performed at

ambient pressure at the boiling point of the

solvent used. A 3.0 g of powered plant material

was extracted with 250 ml of hexane on a water

bath for 6 h in triplicate using the Soxhlet

apparatus (Tarvainen et al., 2010).

The obtained plant extracts were cleaned

from oily materials by saponifying (Daruházi et

al., 2008). To conduct this procedure, the

hexane extracts were concentrated under

vacuum. The residues were then saponified

with 50 ml of 95% ethanol and 2 g of KOH in

50 ml ethanol solution in a hot water bath. The

extracts were diluted with 100 ml of distilled

water and were shaken with 75 ml and then 2 ×

50 ml portions of petroleum ether. The organic

phases were collected and the solution obtained

was washed with 2 × 50 ml portions of distilled

water until neutral pH then evaporated in a

Büchi Rotavapor R-205 under vacuum. The dry

extract was weighed and dissolved in 4 ml of

chloroform. These unsaponified extracts were

stored in the refrigerator at 4o C until analysis.

Determination of alkaloids

Alkaloids were determined following the

method of Fazal et al. (2011). Ten grams of

grounded plant material was extracted with 100

ml of 100% ethanol. Once the ethanol was

evaporated, 2 g of dried extract was dissolved

in 20 ml of 5% HCL. The mixture was

centrifuged for 10 minutes and the aqueous

portion was basified with NH4OH. The basic

solution was extracted three times with CH2Cl2

and concentrated under reduced pressure by

using a Büchi Rotavapor R-205. Once dried,

the sample was weighed to determine the

amount of alkaloid residues.

Determination of saponins

Saponins were determined following the

method of Fazal et al. (2011). Ten grams of

grounded plant material were defatted with 100

ml of hexane and incubated for 10–15 minutes.

Hexane was separated from the plant extract,

which was extracted three times with 30 ml of

methanol. The resulting solution was

concentrated to one third of its original volume

and 100 ml cold acetone was added to this

extract. The extract and acetone solution was

refrigerated for 50 minutes. The extract was

then filtered by pressure filtration using pre-

weighed filter paper (Whatman No. 1

Qualitative Circles 125 mm). The weight of the

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saponins was determined by subtracting the

weight of the pre-weighted filter paper to the

weight of the filter paper with saponins.

Statistical analyses

One-way ANOVA and Tukey‟s HSD were

used to analyze the differences between the

diameters of zones of inhibition using the Kirby

Bauer disc diffusion test with P. major. One-

way ANOVA and Tukey-Kramer‟s were used

to analyze spectrophotometry data. Alkaloid

and saponin mean percentages in P. major at

different times were compared by using a t-test.

Statistical analyses were carried out with the

statistical analysis softwares R (version 3.0.1)

and NCSS and values of p < 0.05 were noted

as statistically significant.

RESULTS

Spectrophotometry

Plantago major / A. millefolium combined

treatment vs. P. aeruginosa:

Combined P. major with A. millefolium

treatments were used to observe possible

synergistic antibacterial effects on bacterial

growth. Treatments with P. major / A.

millefolium at 0.08 and 5.38 g, with 6 ml saliva,

showed absorbance results at 0.65 and 0.77,

respectively, and were greater than and

significantly different than the control result at

0.31 (Table 1). At the 10.76 g P. major / A.

millefolium treatment, absorbance results at

0.26 showed a lower result, significantly

different than the other treatments, but not

significantly different than the control

treatment.

Plantago major / A. millefolium combined

treatment vs. S. aureus

When P. major / A. millefolium treatments

at 0.08, 5.38 and 10.76 g each, with 6 ml of

saliva were used, absorbance results at 0.97,

0.75 and 1.05 showed no significant difference

between plant medicine treatments (Table 1).

All P. major / A. millefolium treatments showed

greater absorbance readings and a significant

difference when compared with the control

treatment at 0.37.

Plantago major treatment vs. P. aeruginosa

Plantago major treatments of 0.5, 5.0 and

10.0 g results showed no significant differences

in absorbance between the plant treatments and

were 0.65, 0.71, and 0.71, respectively (Table

2). All plant treatments showed greater and

significantly different absorbance results than

the control at 0.31.

Plantago major treatment vs. S. aureus

Plantago major treatments at 0.5, 5.0 and

10.0 g showed absorbance results at 0.91, 0.81

and 0.91, respectively, and were greater than

the control at 0.37, but not significantly

different than the control. Results between

plant treatments were not significantly different

(Table 2).

Table 1. Spectrophotometry results for the different combined P. major / A. millefolium plant

medicine treatments when testing effectiveness against P. aeruginosa and S. aureus.

Treatments P. major / A. millefolium

P. aeruginosa S. aureus

Control 0.31a 0.37

a

0.08 g 0.65b 0.97

b

5.38 g 0.77b 0.75

b

10.76 g 0.26a 1.05

b

Means within each column with the same letter (a) are not significantly different (p < 0.05).

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Table 2. Spectrophotometry results for Plantain (P. major) treatments when testing

effectiveness against P. aeruginosa and S. aureus.

Treatments P. major P. aeruginosa

P. major S. aureus

Control 0.31a 0.37

a

0.5 g 0.65b 0.91

a

5.0 g 0.71b 0.81

a

10.0 g 0.71b 0.91

a

Means within each column with the same letter (a) are not significantly different (p < 0.05).

Achillea millefolium treatment vs. P.

aeruginosa

Absorbance results for A. millefolium

treatments at concentrations of 0.08, 5.38 and

10.76 g were observed (Table 3). For plant

medicine treatments, 0.08 and 5.38 g,

absorbance results of 0.97 and 1.24,

respectively, were significantly different and

greater than the control at −0.17. When the

10.76 g treatment was applied, the absorbance

reading at 0.45 showed no significant

difference with the control group.

Achillea millefolium treatments vs. S. aureus

Achillea millefolium treatments at 0.08 and

5.38 g showed greater and significantly

different absorbance values at 1.60 and 0.77

when compared with control at 0.41 (Table 3).

Achillea millefolium treatment of 10.76 g

showed absorbance results at 0.35 and no

significant difference when compared with the

control group.

Table 3. Spectrophotometry results for Yarrow (A. millefolium) treatments when testing

effectiveness against P. aeruginosa and S. aureus.

Treatments A. millefolium P. aeruginosa

A. millefolium S. aureus

Control -0.17a 0.41

a

0.08 g 0.97b 1.60

b

5.38 g 1.24b 0.77

b

10.76 g 0.45a 0.35

a

Means within each column with the same letter (a) are not significantly different (p < 0.05).

Kirby Bauer disc diffusion test with

Plantago major

Plantago major extract vs. P. aeruginosa

According to Nascimento et al. (2000),

zones of inhibition measurements that measure

1 mm greater than the 6 mm filter discs indicate

effectiveness against the bacterial populations

tested. The undiluted P. major extract (500

mg/ml) against P. aeruginosa showed zones of

inhibition at 8.10 mm for picking times at

11:30 a.m. and 6 mm zone of inhibition for

picking times at 7:00 p.m. (Table 4). The zone

of inhibition in the morning was statistically

higher than the negative controls DMSO and

methanol. Results for the more dilute extract

treatments showed a lack of antibacterial

effectiveness against P. aeruginosa, showing

no zones of inhibition for both picking times.

The antibiotics Ciprofloxacin and Gentimicin

resulted in zones of inhibition greater than 17

mm. In summary, the undiluted P. major

extract (500 mg/ml) treatment picked in the

morning showed antibacterial effectiveness

against P. aeruginosa.

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Table 4. The diameters (mm) of inhibition zones using the Kirby Bauer disc diffusion test with

P. major at different times during the day with the bacteria P. aeruginosa and S. aureus.

Treatments 11:30 a.m.

P. aeruginosa

11:30 a.m.

S. aureus

7:00 p.m.

P. aeruginosa

7:00 p.m.

S. aureus

Plant Extraction

500 mg/ml 8.10b 9.05

b 6.0

a 7.95

b

50 mg/ml 6.0a 6.86

ab 6.0

a 6.19

a

5 mg/ml 6.0a 6.0

a 6.0

a 6.0

a

0.5 mg/ml 6.0a 6.24

ab 6.0

a 6.0

a

0.05 mg/ml 6.0a 6.0

a 6.0

a 6.0

a

DMSO 6.0a 6.0

a N/A 6.0

a

Methanol 6.0a 6.0

a 6.0

a 6.0

a

Ciprofloxacin 28.81e 24.67

d 29.33

c 23.38

d

Gentimicin 19.90d 20.14

c 17.52

b 19.38

c

Means within each column with different letters (a–e) differ significantly (p < 0.05)

Plantago major extract vs. S. aureus

Plantago major extract (500 mg/ml) against S. aureus showed zones of inhibition significantly greater than the negative controls in both morning and evening picking times (Table 4). Results for the more diluted extract treatments used on S. aureus showed weak or no antibacterial activity. The antibiotics Ciprofloxacin and Gentimicin resulted in zones of inhibition greater than 19 mm in both morning and evening and showed a significant difference vs. P. major extract at 500 mg/ml. In summary, the undiluted P. major extract at 500 mg/ml showed effectiveness against S. aureus for treatments using plants picked during both morning and evening picking times. Bacterial population counts

Plantago major vs. P. aeruginosa

The P. aeruginosa population counts for P. major treatments conducted at 0.5 and 10 g were similar to the control at too numerous to count (TNTC) (Table 5). Therefore, when considering effectiveness of P. major against P. aeruginosa, P. major showed little effectiveness against P. aeruginosa.

Plantago major vs. S. aureus

Bacterial population counts for S. aureus, following application of P. major treatments of 0.5 and 10.0 g showed viable bacterial cell

counts of 1.20 log cfu ml-1

and too few to count (TFTC), respectively, while the control treatments showed bacterial cell counts at TNTC (Table 5). These results indicate that both P. major treatments were effective at reducing the number of viable S. aureus bacterial cells.

Achillea millefolium vs. P. aeruginosa

The plant A. millefolium treatment at 0.08 g showed P. aeruginosa viable bacterial cell counts at TNTC, while the A. millefolium treatment of 10.57g showed 2.44 log cfu ml

-1

(Table 6). Control results were TFTC. Results indicate that A. millefolium treatment at 10.57 g is more effective against P. aeruginosa when compared to the 0.08 g treatment. However, both of these treatments showed less effectiveness when compared to the control, and thus indicate a lack of effectiveness against P. aeruginosa.

Achillea millefolium vs. S. aureus

When A. millefolium treatment at 0.08 g was used for S. aureus, 0.90 log cfu ml

-1 were

recorded (Table 6). The A. millefolium treatment at 10.57 g yielded results at TFTC. Both of these treatment results showed a lower number of viable S. aureus bacterial cells when compared to the control, at 2.28 log cfu ml

-1. This indicates that both A. millefolium treatments were effective at reducing the number of viable S. aureus bacterial cells.

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Table 5. Numbers of bacteria grown on TSA (log cfu ml-1

) for Plantain (P. major) versus P.

aeruginosa and S. aureus.

Treatments Log cfu ml-1

P. aeruginosa Log cfu ml

-1 S. aureus

Control TNTC TNTC

0.5 g TNTC 1.20

10.0 g TNTC TFTC Abbreviations: TFTC= too few to count, TNTC= too numerous to count.

