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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 389–401
Essien E B, Onwuka F C, Odjoh O, Odeghe O B
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 PROPERTIES OF ARGANIA SPINOSA (L.) SKEELS. LEAF EXTRACTS
AND
ANTIOXIDANT
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 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal
Research Article 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 C2, Odjoh O3, Odeghe O B4 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 twentyfive 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.
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 Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 389–401
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
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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 389–401
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
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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 389–401
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.
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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 389–401
Table 1. Proximate analysis of milk samples and soya milk analyzed Parameter Energy (kcal) Protein (%) Ash (%) Fats (%) Moisture (%) Fiber (%) Carbohydrate (%) Vitamin A (IU) Vitamin B1(mg) Vitamin B2 (mg) Vitamin B3 (mg) Vitamin B6 (mg) Vitamin B12(mg) Vitamin C (mg) Vitamin E (mg) Calcium (mg) Phosphorus (mg) Magnesium (mg) Potassium (mg) Sodium (mg) Iron (mg) Zinc (mg) Copper (mg) Selenium (µg) Manganese (mg)
Skimmed milk 261.5 28.13 ± 0.15 8.70 ± 0.01 2.52 ± 0.08 3.96 ± 0.03 0.00 31.57 ± 0.82 2500* NI 1.40 NI NI 0.003 NI NI 1800 900 120 1600 19.87 NI 0.08 0.12 NI 0.04
whole milk 393.2 24.01 ± 0.03 5.44 ± 0.03 23.16 ± 0.23 0.81 ± 0.01 0.00 22.21 ± 0.03 2700 0.99 1.10 0.60 0.90 0.0024 90 0.60 930 750 85 1200 340 0.17 31 0.02 10 0.02
Flavoured milk 339.2 12.36 ± 0.64 2.13 ± 0.13 7.11 ± 0.20 21.52 ± 0.57 0.00 56.44 ± 2.23 3750 0.90 1.40 11.00 1.50 0.0045 30 4.00 380 312 121 523 106 13.50 3.8 0.1 17.5 0.10
Soya milk 339.2 21.44 ± 0.29 6.50 ± 0.02 21.90 ± 0.42 1.20 ± 0.01 36.62 ± 0.03 12.34 ± 0.17 23.21 ± 0.02 0.19 ± 0.02 0.15 ± 0.03 0.98 ± 0.08 5.35 ± 0.04 3.47 ± 0.07 3.63 ± 0.03 0.34 ± 0.05 81.5 ± 0.06 5.50 ± 0.02 192.69 ± 0.07 12.00 ± 0.06 2.59 ± 0.11 15.30 ± 0.06 0.20 ± 0.03 0.20 ± 0.03 7.15 ± 0.02mg 0.01 ± 0.02
*Enriched, NI= Not indicated
Table 2. Phytochemical Screening Of Soya Milk Secondary metabolites Alkaloids Flavonoids Glycosides Saponins Steroids Terpenioids Carbohydrates Reducing sugar Resin Tannins Proteins Oils Acid compounds
Relative abundance − − ++ ND ++ +++ ND ++ ND − ND ND ND
Key: − = Absent; + = Low in concentration; ++ = Moderate in concentration; +++ = High in concentration; ND = Not determined
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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 389–401
Table 3. Mean body weights of rats (grams) fed the milk samples Groups Control Skimmed milk Whole milk Soya milk Flavoured milk
Initial body weight 218.20 ± 12.31b 208.00 ± 9.43b 206.60 ± 6.79b 209.80 ± 10.05b 209.60 ± 8.61b
Final body weight 266.00 ± 14.50b 240.40 ± 12.25b 242.20 ± 7.45b 271.00 ± 10.95b 245.60 ± 9.26b
Weight gained (%) 47.80 32.40 35.60 61.20 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 Control Skimmed milk Whole milk Soya milk Flavoured milk
Hemoglobin (g/dl) 10.04 ± 0.98b 11.22 ± 0.44b 10.47 ± 0.77b 13.24 ± 0.42a 11.50 ± 0.16b
Packed cell volume (%) 30.00 ± 2.94b 33.60 ± 1.20a 31.50 ± 2.32b 39.80 ± 1.28a 34.80 ± 0.37a
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
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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 389–401