Table 6. Numbers of bacteria grown on TSA (log cfu ml-1

) for Yarrow (A. millefolium)

treatments versus P. aeruginosa and S. aureus

Treatments Log cfu ml-1 Log cfu ml

-1

P. aeruginosa S. aureus

Control TFTC 2.28

0.08 g TNTC 0.90

10.57 g 2.44 TFTC Abbreviations: TFTC= too few to count, TNTC= too numerous to count.

Alkaloids and saponins

Results from British Columbia P. major

analyses show low alkaloid levels for plants

picked during 11:30 a.m., at 0.07% (Table 7).

Alkaloid levels for plants picked at 7:00 p.m.

were recorded at 0.24%. The difference was not

statistically different (p = 0.2742). Saponins

showed similar results for the 11:30 a.m. and

7:00 p.m. picking times at 0.18% and 0.13%,

respectively (p = 0.1776). Although values for

alkaloids and saponins were not significantly

different, the increase in % for alkaloids at the

7:00 p.m. picking time coincided with the

elder‟s Indigenous science knowledge.

Table 7. Alkaloid and saponin mean percentage (%) in Plantain (P. major) with t-test

comparisons.

Sample Alkaloids (%) Saponins (%)

Plantain, BC, 2012, 11:30 a.m. 0.0656 ± 0.0223a 0.1807 ± 0.0663

a

Plantain, BC, 2012, 7:00 p.m. 0.2373 ± 0.3771a 0.1337 ± 0.0327

a

Values are means ± SD of three or more measurements. Means within each column with the same letter (a) are not

significantly different (p < 0.05).

DISCUSSION

Spectrophotometry

Our spectrophotometry results for the two

most diluted plant treatments (in plant

treatment combinations or on their own)

indicate that an increase in bacterial biomass

occurred and thus these results show

ineffectiveness against both P. aeruginosa and

S. aureus. The more concentrated plant

treatments often showed absorbance results that

were lower than the less concentrated plant

extracts, indicating a lower bacterial biomass in

those cultures and possible effectiveness

against the bacterial populations. For example,

A. millefolium appeared more effective against

both bacterial species at the most concentrated

plant treatment. The controls, however,

consistently showed lower absorbance results,

which indicates that the plant treatments

initially allowed bacterial growth to occur. This

also indicates that specific time frames may be

required before specific plant treatments

effectively kill bacterial cells. The resulting

higher absorbance values recorded for plant

treated samples are attributed to dead and

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viable bacterial cells (Hazan et al., 2012) with

the most effective plant treatments further

determined by conducting bacterial population

counts. Therefore, for the plant treatments

performed on the selected bacteria, this method

provided results that were ineffective at

determining viable bacterial biomass.

The effect of plant picking times on

antibacterial effectiveness and plant

biological compounds

Plants were picked at two different times

(11:30 a.m. and 7:00 p.m.) to follow the elder‟s

recommendations that late plant picking times

increase effectiveness of the plant medicine

when using P. major. Results for the Kirby

Bauer disc diffusion method showed that P.

major treatment resulted in a greater and more

consistent level of antibacterial effectiveness

against the known skin pathogen S. aureus,

when compared with results for P. aeruginosa.

Our results also indicate that the highest P.

major concentration was effective at reducing

the growth of S. aureus at both morning and

evening plant picking times. Results showed

that P. major was less effective against P.

aeruginosa with measured zones of inhibition

observed for the 11:30 a.m. plant picking time

only. Lack of zones of inhibition may also

occur if some plant biochemical components

are unable to effectively diffuse through the

agar medium (in spite of addition of DMSO to

facilitate movement).

In accordance with Indigenous science

knowledge, the concentration of alkaloids was

greater at the 7:00 p.m. picking time compared

to the 11:30 a.m. picking time in P. major. This

was not the case for the saponins, which

showed similar, yet slightly higher, saponin

levels for P. major at the 11:30 a.m. picking

time. The higher alkaloid levels determined for

P. major suggest that picking plant material

later in the day for use as plant medicines may

improve the effectiveness of P. major against

specific bacterial species. Thus, for the alkaloid

results, although not significant, the higher

alkaloid levels at the 7:00 p.m. picking time

coincide with local Indigenous science

knowledge. Plant picking times for this study

were arranged on a day when the weather was

cloudy and rainy at the 11:30 a.m. hour, with

similar conditions at 7:00 p.m. Future studies

that focus on the elder‟s guidance relative to

picking times should happen at mid-day when

conditions are distinctly hotter vs. a cooler

evening hour to contribute further to result

outcomes for alkaloids and saponins which

may lead to correlations with zones of

inhibition.

Plant / antibiotic treatments and

antibacterial effectiveness

The plant antibacterial compounds

associated with P. major and A. millefolium

appear to be more effective against the gram

positive S. aureus bacteria tested in this study.

This is supported by previous studies (Pistelli et

al., 2002, Avato et al., 2006, Soetan et al.,

2006). This indicates that the medicinal plants

selected for study may show stronger

antibacterial effectiveness against gram

positive bacteria, possibly due to lack of an

outer cell membrane. Results for the

commercial antibiotics used as treatments

showed strong antibacterial effectiveness

against both the gram positive and gram

negative bacteria used during this study.

It may also be important to consider the

heightened awareness of the medical

community to increasing patterns of resistance

to antibiotics by bacterial populations. When

also considering antibiotic treatment of skin

infections, topical applications of antibiotics

have been shown to cause contact dermatitis

(Sasseville, 2011) and contribute to drug-

resistant gram negative strains of skin

microflora. Some bacterial strains are known to

cause gram-negative folliculitis following

topical application of the antibiotic

Clindamycin (Worret and Fluhr, 2006), while

according to Blumenthal and colleagues

(1998), P. major shows a lack of toxicity on the

human body.

The plant treatments tested may show

antibacterial effectiveness while additionally

contributing to wound healing activity. For

example, to fight bacterial infection, the

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 402–415

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immune system increases levels of one type of

leukocyte, the neutrophils, at the wound site.

While neutrophils phagocytize bacteria,

reactive oxygen species form, often leading to

damaging effects on tissues. Plantago major

extracts significantly inhibit the production of

reactive oxygen species by human neutrophils,

limiting potential damage to tissues (Reina et

al., 2013). Plantago major has also been shown

to contribute to the healing process of wounded

tissues. One study showed that P. major water

and ethanol leaf extracts stimulated

proliferation and migration of cells during

wound healing (Zubair et al., 2012) indicating

that during the healing of wounds, P. major

may effectively contribute to fighting infection

while serving to limit tissue damage and by

contributing to the repair of skin tissue.

CONCLUSION: INDIGENOUS SCIENCE

KNOWLEDGE AND WESTERN SCIENCE

KNOWLEDGE COMING TOGETHER

The coming together of Indigenous science

knowledge with Western science knowledge

may highlight a responsible, positive

mechanism for effectively treating skin

infections. The issue of antibacterial

effectiveness of plant medicines also further

contributes to discussion on the need to reduce

antibiotic usage resulting from the documented

increase in bacterial resistance to a wide

spectrum of antibiotics frequently used today.

Like antibiotics, plant medicines must be

respected for their potentially powerful

medicinal abilities and individuals should talk

with elders or others who have an

understanding of Indigenous science

knowledge prior to preparing and using plant

medicines to heal wounds.

Future collaborative studies between

Indigenous science knowledge and Western

science knowledge need to be further expanded

in partnership with First Nations elders /

traditional knowledge keepers (Nilson et al.,

2008; Ferreira and Gendron, 2011; Gendron et

al., 2013) with a continued focus on picking

times, plant species, antibacterial plant

chemical components and an increase in

bacterial species studied to further investigate

the natural abilities of plants to fight infection.

ACKNOWLEDGEMENTS

The researchers worked in partnership

(western science and indigenous science) when

formulating the experimental design for this

project and would like to thank the following

individuals for their contributions to the

research activity: research assistants: Amie

Oxler, Serena Richardson, and Shanice Manson

(Vancouver Island University); laboratory

technicians: Shelley Corrin and Peter Diamente

(Vancouver Island University) and Simon N.

Makubudi and Raymond McNabb (First

Nations University of Canada) for their help

with the laboratory analyses. This work was

supported by a Social Sciences and Humanities

Research Council (SSHRC) President's Fund

grant, a Vancouver Island University Research

Award grant, and a Canada Summer Jobs grant.

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Source of Support: Social Sciences and

Humanities Research Council (SSHRC)

President's Fund grant, a Vancouver Island

University Research Award grant, and a Canada

Summer Jobs grant.

Conflict of Interest: None Declared

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Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

EVALUATION OF PHENOLIC COMPOUNDS, FLAVONOIDS AND

ANTIOXIDANT PROPERTIES OF ARGANIA SPINOSA (L.) SKEELS

LEAF EXTRACTS

Saliha DJIDEL1, Choubaila -Feriel CHATER

2, Seddik KHENNOUF

3*,

Abderrahmane BAGHIANI4, Daoud HARZALLAH

5

1,2,3Laboratory of Phytotherapy Applied to Chronic Diseases, Department of Animal Biology and Physiology,

Faculty of Natural and Life Sciences, University Sétif 1, 19000 Algeria. 4Laboratory of Applied Biochemistry, Department of Biochemistry, Faculty of Natural and Life Sciences,

University Sétif 1, 19000 Algeria. 5 Laboratory of applied microbiology. Faculty of Natural and Life Sciences, University Sétif 1, 19000 Algeria.

*Corresponding Author: E_mail: Khennouf _sed@yahoo.fr

Received: 28/08/2014; Revised: 25/10/2014; Accepted: 14/11/2014

ABSTRACT

Argania spinosa (L.) Skeels (Sapotaceae family) is an endemic species from Algeria (Tindouf

region) and south-west of Morocco and is a Saharan affinity medicinal plant. In this study, the

extraction of phenolic compounds from the leaves of Argania spinosa (L.) Skeels is carried out using

solvents of different polarity. The yields of extraction from the leaves were 27.7%, 0.7%, 2.1% and

18.1% for the crude, chloroform, ethyl acetate and aqueous extracts respectively. The levels of total

polyphenols, determined by Folin–Ciocalteu’s reagent, in plant extracts varied from 447 ± 0.028 to

106.33 ± 0.062 mg/g dry weight, expressed as gallic acid equivalents (GAE) for ethyl acetate and

chloroform extracts respectively. Total flavonoid contents were determined using aluminum chloride

and the crude extract contained the highest content with 185.93 ± 0.009 mg quercetin Eq/ g of dry

extract. Different antioxidant tests were employed to evaluate the antioxidant activities of these

extracts. The EAE showed the highest antioxidant activity using β-carotene/ linoleic acid, DPPH,

phenanthroline-Fe (II) oxidation and reducing power assays with 90%, 0.014 ± 0.0001 mg/ml, 0.13 ±

0.0008 mg/ml and 1.859 ± 0.037 respectively. The results were compared with natural and synthetic

antioxidants.

KEY WORDS: Argania spinosa (L.) Skeels, antioxidants, Polyphenols, Flavonoids.