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
Whole milk
Soya milk
67.40 ± 0 .86b 40.20 ± 0.49b 58.00 ± 2.00b 2.90 ± 0.10b 3.66 ± 0.24b
Skimmed milk 65.40 ± 0.29b 39.40 ± 0.00b 59.33 ± 1.76b 2.96 ± 0.08b 3.93 ± 0.06b
52.83 ± 5.13a 31.70 ± 3.09a 59.33 ± 0.66b 2.96 ± 0.03b 4.00 ± 0.57b
44.73 ± 4.78a 27.13 ± 2.70a 60.66 ± 0.33b 3.10 ± 0.05b 3.26 ± 0.14b
Flavoured milk 55.23 ± 4.76a 31.13 ± 2.54a 57.33 ± 0.66b 2.80 ± 0.05b 3.70 ± 0.35b
Total protein (g/l) Albumin (g/l) Creatinine (µmol/l) Urea (mmol/l) Direct bilirubin (µmol/l) Total bilirubin (µmol/l)
7.53 ± 0.37b
6.70 ± 0.20b
6.96 ± 0.32b
7.26 ± 0.21b
6.73 ± 0.83b
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
5.90 ± 0.05b Aspartate aminotransaminase 6.10 ± 0.05b Alanine aminotransaminase Alkaline phosphatase 17.60 ± 0.30b
Skimmed milk 5.66 ± 0.33b
Whole milk
Soya milk
6.00 ± 0.57b
6.03 ± 0.03b
Flavoured milk 5.86 ± 0.13b
6.00 ± 0.57b
6.00 ± 0.00b
5.30 ± 0.40b
5.33 ± 0.33b
18.03 ± 0.03b
17.33 ± 1.45b
15.26 ± 0.37b
16.00 ± 1.05b
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
Cholesterol
3.23 ± 0.03b 0.83 ± 0.01b 1.96 ± 0.03b 1.73 ± 0.03a
High density lipoprotein Low density lipoprotein
Triglycerides
Skimmed milk 3.70 ± 0.05b 0.91 ± 0.00b 2.33 ± 0.06b 2.26 ± 0.08a
Whole milk
Soya milk
1.76 ± 0.08a 0.47 ± 0.03b 0.77 ± 0.08a 2.43 ± 0.12a
2.10 ± 0.15b 0.58 ± 0.04b 1.26 ± 0.08b 1.06 ± 0.08a
Flavoured milk 3.13 ± 0.78b 0.64 ± 0.17b 2.03 ± 0.61b 1.60 ± 0.05a
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
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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
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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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Friedewald, W.T., Levy, R.T. and Fredickson, D.S. (1972). Estimation of the concentration of low density lipoprotein cholesterol in plasma, without use of preparative ultracentrifuge. Clin. Chem. 18: 499– 502. Harborne, J.B. (1973). Phytochemical method. A guide to modern techniques for plant analysis. Chapman and Hill, London. Pp.182–201. Jendrassik, L. and Grof, P. (1938). Colorimetric method and determination of bilirubin. Biochem. Z. 297: 81. Jensen, R.G. (1998). Handbook of Milk Composition. Academic Press, New York. Kataoka, K., Nakae, T. and Imamura, T. (1991). Comparative studies on the milk constituents of various mammals in Japan. J. Dairy Sci. 20:222–232. Klein, B., Read, P.A. and Babson, L.A. (1960). Rapid method for quantitative determination of serum alkaline phosphatase. Clin. Chem. 6: 269–275. Kristi,
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Murray-Kolb, L.E., Welch, R., Theil, E.C. and Beard, J.L. (2003). Women with low iron stores absorb iron from soybeans. Am. J. Clin. Nutr. 77:180–4. Ness, A.R., Smith, D.G. and Hart, C. (2001). Milk, Coronary Heart Disease and Mortality. J. Epidemiol. 55: 379–382. Nutritional Aspects of Cardiovascular Disease (1994). Report of the Cardiovascular Review Group: the Committee on Medical Aspects of Food Policy, London. Pamplona-Roger, G.D. (2004). Encyclopedia of foods and their healing power. A guide to Food Science and Diet Therapy. Vol.1. Editorial safeliz. pp. 183. Ramnik,
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Source of Support: NIL
Conflict of Interest: None Declared
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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 402–415 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal
Research Article 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 Gendron2*, Jody Bellegarde3, Betty McKenna4, Delores Louie5, Geraldine Manson6, Harvey Alphonse7 1,5,6,7
Biology 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: 306-7905994
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.
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 Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
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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 soilborne 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).
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.
This preliminary study has employed selected standard methods including Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
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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 preweighed 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 Tukeyâ&#x20AC;&#x;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. Oneway ANOVA and Tukey-Kramerâ&#x20AC;&#x;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 Control 0.08 g 5.38 g 10.76 g
P. major / A. millefolium P. aeruginosa S. aureus 0.31a 0.37a 0.65b 0.97b b 0.77 0.75b 0.26a 1.05b
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. major P. aeruginosa S. aureus
0.31a 0.65b 0.71b 0.71b
Control 0.5 g 5.0 g 10.0 g
0.37a 0.91a 0.81a 0.91a
Means within each column with the same letter (a) are not significantly different (p < 0.05).
Achillea millefolium aeruginosa
treatment
vs.
P.
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 â&#x2C6;&#x2019;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 A. millefolium P. aeruginosa S. aureus
-0.17a 0.97b 1.24b 0.45a
Control 0.08 g 5.38 g 10.76 g
0.41a 1.60b 0.77b 0.35a
Means within each column with the same letter (a) are not significantly different (p < 0.05).
Kirby Bauer disc Plantago major
diffusion
test
with
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. 11:30 a.m. 7:00 p.m. 7:00 p.m. P. aeruginosa S. aureus P. aeruginosa S. aureus
Plant Extraction 500 mg/ml 50 mg/ml 5 mg/ml 0.5 mg/ml 0.05 mg/ml DMSO Methanol Ciprofloxacin Gentimicin
8.10b 6.0a 6.0a 6.0a 6.0a 6.0a 6.0a 28.81e 19.90d
9.05b 6.86ab 6.0a 6.24ab 6.0a 6.0a 6.0a 24.67d 20.14c
6.0a 6.0a 6.0a 6.0a 6.0a N/A 6.0a 29.33c 17.52b
7.95b 6.19a 6.0a 6.0a 6.0a 6.0a 6.0a 23.38d 19.38c
Means within each column with different letters (aâ&#x20AC;&#x201C;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 Control 0.5 g 10.0 g
Log cfu ml-1 Log cfu ml-1 P. aeruginosa S. aureus TNTC TNTC TNTC 1.20 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 TFTC 2.28 Control TNTC 0.90 0.08 g 2.44 TFTC 10.57 g 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 (%) a Plantain, BC, 2012, 11:30 a.m. 0.0656 ± 0.0223 0.1807 ± 0.0663a Plantain, BC, 2012, 7:00 p.m. 0.2373 ± 0.3771a 0.1337 ± 0.0327a 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 elderâ&#x20AC;&#x;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 elderâ&#x20AC;&#x;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 antibacterial effectiveness
and
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 drugresistant 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
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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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Mojab, F., Kamalinejad, M., Ghaderi, N., & Vahidipour, H.R. (2003). Phytochemical screening of some species of Iranian plants. Iran. J. Pharm. Res., 2, 77–82. Nascimento, G.G.F., Locatelli, J., Freitas, P.C., & Silva, G.L. (2000). Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria. Braz. J. Microbiol., 31, 247– 256. Nilson, A.M., & Holley, R.A. (2012). Use of deodorized yellow mustard powder to control Escherichia coli O157:H7 in dry cured Westphalian ham. Food Microbiol., 30, 400–407. Nilson, S.M., Bharadwaj, L.A., Knockwood, D., & Hill, V. (2008). Science in a circle: forming „community links‟ to conduct health research in partnership with communities. Pimatisiwin, 6, 123– 135. Nychas, G.J.E., Tassou, S.C., & Board, R.G. (1990). Phenolic extract from olives: inhibition of Staphylococcus aureus. Lett. Appl. Microbiol., 10, 217–220. Pistelli, L., Bertoli, A., Lepori, E., Morelli, I., & Panizzi, L. (2002). Antimicrobial and antifungal activity of crude extracts and isolated saponins from Astragalus verrucosus. Fitoterapia, 73, 336–339. Reina, E., Al-Shibani, N., Allam, E., Gregson, K.S., Kowolik, M., & Windsor, L.J. (2013). The effects of Plantago major on the activation of the neutrophil respiratory burst. J. Tradit. Complement. Med., 3, 268–272. Samuelsen, A.B. (2000). The traditional uses, chemical constituents and biological activities of Plantago major L. A review. J. Ethnopharmacol., 71, 1–21.