Research Article

Cite this article:

Saliha DJIDEL, Choubaila -Feriel CHATER, Seddik KHENNOUF,

Abderrahmane BAGHIANI, Daoud HARZALLAH (2014), Evaluation of Phenolic compounds,

Flavonoids and antioxidant properties of Argania spinosa (L.) Skeels leaf extracts,

Global J Res. Med. Plants & Indigen. Med., Volume 3(11): 416–426

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Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

INTRODUCTION

Reactive oxygen species (ROS), also called

active oxygen species, are various forms of

activated oxygen, which include free radicals

such as superoxide ions (O2°-) and hydroxyl

radicals (OH), as well as non free-radical

species such as hydrogen peroxide (H2O2)

(Favier, 2003). The excess of these oxygen

radicals can implicate in several diseases,

including cancer, diabetes, cardiovascular

diseases, ageing etc. (Halliwell and Gutteridge,

1999). Antioxidants are vital substances which

possess the ability to protect the body from

damage caused by free radical induced

oxidative stress. There is an increasing interest

in natural antioxidants. Polyphenols, present in

medicinal plants and dietary plants, might

prevent oxidative damage (Shahidi and Naczk,

1995). Antioxidant properties of polyphenols

coud be due to their high reactivity as hydrogen

or electron donors, to the ability of the

polyphenol derived radical to stabilise and

delocalise the unpaired electron (chain-

breaking function), and to their ability to

chelate transition metal ions (termination of the

Fenton reaction) (Rice-Evans et al., 1997).

Argania spinosa L. Skeels (Sapotaceae

family) is a medicinal plant with Saharan

affinity. It is an endemic species to Algeria

(Tindouf region) and south-west of Morocco.

In Algeria, Argania is found and distributed

precisely in the west part of Algerian sahara

between jibel Ouarkziz and hamada of Tindouf

(28° N and 8° W) (Morsli, 1999).

Traditionally, the Argan tree is mainly used

for the preparation of an oil that is extensively

utilized for nutritional purposes but also

recommended to cure some therapeutic

disorders and can be used as a cosmetic

(Bellakhdar et al., 1997). Argan oil is very rich

in antioxidants (Charrouf & Guillaume, 1999),

it reduced blood pressure in an experimentally

induced hypertension (Chen et al., 2001).

Moreover, Berrada et al. (2000) showed a

decrease in blood pressure after ingestion of

argan oil. Antidiabetic activity of argan oil has

been also demonstrated in animals (Bnouham

et al., 1988). These pathologies have a strong

relationship with oxidative stress. Fresh leaves

are used for sheep and goat’s nutrition.

Chemical composition of argan leaves showed

that lipids constitute 4.4% (Chahboun, 1993).

Quercetin and myricetin have also been

isolated from these leaves (Aumente Rubio,

1988). A. spinosa was investigated for

condensed tannins and flavonoids, flavonol

glycosides were identified in this plant by

HPLC, the main flavonols were myricitrin and

its derivative myricetin-3-O-galactoside and

quercetin and its derivative hyperoside

(Tahrouch et al., 2000, Tahrouch et al., 2011).

Few studies were concerned with leaves and

most of the research works was conducted on

Argan oil. In this work, we have evaluated

phenolic contents and antioxidant activity of

different extracts from Argania spinosa leaves;

the radical scavenging capacity, the anti-lipid

peroxidation and reducing power of crude,

chloroform, ethyl acetate and aqueous

fractions.

MATERIAL AND METHODS

Chemicals

Linoleic acid, β-carotene, butylated

hydroxytoluene (BHT), were purchased from

Fluka Chemical Co. (Buchs, Switzerland). 2, 2-

diphenyl-1-picryl-hydrazyl (DPPH),

ethylenediamine tetraacetic acid (EDTA), gallic

acid were obtained from Sigma Chemical Co.

(St. Louis, MO). Potassium ferricyanide,

trichloroacetic acid (TCA), ferrous and ferric

chloride were obtained from Merck. All other

reagents were of analytical grade.

Plant material

The leaves of A. spinosa L. were collected

from Tindouf, south west of Algeria. This plant

was identified by Pr. Laouar Hocine from the

laboratory of Botanical Sciences, Faculty of

Natural and Life Sciences, University Ferhat

Abbas, Setif 1, Algeria. A voucher specimen

was deposited in the laboratory (number

BAAS0314).

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Preparation of A. spinosa leaves extracts

The extraction of phenolic compounds was

reported by Markham (1982). Leaves of A.

spinosa (100 g) were powdered, mixed with

one liter of methanol-water solution (85: 15

v/v) and kept at room temperature for 3 days.

After 3 days it was filtered and the solvent was

evaporated to get crude extract (CE). The

aqueous solution was washed with hexane

several times until a clear upper layer of hexane

was obtained. The lower layer was then treated

with chloroform and ethyl acetate to obtain

chloroform (CHE), ethyl acetate (EAE) and

aqueous extracts (AQE). Each fraction was

dehydrated and stored at −20 °C. A fractions

extract was prepared and used for in vitro

studies.

Total phenolic content

Total phenolic compounds were estimated

using the Folin-Ciocalteu method (li et al.,

2007). 200 μl of each extract was mixed with

1000 μl of Folin-Ciocalteu phenol reagent.

After 4 minutes, 800 µl of 7.5 % sodium

carbonate was added. The mixture was allowed

to stand at room temperature for 1 hour and 30

minutes. The resulting blue complex was then

measured at 760 nm. The TPC was expressed

as mg gallic acid equivalent/g dry weight by

reference to the gallic acid standard calibration

curve.

Total flavonoid content

The AlCl3 method (Bahorun et al., 1996)

was used for estimation of the total flavonoids

content of plant extracts. An aliquot of 100 μl

of each extract (100 μg/ ml) was added

individually to equal volumes of solution of 2%

AlCl3. The mixture was vigorously shaken, and

after 10 minutes of incubation, absorbance was

taken at 430 nm. Flavonoids contents were

calculated from the calibration curve of

quercetin standard solution, and expressed as

mg Quercetin equivalent (QE) / mg dry weight

of plant.

Antioxidant activity by DPPH° assay

The antioxidant activities of different

concentrations of each phenolic extracts were

determined by hydrogen-donating ability of

phenolic compounds of the A. spinosa extracts

to free radical stable DPPH (Burits and Bucar,

2000) with some modifications. 50 µl of

different dilutions of the extracts were added to

1250 µl of 4% solution DPPH dissolved in

methanol. After 30 minutes at room

temperature, the absorbance was measured at

517 nm. The ability to scavenge DPPH radical

was calculated by the following equation:

DPPH radical scavenging activity (%) = (A

control – A sample / A control) ×100

where Abs control is the absorbance without

extract and Abs sample is the absorbance in the

presence of sample. IC50 value (the

concentration required to scavenge 50% DPPH

free radicals) was calculated. Rutin was used as

the standard in the procedure.

Hydroxyl radical-scavenging by

phenanthroline-Fe (II) oxidation assay

The scavenging activity of extracts on

hydroxyl radical was measured according to the

method of of Li et al. (2008). In this system,

hydroxyl radicals were generated by the Fenton

reaction. Hydroxyl radicals could oxidize Fe2+

into Fe3+

, and only Fe2+

could combine with

1,10-phenanthroline to form a red compound

(1,10-phenanthroline-Fe2+

) with the maximum

absorbance at 536 nm. The concentration of

hydroxyl radical was reflected by the degree of

decolourization of the reaction solution.

Briefly, 600 µL of (5 mM) phenanthroline, 600

µL (5 mM) FeSO4, 600 µl of EDTA (15 Mm),

400 µl phosphate buffer (0.2 M, pH= 7.4) and

800 µl (0.01%) H2O2 were added into 600 µl of

extract. After 1 hour of incubation at 37°C, the

absorbance of reaction mixture was measured

at 536 nm against reagent blank. The reaction

mixture without any antioxidant was used as

the negative control, and without H2O2 was

used as the blank. The hydroxyl radical

scavenging activity (HRSA) was calculated by

the following formula: Asample/Acontrol*100

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Determination of reducing power

The reducing power of samples was

determined using the method of Chung et al.

(2005). The assay medium contained 0.1 ml of

sample/standard in a 0.1ml 0.2 M phosphate

buffer (pH 6.6) and 0.1 ml of 1% potassium

ferricyanide. After incubation at 50° C for 20

min, 0.25 ml of 1% trichloroacetic acid were

added to the mixture followed by centrifugation

at 3000 rpm for 10 min. 0.25 ml of the

supernatant was mixed with 0.25 ml distilled

water and 0.5 ml of 0.1% ferric chloride, and

the absorbance of the resultant solution was

read at 700 nm. A standard was prepared using

various concentrations of BHT.

Beta-carotene-linoleic acid assay

In this assay, antioxidant capacity is

determined by measuring the inhibition of the

volatile organic compounds and the conjugated

diene hydroperoxide formation from linoleic

acid oxidation Koleva et al. (2002). A stock

solution of β -carotene-linoleic acid mixture

was prepared as follows: 0.5 mg of β -carotene

was dissolved in 1 ml of chloroform, and 25 μl

of linoleic acid and 200 mg of Tween 40 were

added. Chloroform was completely evaporated

using a vacuum evaporator. Then, 100 ml of

oxygen-saturated distilled water was added;

2500 μl of this reaction mixture was dispensed

into test tubes, and 350 μl volumes of extracts,

prepared in 2 mg/ml concentrations, were

added. The emulsions were incubated for up to

24 h at room temperature. The same procedure

was repeated with a positive control BHT and a

blank. After this incubation time, the

absorbance of the mixture was measured at 490

nm. Antioxidant capacities of the extracts were

compared with BHT.

Chelating activity on Fe2+

The iron (II)-chelating ability of the extract

was assessed by the method of (Decker and

Welch, 1990). In brief, 0.25 ml aliquot of

dissolved extract was added to 0.05 ml (0.6

mM) aqueous FeCl2 - 4H2O and 0.45 ml

Methanol. After 5 min, the reaction was

initiated by the addition of 0.05 mL (5.0 mM)

ferrozine solution. After 10 min, the

absorbance at 562 nm was recorded. The

control contained all the reagents except the

extract or positive control. EDTA was used as a

positive control. The percentage of inhibition of

ferrozine-Fe2+

complex formation was

calculated using the formula:

Chelating activity % = ((A control – A

sample)/Acontrol)*100

EC50 values were calculated by linear

regression analysis; linearity range between

antioxidant concentration and chelating

activity.

2,2'-azino-bis(3-ethylbenzothiazoline-6-

sulphonic acid (ABTS) assay

The ABTS assay was employed to measure

the antioxidant activity of the plant extracts (Re

et al., 1999). ABTS was dissolved in distilled

water to 7 mM concentration, and potassium

persulphate added to a concentration of 2.45

mM. The reaction mixture was left to stand at

room temperature overnight (12 to 16 h) in the

dark before usage. In the assay, 50 µl extract,

standard (Trolox), or control (methanol) and 1

ml ABTS solution were mixed. The absorbance

at 734 nm was determined after 30 min. The

ability to scavenge ABTS radical was

calculated by the following equation:

ABTS radical scavenging activity (%) = (A

control – A sample / A control) x100

IC50 value (the concentration required to

scavenge 50% ABTS free radicals) was

calculated.