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Sasseville, D. (2011). Contact dermatitis from topical antibiotics. Eur. J. Dermatol., 21, 311–322.
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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.
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Conflict of Interest: None Declared
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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 416–426 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal
Research Article EVALUATION OF PHENOLIC COMPOUNDS, FLAVONOIDS AND ANTIOXIDANT PROPERTIES OF ARGANIA SPINOSA (L.) SKEELS LEAF EXTRACTS Saliha DJIDEL1, Choubaila -Feriel CHATER2, Seddik KHENNOUF3*, Abderrahmane BAGHIANI4, Daoud HARZALLAH5 1,2,3
Laboratory of Phytotherapy Applied to Chronic Diseases, Department of Animal Biology and Physiology, Faculty of Natural and Life Sciences, University Sétif 1, 19000 Algeria. 4 Laboratory 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.
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 J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 416–426
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 (chainbreaking 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, 2diphenyl-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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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 416–426
Preparation of A. spinosa leaves extracts
Antioxidant activity by DPPH° assay
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.
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.
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.
Hydroxyl radical-scavenging phenanthroline-Fe (II) oxidation assay
by
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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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 416–426
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-6sulphonic 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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We can deduce that all these extracts are rich in polyphenols and flavonoids.
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.
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) CE 27.7 256.16 ± 0.02 CHE 0.7 106.33 ± 0.062 EAE 2.1 447.2 ±0.028 AQE 18.1 216.33 ±0.004
Total flavonoids (mg QEq/g) 18.8 ± 0.007 30.76 ± 0.004 185.93 ± 0.009 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. Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
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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 doseresponse 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. IC 50 mg/ml
0.5 0.4
***
***
0.3
***
0.2
**
0.1 0 CE
CHE
EAE
AQE
VIT C
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. Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
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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. ns
Inhibition %
100
ns
**
80
** 60 40 20
***
***
0
BHT
CE
CHE
EAE
AQ
MOH
H2O
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 dosedependent 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
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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
IC 50 mg/ml
0.012
0.009 0.006
*** µ*** *µµ
*** µ** **µ µ
0.003 -2.26E-17
CE
CHE
** µ ** ** EAE µ µ
*** µ* *** µµ
AQE
Trolox
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.
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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 (RiceEvans 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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Bnouham, M., Bellahcen, S. Benalla, W. (2008). J. Compl. Integ.r Med. 5: 1–32 Burits M, Bucar F. (2000). Antioxidant activity of Nigella sativa essential oil. Phytotheraphy Research. 14: 323–328 Chahboun J. (1993). La filière triterpénique dans les lipides des feuilles d'Argania spinosa, PhD Thesis, Perpignan, France, Chang CC, Yang MH, Wen HM, Chern JC. (2002). Estimation of total flavonoid content in Propolis by two complementary colorimetric methods. J. Food Drug Anal. 10:178–182. Charrouf Z, Guillaume D. (1999). Ethnoeconomical, ethnomedical, and phytochemical study of Argania spinosa (L.) Skeels. J Ethnopharmacol. 67: 7– 14. Chung YC, Chen SJ, Hsu CK, Chang CT, and Chou ST. (2005). Studies on the paraguayense, Food Chemistry. 91: 419–424.
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Decker EA, Welch B. (1990). Role of ferritin as a lipid oxidation catalyst in muscle food. Journal of Agricultural and Food Chemistry. 38: 674–677. Djeridane A, Yousfi M, Nadjemi B, Boutassouna D, Stocker P and Vidal N. (2006) Antioxidant activity of some algerian medicinal plants extracts containing phenolic compounds. Food Chemistry. 97: 654–660.