Statistical analysis

All the experiments were carried out in

triplicate. The IC50 were presented by their

respective 95% confidence limits. The TPC

(mg/g) were shown as mean ± SD. One-way

analysis of variance (ANOVA) followed by

Dunnet’s test was used to assess significant

differences (p<0.05) between extracts and

standards.

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RESULTS AND DISCUSSION

Total phenolic and flavonoid content

The amount of total phenolics, measured by

Folin–Ciocalteu method, varied in four extracts

of A. spinosa and ranged from 447.22 ± 0.028

to 106.33 ± 0.062 mg Gallic Acid Equivalent

(GAE)/ g dry weight (dw) (Table 1).

The highest level of phenolics was found in

crude extract, while the lowest was in

chloroform extract. Phenolic compounds,

especially flavonoids, constitute one of the

most diverse and widespread group of natural

compounds. These compounds possess a broad

spectrum of biological activities including

antioxidant and radical scavenging properties

(Parejo et al., 2002; Galvez et al., 2005),

therefore the total phenolic compounds in the

extracts was determined in (Table 1). The total

flavonoid content, in quercetin equivalent,

varied from 185.93 ± 0.009 to 1.18 ± 0.003 mg

quercetin equivalent (QE)/ g of dry extract. The

highest amount of the total flavonoid was found

in the EAE of A. spinosa, while AQE contained

remarkably lower amount of these compounds.

We can deduce that all these extracts are rich in

polyphenols and flavonoids.

Antioxidant activity by DPPH° Assay

DPPH stable free radical method is an easy,

rapid and sensitive way to survey the

antioxidant activity of a specific compound or

plant extracts (Koleva et al., 2002). In this

assay the scavenging of the DPPH radical is

followed by monitoring the decrease in

absorbance at 517 nm. Figure1 shows the

amount of each extract needed for 50%

inhibition (IC50).

The IC50 of the standard compound Rutin

was (0.004 ± 0.0008 mg/ml). The highest

radical scavenging activity was showed by

EAE with IC50=0.014 mg/ ml which is lower

than that of rutin (P<0.05). The IC50 (DPPH°)

values of the extracts increased in the following

order: EAE<CE<AQE<CHE. Our results are

supported by the fact that 70% aqueous ethanol

solution from A. spinosa leaves exhibited a

DPPH scavenging activity with IC50 = 0.028

µl/ml (Joguet and Maugard, 2013).

Table 1.0 Total phenolic and flavonoid contents of Argania spinosa L leaves extracts

Extracts % Yield w/w Total phenolic ( mg GA.Eq/g)

Total flavonoids (mg QEq/g)

CE 27.7 256.16 ± 0.02 18.8 ± 0.007

CHE 0.7 106.33 ± 0.062 30.76 ± 0.004

EAE 2.1 447.2 ±0.028 185.93 ± 0.009

AQE 18.1 216.33 ±0.004 1.18 ±0.003 (CE) crude extract (CHE) chloroform, (EAE) ethyl acetate and (AQE) aqueous extracts. Results are expressed as

means ± standard deviation (n = 3).

Figure 1. IC50 values of plant extracts for free radical scavenging activity by DPPH method.

Lower IC50 value indicates higher antioxidant activity. CE; crud extract, CHE; chloroform extract, EAE; ethyl acetate

extract, AQE; aqueous extract. *** significant difference (P<0.01) **(P<0.05) compared to rutin.

**

***

**

**

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Hydroxyl radical scavenging activity

Among the oxygen radicals, hydroxyl

radical is the most reactive and induces severe

damage to biomolecules (Sakanaka et al.,

2005). Figure 2 shows that all extracts and

reference antioxidant (vitamin C) had radical

scavenging activities on the hydroxyl radicals.

Among the plant extracts there were

differences in their activities in scavenging

hydroxyl radicals. EAE had a highest radical

scavenging with a value IC50= 0.13 ± 0008

followed by CHE (0.2 ± 0.005 mg/ml) and CE,

AQE with a same value (0.41 mg/ml). The

scavenging effect of vitamin C (0.13 ± 1.76

mg/ml) was nearly equal to that of EAE, but in

significant difference between them (p<0.01).

The scavenging abilities on hydroxyl radicals

were in descending order: EAE > CHE > CE >

AQE. Antiradical effect can be due to the

reduction by antioxidants and has been used to

assess the ability of phenolic compounds to

transfer labile H atoms to radicals (Djerdiane et

al., 2006).

Reducing power

Fe (III) reduction is often used as an

indicator of electron- donating activity, which

is an important mechanism of phenolic

antioxidant action, and can be strongly

correlated with other antioxidant properties

(Dorman et al., 2003). Figure 3 shows the dose-

response curves for the reducing powers of the

extracts from A. spinosa leaves. All the extracts

showed degree of electron donation capacity in

a concentration-dependent manner, but the

capacities were inferior to that of BHT.

Figure 2. IC50 values of plant extracts for hydroxyl radical scavenging activity by phenantrine

method.

Lower IC50 value indicates higher antioxidant activity. CE; crud extract, CHE; chloroform extract, EAE; ethyl acetate

extract, AQE; aqueous extract, VIT C; vitamin C. *** significant difference (P<0.01) **(P<0.05) compared to vit. C.

Figure 3. Antioxidant activity of A. spinosa extracts expressed as reducing power.

CE; crude extract, CHE; chloroform extract, EAE; ethyl acetate extract, AQE; aqueous extract, BHT.

0

0.1

0.2

0.3

0.4

0.5

CE CHE EAE AQE VIT C

IC 5

0m

g/m

l

**

***

***

***

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The reducing power of the CE, AQE and

CHE increased from 0.123±0.0007,

0.129±0.001 and 0.056±0.002 at 0.003 mg/ml,

respectively to 1.47 ± 0.15, 1.342 ± 0.04 and

0.686 ± 0.01 at 0.2 mg/ml, respectively. The

reducing power of EAE increased from 0.149 ±

0.004 at 0.002 mg/ml to 1.859 ± 0.037 at 0.08

mg/ml. BHT (1.163 ± 0.06) at a concentration

of 0.03 mg/ml.

The reducing power of extracts may be due

by the presence of phenolic compounds in these

fractions. Similar relations between Fe3+

reducing activity and total phenol content have

been reported in the literature (Negi and

Jayaprakasha, 2003).

β-carotene/linoleic acid assay

The antioxidant activities of the extracts

determined by the β -carotene linoleic acid

system assay are presented in figure 4. The

antioxidant activity of samples at the

concentration of 2 mg/mL was reflected in their

ability to inhibit the bleaching of β -carotene.

In this assay, the ethyl acetate and aqueous

extracts also possessed better antioxidant

activity than other extracts and similar to BHT

(96%) (p > 0.05). Other extracts were also

effective in inhibiting lipid peroxidation, but

not as active as BHT (p < 0.01).

Figure 4. Antioxidant activities of A. spinosa extracts (2 mg/ml at 24 hours of incubation)

measured by β-carotene bleaching method.

CE; crude extract, CHE; chloroform extract, EAE; ethyl acetate extract, AQE; aqueous extract. BHT was used as

reference antioxidant. Values are means ± SD (n = 3) *** significant difference (P<0.01) ** (P<0.05) compared to BHT,

ns: no significant difference from BHT

Chelating activity on Fe2+

Crude, chloroform, ethyl acetate and

aqueous extracts were assessed for their ability

to compete with ferrozine for iron (II) ions in

free solution. All the extracts demonstrated an

ability to chelate iron (II) ions in a dose-

dependent manner (Figure 5). At 0.15 mg/ml

CE, CHE and AQE extracts chelated ferrous

ions by 18.5 ± 2.25 %, 14.4 ± 2.25 % and

16.4 ± 4.19% respectively, whereas at 1.56

mg/ml, this extract showed an excellent

chelating ability of 93.9 ± 3.24 %, 71.1 ± 2.99

% and 84.9 ± 1.14% in the same order. The

chelating effect of the EAE on ferrous ions was

low (24.14 ± 4.08 %) at 9.37 mg/ml. None of

the extracts appeared to be better chelators of

iron (II) ions than the positive control EDTA in

this assay system. EDTA showed excellent

chelating ability of 95.7 ± 2.3% at a

concentration of 0.015 mg/ml.

EC50 value (the effective concentration at

which ferrous ions were chelated by 50%) of

0

20

40

60

80

100

BHT CE CHE EAE AQ MOH H2O

Inh

ibit

ion

%

**

**

*** ***

ns ns

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CE (0.47 ± 0.01 mg/ml) was significantly

higher (p > 0.05) than that of AQE (0.61 ± 0.04

mg/ml) and CHE (1.05 ± 0.07 mg/ml) extracts,

which were comparable. The chelating abilities

on ferrous ions were in descending order: CE >

AQE > CHE > EAE.

Figure 5. Chelating activity of the extracts from A. spinosa leaves.

EDTA was used as the positive control. Values are means ± SD (n = 3).

ABTS assay

ABTS+ is a blue colored chromophore

which is reduced to ABTS on a concentration

dependant manner upon addition of the plant

extracts. The results are compared with trolox

and the IC50 value demonstrates the extracts as

a potent antioxidant, with their IC50 values

following the order: EAE >CE >AQE>CHE.

Figure 6 showed that EAE had a highest

scavenging activity with IC50 =

0.0052 ± 0.0001 mg/ml, while lowest for CHE

with IC50= 0.0095 ± 0.0002 mg/ml. All extracts

were significantly (p < 0.05) lower than that of

antioxidant reference (Trolox: 0.0014 mg/ml).

Figure 6. IC50 values of plant extracts for ABTS radical scavenging activity

Lower IC50 value indicates higher antioxidant activity. CE; crude extract, CHE; chloroform extract, EAE; ethyl acetate

extract, AQE; aqueous extract. *** Significant difference (P<0.01) ** (P<0.05) compared to Torox.

-2.26E-17

0.003

0.006

0.009

0.012

CE CHE EAE AQE Trolox

IC 5

0m

g/m

l

***

µ***

*µµ

***

µ**

**µ

µ

**

µ

**

**

µ

µ

***

µ*

***

µµ

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Reduction of the radicals of leaves extracts

may be due to the phenolic compounds witch

they possess ideal structural chemistry for free

radical scavenging activity. Antioxidant

properties of these molecules arise from their

high reactivity as hydrogen or electron donors,

and from the ability of the polyphenol derived

radical to stabilize the unpaired electron (Rice-

Evans et al., 1997).

CONCLUSION

We have demonstrated that extracts of A.

spinosa leaves contain high levels of total

phenolic compounds and were capable of

inhibiting lipid peroxidation, directly

quenching free radicals to terminate the radical

chain reaction, acting as reducing agents, and

chelating transition metals to suppress the

initiation of radical formation. It is well-known

that phenolic compounds present in the plant

kingdom are mainly responsible for the

antioxidant potential of plants. Argania leaves

are good source of antioxidants, it is therefore,

possible to valorise these leaves in the

pharmaceutical and food industries.

ACKNOWLEDGEMENT

This work was supported by the Algerian

Ministry of Higher Education and Scientific

Research (MERS) and L’ Agence Thématique

de Recherche en Sciences de la Santé

(ATRSS). We express our gratitude to these

organisations.