Li W, Wei CV, White PJ, Beta T. (2007). High-amylose corn exhibits better antioxidant activity than typical and waxy genotypes. J. Agric. Food. Chem. 55(2): 291–298. Li YH, Jiang B, Zhang T, Mu WM, Liu J. (2008). Antioxidant and free radicalscavenging activities of chickpea protein hydrolysate (CPH). Food Chemistry. 106: 444–450.
Dorman HJ, Peltoketo D, Hiltunen AR and Tikkanen MJ. (2003). Characterization of the antioxidant properties of deodourised aqueous extracts from selected Lamiaceae herbs. Food Chemistry. 83: 255–262.
Markham KR. (1982).Techniques of Flavonoid Identification, Academic Press, London, 1–133.
Favier A. (2003). Le stress oxydant : Intérêt conceptuel et expérimental dans la compréhension des mécanismes des maladies et potentiel thérapeutique. Actualité chimique. 108–115.
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Joguet N, Maugard T. (2013). Characterization and quantification of phenolic compounds of Argania spinosa leaves by HPLC-PDA-ESI-MS analyses and their antioxidant activity. Chemistry of Natural Compounds. 48: 1069–1071. Galvez M, Martin-Cordero C, Houghton PJ, Ayuso MJ. (2005). Antioxidant activity of methanol extracts obtained from Plantago species. J. Agric. Food Chem. 53: 1927–1933 Halliwell B, Gutteridge J.(1999). Free radicals in biology and medicine, Oxford University Press, 888 pages Koleva II, Van Beek TA, Linssen JPH, de Groot A, Evstatieva LN. (2002). Screening of plant extracts for antioxidant activity: a comparative study on three testing methods. Phytochemical Analysis. 13: 8–17.
Morsli A. (1999). Thèse de magister, protection de la nature, INA, Algérie, 89 pp. PS, Jayaprakasha GK. (2003). Antioxidant and Antibacterial Activities of Punica granatum Peel Extracts. Journal of Food Science. 68: 1473– 1477.
Parejo I, Viladomat F, Bastida J, RosasRomero A, Flerlage N, Burillo J, and Codina C. (2002). Comparison between the radical scavenging activities and antioxidant activity of six distilled and nondistilled maditerranean herbs and aromatic plants. J. Agric. Food. Chem. 50: 6882–6890. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 26:1231–1237. Rice-Evans CA, Miller NJ, Paganga G. (1997). Antioxidant properties of phenolic compounds. Trends in Plant Science. 2(4):152–159. Sakanaka S, Tachibana Y, Okada Y. (2005). Preparation and antioxidant properties of extracts of Japanese persimmon of Leaf tea (kakinohacha). Food Chemistry. 89: 569–575.
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Shahidi F and Naczk M. (1995). Food Phenolics: Sources, Chemistry, Effects and Applications. Technomic Publishing Company Inc., Lancaster (Pennsylvania). pp. 281–319. Tahrouch S, Andary C, Rapior S, Mondolot L, Gargadennec A, Fruchier A. (2000). Polyphenol investigation of Argania spinosa (Sapotaceae) endemic tree from
Source of Support: Algerian Ministry of Higher Education and Scientific Research (MERS) and L’ Agence Thématique de Recherche en Sciences de la Santé (ATRSS)
Morocco. Acta Botanica Gallica 147 (3) 225–232 Tahrouch SA, Hatimi S, Rapior L, Mondolot A, Gargadennec C, Andary C. (2011). Phenolic compounds of Argan tree, Argania spinosa (endemic species of south western Morocco. The Online Journal of Science and Technology. 1(4): 17–23.
Conflict of Interest: None Declared
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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 11 | November 2014 | 427–434 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal
Research Article ASSESSMENT OF ‘VIPAKA’ (METABOLISM) OF A NEW MEDICINAL PLANT IN ANIMAL MODEL Bidhan Mahajon1*, Ravi Shankar B2, Remadevi R3 1
PG Scholar, Department of Dravyaguna Vijnanam, Vaidyaratnam P. S. Varier Ayurveda College, Kottakkal, Kerala, India 676501. 2 Professor of Experimental Medicine and Director, Department of Pharmacology and Toxicology, SDM Centre for Research in Ayurveda and Allied Sciences, Udupi, India 574 118. 3 Professor 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
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
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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 Strobilifera:
F.