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Source of Support: Algerian Ministry of

Higher Education and Scientific Research

(MERS) and L’ Agence Thématique de

Recherche en Sciences de la Santé (ATRSS)

Conflict of Interest: None Declared

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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 427–434

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

ASSESSMENT OF ‘VIPAKA’ (METABOLISM) OF A NEW MEDICINAL

PLANT IN ANIMAL MODEL

Bidhan Mahajon1*

, Ravi Shankar B2, Remadevi R

3

1PG Scholar, Department of Dravyaguna Vijnanam, Vaidyaratnam P. S. Varier Ayurveda College, Kottakkal,

Kerala, India 676501. 2Professor of Experimental Medicine and Director, Department of Pharmacology and Toxicology, SDM

Centre for Research in Ayurveda and Allied Sciences, Udupi, India 574 118. 3Professor and HOD, Department of Dravyaguna Vijnanam, Vaidyaratnam P. S. Varier Ayurveda College,

Kottakkal, Kerala, India 676501.

* Corresponding Author: Email: bidhanmahajon@gmail.com; Mobile- +918593038842

Received: 11/09/2014; Revised: 10/11/2014; Accepted: 15/11/2014

ABSTRACT

Due to wide range of climatic condition India holds rich variety of flora. Since ancient times,

plants have been widely used as medicine in India. Ayurveda, the Indian system of medicine opines,

there is no such dravya (substance) in the Universe, which has no medicinal value. Systematic

documentation of folklore medicinal practices has introduced many new medicinal plants to the

Ayurvedic system. Comprehensive research on such plant species for their therapeutic properties will

enrich the Ayurvedic pharmacopeia. A thorough study of Rasapanchaka (Ayurvedic

pharmacological property) of a drug is mandatory for its therapeutic use; hence the need for

developing a standard, valid protocol for assessment of Rasa (Taste), Guna (Quality), Veerya (Active

potency) & Vipaka (Metabolism) of a medicinal plant. On this background the present study was

taken up for analysis of Vipaka (Metabolism) of medicinal plant Flemingia strobilifera. This is an

important medicinal plant of Fabaceae family, traditionally used in epilepsy, insomnia and hysteria

in different regions of India. The outcome of the study can be considered as preliminary evidence

and will hopefully inspire more studies with different parameters for further validation.

KEY WORDS: Flemingia strobilifera, Vipaka, Rasapanchaka, Vipaka assessment

Research Article

Cite this article:

Bidhan Mahajon, Ravi Shankar B, Remadevi R (2014), ASSESSMENT OF ‘VIPAKA’

(METABOLISM) OF A NEW MEDICINAL PLANT IN ANIMAL MODEL,

Global J Res. Med. Plants & Indigen. Med., Volume 3(11): 427–434

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 427–434

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

INTRODUCTION

Due to the wide range of climatic

conditions, India holds rich variety of flora.

India is home to more than 45,000 species of

flora, out of which many are found nowhere

else. There are more than 3000 officially

recognized plants in India that hold medicinal

potential (Reddy Janardhana K et al., 2007).

But only a small amount of them are used in

Ayurveda. On the other hand Ancient

Ayurvedic authorities have opined, all the

dravyas (substance) of the universe have

medicinal value & can be utilized as medicine.

Therefore comprehensive research on such new

medicinal plant species will enrich the

Ayurvedic pharmacopeia. For utilising a new

drug in Ayurveda based on Ayurvedic

fundamental principles, the knowledge of Rasa

(Taste), Guna (Property), Veerya (Active

potency), Vipaka (Metabolism) of the drug is

mandatory. For Rasa and Veerya analysis,

present Ayurvedic scholars follow available

experimental methods (Dhyani S.C., 2003) but

for Vipaka analysis no standard protocol is

available. So here is the need for development

of a standard experimental model with protocol

for Vipaka analysis. Vipaka can be assessed

based on Dosa karma (action on humors),

Dhatu karma(action on tissues), Mala karma

(action on metabolic waste products) (Dhyani

S.C., 2003). Though assessment of Dosa karma

and Dhatu karma is complicated, Mala karma

can be easily assessed in animal model.

Flemingia strobilifera (L).WT Aiton is an

important medicinal plant known as Kamalu in

Malayalam and Kusrunt in Hindi (Kirtikar KR

et al., 1935).It is a perennial shrub of Fabaceae

family, commonly available throughout the

tropics of India. Root of this plant is being used

in treating epilepsy, hysteria, insomnia and to

relieve pain (Kirtikar KR et al., 1935) by folk

practitioners. Recent experimental studies have

proved various pharmacological activities of

this plant (Saxena VK, 1995; Madan S et al.,

2010; Anil K et al., 2011 Kavita G et al., 2012;

Gahlot K et al., 2013). Therefore to utilise

these important properties of this medicinal

plant in Ayurveda based on Ayurvedic

fundamental principles, an experiment was

carried out in the following manner. 12 Wistar

strain albino rats were selected and divided into

2 groups; Group A- Control, Group B -Test

group. Each rat was kept in separate metabolic

cages provided with constant amount of water

and food per day. Assessment of Vipaka was

done on the basis of consumption of food;

consumption of water; quantity of faecal

matter, urine output and quantity water content

of expelled faecal matter per day (Dhyani S.C.,

2003).

MATERIALS AND METHODS

Plant Material:

Root of F. strobilifera was collected from

Jagiroad, Assam during December 2013. It was

authenticated by department of Pharmacognosy

at SDM Centre for Research in Ayurveda and

Allied Sciences, Udupi, Karnataka, India. A

voucher specimen (No. 385/14020702) has

been deposited for further future reference.

Preparation of aqueous extract of F.

Strobilifera:

The root of plant F. strobilifera was shade

dried and pulverized, finely sieved and 500g of

plant root powder was soaked in 2 lit of

distilled water for 24 hour, after which it was

filtered. The filtrate was evaporated in a rotator

evaporator and used for the experiment

(Harborne J.B., 1998).

Experimental Model:

Wistar strain albino rats of either sex

between 250–350g body weights were obtained

from animal house attached to department of

Pharmacology, SDM Centre for Research in

Ayurveda and Allied Sciences, Udupi,

Karnataka, India. The animals were fed with

normal rat diet and water ad libitum throughout

the study period. They were acclimatized in the

laboratory condition for two weeks prior to the

study. The housing conditions: controlled

lighting of 12:12h light and dark cycle,

temperature of 25ºC and relative humidity of

about 50%.

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 427–434

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Experimental Procedure:

12 Wistar strain albino rats were selected,

which were divided into 2 groups. The rats

were weighed and group was named as, Group

A- Control; Group B -Test group. Each rat

from two groups was kept in separate metabolic

cages provided with constant amount of water

and food per day. To each rat 200 ml of water

& 50 g of food were provided in the food

hopper and bottle holder per day. After 24 hour

the amount of leftover water and food was

measured to obtain the quantity of water and

food consumed per day, this was recorded for

consecutive 5 days without administering the

drug to the rats in both groups. Sixth day

onwards the test drug was administered at the

dose of 200 mg/kg body weight to the test

group and the same observation procedure was

repeated for 10 more days in both groups.

Quantity of stool and urine was measured every

day. On every alternative day, the weight of

each rat from all the groups was noted down.

The parameters recorded for each rat on a day

were, food consumption, water ingestion,

faecal output (wet faecal-immediate after

collection and dry faecal-after keeping in hot

air oven for 105°C temperature for 4 hours),

faecal water (wet faecal weight - dry faecal

weight), urine output and food conversion ratio

[Food consumption (divided by) dry faecal

weight per day] both in Absolute value and

Relative value.

Statistical analysis:

All the values were expressed as MEAN ±

SEM (standard error of mean) and the data

were analyzed by unpaired‘t’ test. A level of

P<0.05 was considered as statistically

significant. Level of significance was noted and

interpreted accordingly.

RESULTS

Results obtained from the experiment are

summarized in tables 1–15.

Table 1 depicts the following; After

administering the test drug, food intake in

gm/day was increased by 10.89% in test group

when compared to the control group; however

that increased data was statistically not

significant.

Table 2 shows; After administering the test

drug, food intake in gm/100gm body weight of

rats was decreased by 10% in test group when

compared to the control group; however that

decreased data was statistically not significant.

Table 3 depicts; After administration of the

test drug, water intake in ml/day was increased

by 38.81% in test group when compared to the

control group, however that increased data was

statistically not significant.

Table 1: Effect of test drug on food intake with data presented in terms of absolute values:

Group Food intake in grams ( absolute values)

Preliminary phase

MEAN ± SEM

Therapeutic phase

MEAN ± SEM

% change

Control 13.34 ± 0.65 11.93 ± 1.007 −

Test 13.63 ± 1.07 13.23 ± 0.88 10.89↑

Table 2: Effect of test drug on food intake with data presented in terms of relative values:

Group Food intake in gm/100g body weight

Preliminary phase

MEAN ± SEM

Therapeutic phase

MEAN ± SEM

% change

Control 5.16 ± 0.12 5.10 ± 0.28

Test 4.62 ± 0.23 4.59 ± 0.19 10↓

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Table 3.Effect of test drug on water intake with data presented in terms of absolute values:

Group Water intake in ml ( absolute values)

Preliminary phase

MEAN ± SEM

Therapeutic phase

MEAN ± SEM

% change

Control 27.33 ± 1.22 27 ± 1.56

Test 39.7 ± 3.91 37.48 ± 4.45 38.81↑

Table 4 shows; After administration of the

test drug, water intake in ml/100 gm body

weight of rats was 6.77% increased in test

group compared to the control group; however

that increased data was statistically not

significant.

Table 5 shows; After administration of the

test drug urine output in ml/day was

significantly increased (‘P’ value less than

0.05) in test group compared to the control

group.

Table 6 depicts; After administration of the

test drug, urine output in ml/100gm body

weight of rats was significantly increased (‘P’

value less than 0.05) in test group compared to

the control group.

Table 7 shows; After administration of the

test drug wet faecal output in gm/day was

86.74% increased in test group compared to the

control group. The increased data was

statistically highly significant (‘P’ value less

than 0.01).

Table 8 depicts; After administration of the

test drug, wet faecal output in gm/100gm body

weight of rats was 39.19% increased in test

group compared to the control group. The

increased data was statistically highly

significant (‘P’ value less than 0.01).

Table 4. Effect of test drug on water intake with data presented in terms of relative values:

Group Water intake in ml/100gm body weight

Preliminary phase

MEAN ± SEM

Therapeutic phase

MEAN ± SEM

% change

Control 12.62 ± 0.92 11.81 ± 1.02

Test 13.49 ± 0.94 12.61 ± 1.01 6.77↑

Table 5.Effect of test drug on urine output with data presented in terms of absolute values:

Group Urine output in ml (absolute values)

Preliminary phase

MEAN ± SEM

Therapeutic phase

MEAN ± SEM

% change

Control 3.16 ± 0.63 1.90 ± 0.35

Test 9.06 ± 1.23 6.75 ± 1.53* 255.26↑ *P<0.05- unpaired data for comparison of control group with test drug group.

Table 6: Effect of test drug on urine output with data presented in terms of relative values:

Group Urine output in ml/100gm body weight

Preliminary phase

MEAN ± SEM

Therapeutic phase

MEAN ± SEM

% change

Control 1.44 ± 0.28 0.85 ± 0.18

Test 3.06 ± 0.31 2.22 ± 0.42* 161.18↑ *P<0.05- unpaired data for comparison of control group with test drug group.

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Table 7: Effect of test drug on faecal output (wet) with data presented in terms of absolute values:

Group Faecal matter expelled(wet) in grams ( absolute values)

Preliminary phase

MEAN ± SEM

Therapeutic phase

MEAN ± SEM

% change

Control 3.16 ± 0.39 3.47 ± 0.34

Test 7.30 ± 0.78 6.48 ± 0.45** 86.74↑

**P<0.01-unpaired data for comparison of control group with test drug group

Table 8: Effect of test drug on faecal output (wet) with data presented in terms of relative values:

Group Faecal matter expelled(wet) in gm/100gm body weight

Preliminary phase

MEAN ± SEM

Therapeutic phase

MEAN ± SEM

% change

Control 1.62 ± 0.24 1.48 ± 0.09

Test 2.47 ± 0.20 2.06 ± 0.04** 39.19↑ **P<0.01-unpaired data for comparison of control group with test drug group

Table 9 shows; After administration of the

test drug, dry faecal output in gm/day was

79.19 % increased in test group compared to

the control group. The increased data was

statistically highly significant.

Table 10 shows; After administration of the

test drug, dry faecal output in gm/100gm body

weight of rats was 47.46% increased in test

group compared to the control group. The

increased data was statistically highly

significant (‘P’ value less than 0.01).

Table 11 depicts; After administration of

the test drug, faecal water in ml/day was

90.54% increased in test group compared to the

control group. The increased data was

statistically significant(‘P’ value less than

0.05).

Table 12 depicts; After administration of

the test drug, faecal water in ml/100gm body

weight of rats was 50% increased in test group

compared to the control group.

Table 9: Effect of test drug on faecal output (dry) with data presented in terms of relative values:

Group Faecal matter expelled(dry) in grams ( absolute values)

Preliminary phase

MEAN ± SEM

Therapeutic phase

MEAN ± SEM

% change

Control 2.71 ± 0.04 2.76 ± 0.23

Test 5.73 ± 0.36 4.96 ± 0.16** 79.71↑ **P<0.01-(unpaired data for comparison of control group with test drug group).

Table 10: Effect of test drug on faecal output (dry) with data presented in terms of relative values:

Group Faecal matter expelled(dry) in gm/100gm body weight

Preliminary phase

MEAN ± SEM

Therapeutic phase

MEAN ± SEM

% change

Control 1.39 ± 0.21 1.18 ± 0.07

Test 1.96 ± 0.09 1.74 ± 0.07** 47.46↑ **P<0.01-unpaired data for comparison of control group with test drug group

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Table 11: Effect of test drug on faecal water with data presented in terms of absolute values:

Group Faecal water in ml (absolute values)

Preliminary phase

MEAN ± SEM

Therapeutic phase

MEAN ± SEM

% change

Control 0.45 ± 0.05 0.74 ± 0.15

Test 1.57 ± 0.45 1.41 ± 0.24* 90.54↑ *P<0.05 - unpaired data for comparison of control group with test drug group

Table 12: Effect of test drug on faecal water with data presented in terms of relative values:

Group Faecal water in ml/100 gm body weight

Preliminary phase

MEAN ± SEM

Therapeutic phase

MEAN ± SEM

% change

Control 0.23 ± 0.05 0.30 ± 0.04

Test 0.51 ± 0.12 0.45 ± 0.05 50↑

Table 13 depicts; After administration of

the test drug, food conversion ratio in absolute

value was significantly (36.76% ↓) decreased in

test group compared to the control group.

Table 14 depicts; After administration of

the test drug, food conversion ratio in relative

value was significantly (30.79% ↓) decreased in

test group compared to the control group.

Table 15 depicts, After administration of

the test drug, decreased in body weight was

observed in test group compared to the control

group. However the decreased data was

statistically not significant.

Table 13: Effect of test drug on food conversion ratio with data presented in terms of absolute values:

Group Food conversion ratio(absolute value)

Preliminary phase

MEAN ± SEM

Therapeutic phase

MEAN ± SEM

% change

Control 7.53 ± 1.68 4.57 ± 0.34

Test 2.39 ± 0.08 2.89 ± 0.17** 36.76↓ **P<0.01-unpaired data for comparison of control group with test drug group

Table 14: Effect of test drug on food conversion ratio with data presented in terms of relative values:

Group Food conversion ratio relative values

Preliminary phase

MEAN ± SEM

Therapeutic phase

MEAN ± SEM

% change

Control 7.37 ± 1.73 4.58 ± 0.34

Test 2.37 ± 0.08 3.17 ± 0.43* 30.79↓

*P<0.05- unpaired data for comparison of control group with test drug group

Table 15: Effect of test drug on body weight:

Group Change in Body weight

Control group 6.93 ± 3.67

Test group 1.12 ± 1.03

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 427–434

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DISCUSSION

Vipaka is defined by the Ayurvedic

scholars as the final transformation of

substances after digestion. Even though the

Shadvidha (six types), Trividha (three types)

and Dwividha (two types) Vipaka vadas

(different opinions) are discussed in classical

Ayurvedic texts, the most accepted one is

Trividha vipakavada (three types of Vipaka).

This includes Madhura vipaka (sweet

metabolic transformation), Amla vipaka (sour

metabolic transformation) and Katu vipaka

(pungent metabolic transformation). The action

of Vipaka takes place at the level of Dosha,

Dhatu and Mala (Dhyani S.C, 2003). During

the experimental study significant increase in

faecal output and urine output was observed.

Also the water content in the faecal matter was

significantly increased. This helps in the easy

evacuation of faeces. This total effect may be

considered as Sristavinmutrata (loose & easy

evacuation of bowel) which is the action of

both Madhura vipaka and Amla vipaka.

Madhura vipaka is Guru and Shukrala i.e

responsible for increase in body weight and

Shukra dhatu (increased spermatogenesis). On

the other hand Amla vipaka is just the opposite

of it, responsible for decreased spermatogenesis

and body weight. Here after giving the drug in

test group, body weight was decreased. Also

one study (Jain SK Srivastava et al., 2005) has

reported the drug, F. strobilifera used as an anti

fertility agent by traditional healers. On a

preliminary analysis of rasa (taste) and veerya

(active potency) as per available method

(Dhyani S.C, 2003), it was observed that

F.strobilifera has tikta, kashaya rasa (bitter,

astringent taste predominant) & ushna veerya

(hot potency). These data directs to a

conclusion that this drug may be a vichitra

prathyaarabdha (unusual combination of

panchamahabhutas or 5 basic elements

revealed different kind of action) one, having

Amla vipaka. This finding is supported by the

observation by a group of scientists that action

of Amla (sour) is resulted by the presence of

flavonoids and isoflavonoids in a drug (Experts

TBGRI, Kerala, India, Personal

communication). As the plant F. strobilifera

contain flavonoids, as its main active

constituent (Madan S. et al., 2008, 2009),

probability of Amla vipaka (sour metabolism)

is more.

CONCLUSION

From this preliminary assessment and

available limited data it may be concluded that

the drug, F. strobilifera may possess tikta,

kasaya rasa (bitter and astringent taste), ushna

veerya (hot potency) and Amla vipaka (sour

metabolic transformation), thus identifying it as

a vichitra prathyaarabdha drug. The results

obtained can be considered as preliminary

evidence. Based on these findings it can be

suggested that along with critical study of

literature and further experimental studies a set

of parameters can be prescribed for

determining the Rasapancaka (Ayurvedic

pharmacological properties) profile of the test

drug, especially for those plants for which such

profile is unavailable.

ACKNOWLEDGEMENT

The authors are grateful to Mr. Ravi M. and

Mr. Sudhakar, Research Officers- Department

of Pharmacology and Toxicology, SDM Centre

for Research in Ayurveda and Allied Sciences,

Udupi for their technical support.

REFERENCES

Anil Kumar K. V., Veere Gowda K (2011).

Evaluation of hepatoprotective and

antioxidant activity of Flemingia

strobilifera R.Br.against experimentally

induced Liver injury in rats. Int J

Pharm Pharm Sci. 3:115–9.

Anil K., Kavita G., Jyotsna D., Pankaj S

(2011). Analgesic activity of methanolic

extract of Flemingia strobilifera (R.Br).

IJRPC.1:8257

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Dhyani S.C (2003). Rasa-Panchaka Ayurvedic

principle of drug-action. 2nd

edition.

Choukhambha Krishnadas Academi

publisher, Varanasi, India p.60-123.

Gahlot K., Lal VK., Jha S (2013). Anti

convulsant potential of ethanol extracts

and their solvent partitioned fractions

from Flemingia strobelifera. Phcog.

Res.5:265.

Harborne J. B. Method of extraction and

isolation, In: Phytochemical methods,

2nd

ed. London: Chapman & Hall,

1998.p. 60-6.

Kavita G., Lal V. K., Jha S (2012).

Comparative morpho-anatomical and

Preliminary Phytochemical studies of

Flemingia strobilifera (L.) R.Br. and

Flemingia macrophylla (Willd.) Merr

(Fabaceae). International Journal of

PharmTech Research. 4:495.

Kirtikar K.R., Basu B.D (1935). Indian

Medicinal Plants. Vol 1. Lalit Mohan

Basu Publishers Allahabad, India p.

813.

Madan S., Singh GN., Kohli K (2009).

Isoflavonoids from Flemingia

Strobilifera (L) R.Br. roots. Acta Pol

Pharm. 66:297–303.

Madan S., Singh GN., Kumar Y (2008). A New

Flavanone from Flemingia

strobilifera (Linn.) R.Br. and its

Antimicrobial Activity. Trop J Pharm

Res.7:921–927.

Madan S., Singh G. N., Kumar Y., Kohli K

(2010). Phytochemical analysis and

free-radical scavenging activity of

Flemingia strobilifera (Linn.) R. Br.

Research Journal of Pharmaceutical,

Biological and Chemical

Sciences.1:183–90.

Reddy Janardhana K., Bahadur Bir, Bhadraiah

B., Rao MLN (2007). Advances in

Medicinal Plants. 1st edition. University

Press Private Limited, Hydrabad, India

p.3.

Saxena VK (1995). Epoxy chromenes-

Therapeutic agents from Flemingia

strobilifera. Asian J Chem.7: 307–10.

Source of Support: NIL Conflict of Interest: None Declared

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ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

QUESTIONNAIRE DESIGNING AND VALIDATION IN AYURVEDIC

RESEARCH

Ravi Bhat1*, Shivprasad Chiplunkar

2, Suhaskumar Shetty

3, Arhanth Kumar

4

1Assisstant Professor, Dept of Kriya Sharira, SDM College of Ayurveda & Hospital, Udupi, Karnataka, India

2Associate Professor, Dept of Kriya Sharira, SDM College of Ayurveda & Hospital, Hassan, Karnataka, India

3Associate Professor, Dept of Manasa Roga, SDM College of Ayurveda & Hospital, Hassan, Karnataka, India

4Assisstant Professor, Dept of Samhita, SDM College of Ayurveda & Hospital, Udupi, Karnataka, India

*Corresponding Author: drravi1@gmail.com; Mobile: 09632452121

Received: 19/09/2014; Revised: 30/10/2014; Accepted: 10/11/2014

ABSTRACT

Research in Ayurveda is gaining fast momentum now a day. Newer technique and ways are being

designed to revalidate and reestablish the time tested principles of Ayurveda. Questionnaire is one of

the extensively used tools for the collection of data in research. Use of questionnaire eases the study

for both researcher and respondents. Before using the questionnaire in Ayurveda one should know

the steps in the formation of the questionnaire and its validation. A properly framed and validated

questionnaire helps in proper collection and analysis of the data. Questionnaire helps to validate

principle and also to update the knowledge. This article aims to put light on the steps involved in

designing and validation of questionnaire in Ayurveda.