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%.
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Experimental Procedure:
Statistical analysis:
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.
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
Control Test
Food intake in grams ( absolute values) Preliminary phase Therapeutic phase % change MEAN ± SEM MEAN ± SEM 13.34 ± 0.65 11.93 ± 1.007 − 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
Control Test
Food intake in gm/100g body weight Preliminary phase Therapeutic phase % change MEAN ± SEM MEAN ± SEM 5.16 ± 0.12 5.10 ± 0.28 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 Therapeutic phase % change MEAN ± SEM MEAN ± SEM 27.33 ± 1.22 27 ± 1.56 Control 39.7 ± 3.91 37.48 ± 4.45 38.81↑ Test value less than 0.05) in test group compared to Table 4 shows; After administration of the the control group. test drug, water intake in ml/100 gm body Table 7 shows; After administration of the weight of rats was 6.77% increased in test test drug wet faecal output in gm/day was group compared to the control group; however 86.74% increased in test group compared to the that increased data was statistically not control group. The increased data was significant. statistically highly significant (‘P’ value less Table 5 shows; After administration of the than 0.01). test drug urine output in ml/day was Table 8 depicts; After administration of the significantly increased (‘P’ value less than test drug, wet faecal output in gm/100gm body 0.05) in test group compared to the control weight of rats was 39.19% increased in test group. group compared to the control group. The Table 6 depicts; After administration of the increased data was statistically highly test drug, urine output in ml/100gm body significant (‘P’ value less than 0.01). weight of rats was significantly increased (‘P’ 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 Therapeutic phase % change MEAN ± SEM MEAN ± SEM 12.62 ± 0.92 11.81 ± 1.02 Control 13.49 ± 0.94 12.61 ± 1.01 6.77↑ Test 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 Therapeutic phase % change MEAN ± SEM MEAN ± SEM 3.16 ± 0.63 1.90 ± 0.35 Control 9.06 ± 1.23 6.75 ± 1.53* 255.26↑ Test *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 Therapeutic phase % change MEAN ± SEM MEAN ± SEM 1.44 ± 0.28 0.85 ± 0.18 Control 3.06 ± 0.31 2.22 ± 0.42* 161.18↑ Test *P<0.05- unpaired data for comparison of control group with test drug group. Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
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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 Therapeutic phase % change MEAN ± SEM MEAN ± SEM 3.16 ± 0.39 3.47 ± 0.34 Control 7.30 ± 0.78 6.48 ± 0.45** 86.74↑ Test **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
Control Test
Faecal matter expelled(wet) in gm/100gm body weight Preliminary phase MEAN ± SEM 1.62 ± 0.24 2.47 ± 0.20
Therapeutic phase MEAN ± SEM 1.48 ± 0.09 2.06 ± 0.04**
% change
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
Control Test
Faecal matter expelled(dry) in grams ( absolute values) Preliminary phase MEAN ± SEM 2.71 ± 0.04 5.73 ± 0.36
Therapeutic phase MEAN ± SEM 2.76 ± 0.23 4.96 ± 0.16**
% change
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
Control Test
Faecal matter expelled(dry) in gm/100gm body weight Preliminary phase Therapeutic phase % change MEAN ± SEM MEAN ± SEM 1.39 ± 0.21 1.18 ± 0.07 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 Therapeutic phase % change MEAN ± SEM MEAN ± SEM 0.45 ± 0.05 0.74 ± 0.15 Control 1.57 ± 0.45 1.41 ± 0.24* 90.54↑ Test *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 Therapeutic phase % change MEAN ± SEM MEAN ± SEM 0.23 ± 0.05 0.30 ± 0.04 Control 0.51 ± 0.12 0.45 ± 0.05 50↑ Test 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 Therapeutic phase % change MEAN ± SEM MEAN ± SEM 4.57 ± 0.34 Control 7.53 ± 1.68 2.39 ± 0.08 2.89 ± 0.17** 36.76↓ Test **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 Therapeutic phase % change MEAN ± SEM MEAN ± SEM 4.58 ± 0.34 Control 7.37 ± 1.73 2.37 ± 0.08 3.17 ± 0.43* 30.79↓ Test *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 1.12 ± 1.03 Test group Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
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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.