KEY WORDS: Ayurveda research, questionnaire designing, validation

Review Article

Cite this article:

Ravi Bhat, Shivprasad Chiplunkar, Suhaskumar Shetty, Arhanth Kumar (2014),

QUESTIONNAIRE DESIGNING AND VALIDATION IN AYURVEDIC RESEARCH,

Global J Res. Med. Plants & Indigen. Med., Volume 3(11): 435–444

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INTRODUCTION

Ayurveda is the life science and its

foundation is based on multiple basic

principles. A science can be called as doctrine

when it is examined by many scholars and

established with empirical results (Acharya JT,

2000). Measurement is one of the important

tools used in any medical science and its

relative research modules. One of the basic

requirements of research is data collection.

This process is carried out effectively by

incorporating various techniques such as

questionnaire format, examination and

interview. The usage of questionnaire format is

very wide in the field of Ayurvedic research. To

standardize and validate many of the Ayurvedic

principles, these questionnaires are much

needed. Concept of Prakruti (Basic body

constitution), Sāra (Effect of proper body

elements), Samhanana (Physical compactness),

Sātmya (habituation for food and activity),

Satva (Mental status), Āhara shakti (Capacity

for intake of food), Abhyavaharana shakti

(Digestive capacity), Vyāmama shakti (Physical

strength), Vaya (Age factor) and many other

concepts can be brought in to lime light by

adopting the questionnaire format in the

research of the same. These questionnaires can

be used in all sorts of Ayurvedic research. It can

be used widely in basic or pure or fundamental

research in order to formulate basic fixed

definitions for many concepts like Dosha. It

can also be used in drug research, for the

purpose of identification and availability of

some rare species like tha drug Hamsapāda.

All most all the survey studies in Ayurveda are

carried out based mainly on questionnaire.

Many pre clinical trials adopt questionnaire to

asses many clinical parameters. In this article a

sincere effort has been done to show the steps

involved in questionnaire development.

The aim of this paper is to give a basic

introduction to the research scholars about

designing and validating questionnaires in

Ayurveda research.

REVIEW OF QUESTIONNAIRE:

In the present article the entire literature is

explained under the heading of steps of

questionnaire development. Details regarding

the same are given below.

Steps of questionnaire development:

For the development of a questionnaire

following steps can be employed:

1. Decide the information required.

2. Define the target respondents.

3. Choose the method(s) of reaching target

respondents.

4. Decide on question content.

5. Develop the question wording.

6. Put questions into a meaningful order and

format.

7. Check the length of the questionnaire.

8. Pre-test the questionnaire.

9. Develop the final survey form.

1. Literary Review or collection of

information:

The basic requisite of the Questionnaire

development is the deep knowledge about the

subject. One can achieve this through the

literary review. As a first step it is always

recommended to do a literature search on

previously used and validated questionnaires

that can be administered in similar settings and

capture variables that are of interest according

to the study hypothesis. These questionnaires

do not need to be tested for reliability and

results can be compared for different studies

and also combined for meta-analysis. However

one needs to make sure that the mode of

administration should be similar to the original

questionnaire.

Literature searches for articles on

Ayurveda provide special challenges, since

many of the Indian journals in which such

articles appear are not indexed by current

medical databases such as PubMed and

Cochrane Central Register of Controlled Trials.

To solve this problem a literature search

procedure was developed that can recover the

great majority of articles on any given topic

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associated with Ayurveda. This

procedure proposes guidelines enabling

comprehensive searches to locate all types of

Ayurvedic articles, not necessarily only

randomized controlled trials (Narahari S R et

al., 2010). In the similar way there are many

databases which also help in searching the

references about the topic Eg:

http://ayurvedahealthcare.com, http://cdac.in/,

http://dharaonline.org,

http://ayurvedamanuscripts.com/,

http://rria.nic.in/, http://ayushportal.ap.nic.in

and there are many CD’s published by Govt of

India which also help in doing electronic search

of the literary review.

Systematic literature searches in

bibliographic databases are an essential step in

constructing systematic reviews and health

technology assessments. The purpose of this

kind of search is to identify as many relevant

references on a given topic in electronic

databases and other databases as much as

possible.

Most Important is the review about the

topic in the classical texts of the Ayurveda. A

detailed search about the topic should be done

in all the classical and corresponding modern

texts such that it covers all the details about the

topic. In this era of digital age, computer can

also be used for doing the literary review from

the classical texts of Ayurveda. Things are

relatively better and initiatives have already

been started elsewhere but more and more

endeavors are needed to place it on a noticeable

height. As a specialized field this particular

domain requires an integrative approach from

both the field of Ayurveda and Information

Technology. This judicious blend will

definitely be of great help in different facets of

Ayurveda be it clinical medicine, biomedical

research or information storage and retrieval

(Janmejaya S, 2013).

2. Target respondents

Important thing before starting a

research is deciding the respondents and

defining the population. For example, in an

epidemiological survey, researchers often have

to decide whether they should cover educated

population and uneducated or either of the one.

Secondly, researchers have to draw up a

sampling frame. Finally, in the questionnaire

we must take into account factors such as the

age, education, etc. of the target respondents.

3. Method(s) of reaching target respondents

Mode of administration of questionnaire

should be kept in mind at the time of its

development. On the basis of self administered

of interview based questionnaire format the

design and flow should be planned.

The language of questionnaires should be

easily understanding to the participants. It is

essential to frame the questions in a way that

they can easily be understood by participant

and should be according to their level of

education if the questions are interpreted

differently by the participants it will result in

wrong answers and responses will thus be

biased. Easiness of a questionnaire can be

assessed by Flesch reading ease score.

Translation of a questionnaire is essential if

an instrument is not available in a language of

target population. Translation is not a

mechanical work and should not be done on

word to word bases across languages rather it

should be done on the basis of meaning of the

sentence. It is important to understand the

context, specific issues and meanings the

language carries. Back Translation is highly

recommended in health surveys. Back

translation helps in evaluating the quality of the

translation. The original language is translated

in another language and again translated back

into the original language. Translation back to

the original language is done by another

translator who is uninformed of the origin

language version. (Abdul Momin Kazi, Wardah

Khalid, 2012)

There are mainly two modes of

administrating a questionnaire a) self-

administered and b) interviewer administered

questionnaire.

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Self-administered questionnaire only

requires distribution of questionnaire; it is

much easier and doesn't require trained staff. In

this technique there is less chances of

information bias. Through this technique a

large sample can be reached with wide

geographical area and wide population.

Commonly it is administered through direct

distribution or mail or electronic distribution.

Interview based administration provides

direct interaction with the participants but it is

expensive. Interviewer has the opportunity to

explain about the study and motivate the

participants for definite answers. It is the best

method to collect data in epidemiological

studies.

4. Question content or Research Question:

The questionnaire can be framed on the

basis of the hypothesis and the theory

underlying the hypothesis. It should be framed

by using the data available from the

authoritative texts of the field. Example:

Generation of items for manasika prakriti

assessment questionnaire: The questionnaire

consisted of statement on the characteristic

features of Satvika, Rajasika and Tamasika

Prakriti. The questionnaire was designed with a

total of 60 questions, among which 24

questions were related to Satva, 24 questions

for Raja and 12 questions for assessing the

Tama. The Lakshana of the each Prakriti was

converted into English for easy understanding

of the characters (Bhat R, 2013).

Table 1: Showing Satvika Prakriti assessment questionnaire

1. I am a neat and tidy person

Strongly agree Agree Can’t Say Disagree Strongly Disagree

2. I always speak truth

Strongly agree Agree Can’t Say Disagree Strongly Disagree

3. I have firm control over my mind and senses

Strongly agree Agree Can’t Say Disagree Strongly Disagree

4. I am un biased in segregating the things

Strongly agree Agree Can’t Say Disagree Strongly Disagree

5. I am quite knowledge able and talented and I can debate confidently in my area

of specialization

Strongly agree Agree Can’t Say Disagree Strongly Disagree

6. I have got a very good memory

Strongly agree Agree Can’t Say Disagree Strongly Disagree

7. I am devoid of six passions like lust, anger, delusory, emotional attachment,

pride envy

Strongly agree Agree Can’t Say Disagree Strongly Disagree

8. I am always learning or studying new things.

Strongly agree Agree Can’t Say Disagree Strongly Disagree

9. I am very devoted to my work.

Strongly agree Agree Can’t Say Disagree Strongly Disagree

10. I am involved in religious activities.

Strongly agree Agree Can’t Say Disagree Strongly Disagree *Table Courtesy: Bhat R et al., (2013)

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5. Question wording and format

Scale and response format:

It is one of the important parts of designing

a questionnaire. A questionnaire is a written

document to gather information irrespective of

mode of administration. It can be undertaken in

following steps:

Type of Questions and scale:

A questionnaire can be structured or

unstructured, open-ended or close ended. It can

be selected according to need. Structured

questionnaire can be selected if all the

participants are asked same question in the

same way as done in an interview. In

Unstructured format questions may vary with

the judgment of the interviewer. It is of more

useful at clinical settings however structured

questionnaire are more preferred for

epidemiological studies as same data from all

respondents need to be analyzed and measured.

(Nigel Mathers, Nick Fox, Amanda Hunn

2002)

Open-ended questionnaire is suitable for the

study when large number of options are

available and where it is not possible to write

all the answers in advance e.g: height of

patients. It allows respondents to write answers

in any way they want. This kind of

questionnaire might increase the burden on

work and responses have to be individually

reviewed by the investigator before assigning

codes and analysis. Eg: Open-Ended Interview

Questions: 1. Tell me about nature of pain. 2.

Where is pain located? 3. How often you get

sneeze? 4. How many hours you sleep? 5.

Which food stuff increases the itching?

In closed-ended questionnaire the

respondents are said to make choices among a

set of answers in a given question. The

response could be exclusive or may select more

than one option. For measuring variables which

are sharply opposed closed- ended questions

are preferred because possible answers can be

easily pre-coded. Pre-coding of questions is

defined by assigning numbers to an answer. It

saves time as assigning of number latter is

reduced and hence decreases error; however for

open-ended questions coding is done after the

data is collected. Coding helps in data entry, as

information of questionnaires in paper format

are entered in data entry programs by putting in

the numbers rather than writing the whole

answer (Reja U et al., 2003). Eg: In Designing

and validation of ojo kshaya scale; The scale

consisted of statement for subjective

parameters based on the characteristic features

of Ojo Kshaya given in Charaka Samhita. The

appropriate English meanings of Lakshana

(symptom) were referred to and were framed in

the sentence form with five options to each,

e.g., Vyathitendriya means pain/discomfort in

the chest region. It was framed as, “Do you feel

pain or discomfort in chest region?” and the

response format consisted of eight questions

and the maximum score was 32 (Bhat R, 2013).

Two different reasons for using open ended

as opposed to closed ended questions can be

distinguished. One is to discover the response

that individual give spontaneously and the

other is to avoid the bias that may result from

suggesting response to individuals (Vasja V,

2003).

6. Putting questions into a meaningful order

and format

Opening questions: Opening questions should

be easy to answer and not in any way

threatening to the respondents. The first

question is crucial because it is the respondent's

first exposure to the interview and sets the tone

for the nature of the task to be performed. If

they find the first question difficult to

understand, or beyond their knowledge and

experience, or embarrassing in some way, they

are likely to break off immediately. If, on the

other hand, they find the opening question easy

and pleasant to answer, they are encouraged to

continue (Reja U et al., 2003).

Question flow: Questions should flow in some

kind of psychological order, so that one leads

easily and naturally to the next. Questions on

one subject, or one particular aspect of a

subject, should be grouped together.

Respondents may feel it disconcerting to keep

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shifting from one topic to another, or to be

asked to return to some subject they thought

they gave their opinions about earlier.

Question variety: Respondents become bored

quickly and restless when asked similar

questions for half an hour or so. It usually

improves response, therefore, to vary the

respondent's task from time to time. An open-

ended question here and there (even if it is not

analyzed) may provide much-needed relief

from a long series of questions in which

respondents have been forced to limit their

replies to pre-coded categories. Questions

involving showing cards/pictures to

respondents can help vary the pace and increase

interest.

7. Length of the questionnaire

Questionnaire Style and Appearance:

The appearance and style of the

questionnaire plays a very important role

especially in self- administered questionnaire.

Format, order, spacing, fonts used and grouping

of the response are very important features of a

good questionnaire and have a direct effect on

the responses and time spent by the respondent

to provide it. Questions should be simple, clear

and easy to understand, using minimum of

words and space and only things what is

needed should be asked. Lengthy or confusing

questionnaire can also make the interviewer

confused and responses administered by the

interviewers may not be accurate or complete.

The clarity of questionnaire has direct impact

on data collected by the interviewer and

responses given by the responders.

(Questionnaire Design, 2003) Example:

The Hamilton Anxiety Rating Scale (HAM-A)

is a psychological questionnaire used

by clinicians to rate the severity of a

patient's anxiety. The Hamilton Anxiety Rating

Scale is composed of fourteen items. On the

scale, each item is presented in a specific

format. Following the item number, the item

itself is listed along with a brief description of

the criterion. Each criterion on the scale is an

independent feeling that is related to anxiety.

The collaboration of each of these

independently-rated criteria is meant to

evaluate a patient's anxiety severity (Hamilton

M., 1959).

Phraseology

The wordings on the questionnaire are very

important and should be given at most

importance when it is framed. Appropriateness

of the content, sophistication of language,

sequence of question, type, form and how data

is collected from the respondents says about the

quality of study.

8. Testing the questionnaire

Validity:

A questionnaire must be validated to make

sure that it accurately measures what it is

supposed to do, regardless of the responder.

Valid questionnaire helps to collect better

quality data with high comparability which

reduces the effort and increase the reliability of

data. (Questionnaire Design, 2003) A valid

questionnaire must have following

characteristics (i) simplicity and viability (ii)

reliability and precision in the words (iii)

adequate for the problem intended to measure

(iv) reflect underlying theory or concept to be

measured and (v) capable of measuring change.

Validity of a questionnaire is an assessment

measures which checks the quality of the

questionnaire for assessing what is it supposed

to.

A questionnaire can be validated using

following steps,

Content Validity

Content Validation in any tool says how

well the individual items in the tool correspond

to the concept of what are being examined

(Bordens S K &Abott B B., 1998). It is usually

tested using the qualitative technique. Eg:

Content validation of the Manasika Prakriti

assessing Questionnaire was done by studying

the references available in Charaka Samhita.

Considering their measuring feasibility and the

selected variable were also cross – validated by

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 435–444

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Ayurvedic Experts for their suitability as a

dependable expression to identify the

dominance of particular Prakriti (Bhat R,

2013).

Criterion/face validity:

Face validity is the extent to which a test

is subjectively viewed as covering the concept

it purports to measure. It refers to the

transparency or relevance of a test as they

appear to test participants. In other words, a test

can be said to have face validity if it "looks

like" it is going to measure what it is supposed

to measure (Sirkin M, 2012). For instance, if

you prepare a test to measure whether students

can perform multiplication and the people you

show it to all agree that it looks like a good test

of multiplication ability; you have shown the

face validity of your test.

Construct Validity:

Construct validity is “the degree to which a

test measures what it claims, or purports, to be

measuring.” In the classical model of validity,

construct validity is one of three main types of

validity evidence, alongside content

validity and criterion validity

(Hegan E F,

2012)

Construct validity is the appropriateness of

inferences made on the basis of observations or

measurements (often test scores), specifically

whether a test measures the intended construct.

Constructs are abstractions that are deliberately

created by researchers in order to conceptualize

the latent variable, which is the cause of scores

on a given measure (although it is not directly

observable).

Construct validity is essential to the

perceived overall validity of the test. Construct

validity is particularly important in the social

sciences, psychology, psychometrics and

language studies.

Internal consistency:

In statistics and research, internal

consistency is typically a measure based on

the correlations between different items on the

same test (or the same subscale on a larger

test). It measures whether several items that

propose to measure the same general construct

produce similar scores. Internal consistency is

usually measured with Cronbach's alpha, a

statistic calculated from the pair wise

correlations between items. Internal

consistency ranges between negative infinity

and one. Coefficient alpha will be negative

whenever there is greater within-subject

variability than between-subject variability. For

example, In assessing personality scale validity

using internal consistency and retest reliability

data (N = 34,108) was examined on the

differential reliability and validity of facet

scales from the NEO Inventories. We evaluated

the extent to which (a) psychometric properties

of facet scales are generalizable across ages,

cultures, and methods of measurement; and (b)

validity criteria are associated with different

forms of reliability. In the study the Cronbach’s

alpha value was 0.88 indicating good internal

consistency (McCrae R E & Kurtz JV, 2011).

Factor analysis:

Factor analysis is a statistical method used

to describe variability among observed,

correlated variables in terms of a potentially

lower number of unobserved variables called

factors. (Wikipedia, 2012) For example, it is

possible that variations in four observed

variables mainly reflect the variations in two

unobserved variables. Factor analysis searches

for such joint variations in response to

unobserved latent variables.

Inter rater reliability

Inter-rater reliability, inter-rater agreement,

or concordance is the degree of agreement

among raters. It gives a score of how

much homogeneity, or consensus, there is in

the ratings given by judges. It is useful in

refining the tools given to human judges, for

example by determining if a particular scale is

appropriate for measuring a particular variable.

If various raters do not agree, either the scale is

defective or the raters need to be re-trained

(Wikipedia 2012).

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There are a number of statistics which can

be used to determine inter-rater reliability.

Different statistics are appropriate for different

types of measurement. Some options are: joint-

probability of agreement, Cohen's kappa and

the related Fleiss' kappa, inter-rater correlation,

concordance correlation coefficient and intra-

class correlation.

Pilot Study

Designing a questionnaire is incomplete

without pilot study, it is impossible even for the

experts to get it right the first time round.

Questionnaires must be pretested that is, piloted

on a small sample of people characteristic of

those in the survey. In a small survey, there

might be only pretesting of the drafted

questionnaire. In a large survey, there may be

three phases of piloting. In the first phase we

might ask each respondent in great detail about

a limited number of questions: effects of

different wordings, what they have in mind

when they give a particular answer, how they

understand a particular word, etc. In the second

phase the whole questionnaire is administered

by interviewers. Analysis of the responses and

the interviewers’ comments are used to

improve the questionnaire. Ideally, there should

be sufficient variations in responses among

respondents; each question should measure a

different quality that is, the responses between

any two items should not be very strongly

correlated and the non-response rate should be

low. In the third phase the pilot test is polished

to improve the question order, filter questions,

and layout (Branacto G, Macchina S, Sigore M,

2012).

Final survey form:

If the questionnaire has been subjected to a

thorough pilot test, the final form of the

questions and questionnaire will have evolved

into its final form. All that remains to be done

is the mechanical process of laying out and

setting up the questionnaire in its final form.

This will involve grouping and sequencing

questions into an appropriate order, numbering

questions, and inserting interviewer

instructions.

DISCUSSION

The qualities of a good questionnaire

It is extremely significant for a Ayurvedic

researcher to know the importance of a proper

questionnaire formation and to know whether it

measures what it is intended to measure.

Composing of a questionnaire is always much

more complex than expected. Great attention is

required to its flow, format and length. Making

an individual question is a tedious task and

validating this questionnaire is another

challenge which at times is over looked.

Importance should be given on whether the

questionnaire will measure quantitative or

qualitative data, and what would be its mode of

administration.

Considering all these views here is an

attempt made to explore and explain the

importance of questionnaire in Ayurveda

through an example.

Considering the deficit in the tools for the

analysis of Satvika Prakriti, and its importance

in the maintenance of health and in treating the

disease, a Questionnaire for assessing the same

was designed.

To frame the questionnaire literary data is

collected from all classical text books of

Ayurveda, electronic media and web media.

The collected literary information is analyzed

and systematically arranged. The target

respondents will be educated individuals who

are apparently healthy. Sample size is decided

statically.

The mode of reaching target is self

administrable questionnaire for the assessment

of Satva. This mode is selected because target

sample is educated and to make the sample

comfortable.

Contents of the questionnaire are decided

based on the literary information available in

classical text books of Ayurveda mainly

Charaka Samhita.

The questionnaire was framed in a close

ended Likert format with 5 options for each

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 435–444

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question i.e always, occasionally, can’t say, no

and never. Always was graded as 4,

occasionally as 3, can’t say 2, no as 1 and never

was graded 0.

The questionnaire consisted of statement on

the characteristic features of Satvika Prakriti.

The literary information regarding the features

of Satvika Prakriti was converted into English

for easy understanding of the characters.

The questionnaire is then subjected for

validation. For this purpose the questionnaire

can be subjected for content validation, face

validation, construct validation and statistical

tests such as internal consistency, factor

analysis and inter rater reliability. Table 1

shows the questionnaire format for the

assessment of Satvika Prakriti (Bhat R, 2013)

After the completion of the steps of

validation, the questionnaire is subjected for

further process. In case of positive validation

the questionnaire is subjected for the process of

final survey form. If the questionnaire is

negatively validated then the questionnaire

should be revised from the beginning.

CONCLUSION

A good questionnaire is one which helps

the researcher to achieve the objectives,

provides complete and accurate information. In

Ayurveda validation of many concepts is need

of the era. For the process of validation,

framing a suitable tool is much more essential.

In this regard questionnaire development plays

a major role. In the present study questionnaire

development techniques has been explained in

detail. Along with this a suitable example of

Satvika Prakriti assessment is also described.

In total a complete ideology is given with an

intension of enlightening the concept of

Ayurveda through questionnaire development.

REFERENCES

Acharya JT (2000), Charaka Samhita of

Agnivesa elaborated by Charaka &

Dridhabala with the Ayurveda dipika

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Chaukhambha Surbharati Prakashan,

Varanasi, p.267

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