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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.
Madan S., Singh GN., Kohli K (2009). Isoflavonoids from Flemingia Strobilifera (L) R.Br. roots. Acta Pol Pharm. 66:297–303.
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.
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Harborne J. B. Method of extraction and isolation, In: Phytochemical methods, 2nd ed. London: Chapman & Hall, 1998.p. 60-6.
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.
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.
Source of Support:
NIL
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 chromenesTherapeutic agents from Flemingia strobilifera. Asian J Chem.7: 307–10.
Conflict of Interest: None Declared
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Review Article QUESTIONNAIRE DESIGNING AND VALIDATION IN AYURVEDIC RESEARCH Ravi Bhat1*, Shivprasad Chiplunkar2, Suhaskumar Shetty3, Arhanth Kumar4 1
Assisstant Professor, Dept of Kriya Sharira, SDM College of Ayurveda & Hospital, Udupi, Karnataka, India Associate Professor, Dept of Kriya Sharira, SDM College of Ayurveda & Hospital, Hassan, Karnataka, India 3 Associate Professor, Dept of Manasa Roga, SDM College of Ayurveda & Hospital, Hassan, Karnataka, India 4 Assisstant Professor, Dept of Samhita, SDM College of Ayurveda & Hospital, Udupi, Karnataka, India *Corresponding Author: drravi1@gmail.com; Mobile: 09632452121 2
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
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): 435â&#x20AC;&#x201C;444
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INTRODUCTION
REVIEW OF QUESTIONNAIRE:
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.
In the present article the entire literature is explained under the heading of steps of questionnaire development. Details regarding the same are given below.
The aim of this paper is to give a basic introduction to the research scholars about designing and validating questionnaires in Ayurveda research.
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 information:
Review
or
collection
of
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 CDâ&#x20AC;&#x2122;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) selfadministered and b) interviewer administered questionnaire.
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4. Question content or Research Question: 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.
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 I am always learning or studying new things. 8. 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.
independently-rated criteria is meant to evaluate a patient's anxiety severity (Hamilton M., 1959).
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 openended 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.
Phraseology
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
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 â&#x20AC;&#x201C; validated by
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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: jointprobability of agreement, Cohen's kappa and the related Fleiss' kappa, inter-rater correlation, concordance correlation coefficient and intraclass 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 interviewersâ&#x20AC;&#x2122; 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
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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 commentary by Chakrapani,; Chaukhambha Surbharati Prakashan, Varanasi, p.267 Bhat
R, (2013). DESIGNING AND VALIDATION OF OJO KSHAYA SCALE. Journal of AYUSH: Ayurveda, Yoga, Unani, Siddha and Homeopathy. 2(3):36–42.
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Branacto G, Macchina S, Sigore M (2012). Hand book of recommended practice for questionnaire development. Version 1 Hamilton M. (1959). The assessment of anxiety states by rating. Br J Med Psychol; 32:50–55. Hegan E F (2012) Essential of research method for criminal justice. 2nd ed. US: Content Technology Inc. Janmejaya, S, (2013). ADVANCEMENTS IN INDIAN SYSTEM OF MEDICINE (ISM) INFORMATICS: AN OVERVIEW. Global J Res. Med. Plants & Indigen. Med, 2(7): 546–553. Kazi A, Khalid W, (2012). JOURNAL OF PAKISTAN MEDICAL ASSOCIATION(JPMA) Questionnaire designing and validation. 1:514-516
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Med.(2010). Conducting literature searches on Ayurveda in PubMed, Indian, and other databases. [Online] Available at http://www.ncbi.nlm.nih.gov. [Accessed 31May 14.] Questionnaire Design[Online] Available at http://www.cdc.gov/reproductivehealth/ ProductsPubs/PDFs/Epi_Module_04_T ag508.pdf [Accessed 31May14]. Reja U, Manfreda L, Hlebec V, Vehovar V, (2003) DEVELOPMENTS IN APPLIED STATISTICS: Open-ended vs. Close-ended Questions in Web Questionnaires. 19:161-176 Robert R. McCrae, John E. Kurtz, v. (2011). Internal Consistency, Retest Reliability, and their Implications For Personality
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Conflict of Interest: None Declared
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