14 Chapter 5

1.
CHEMICAL COMPOSITION
The nutritional composition of all samples was determined with standard techniques and
results are presented in Table 17. A wide range of variations was found in different
constituents as the selected herbs and spices come from different parts of plant, borage
being flower, valerian and ginger, the root portion, lime, the fruit and shallot, the bulb
root. In herbs, protein content of borage and valerian was found to be 8.10 and 4.63 and
fat content was 1.35 and 1.17 g/100g respectively. In spices, protein content was found to
be 5.24, 7.80 and 6.90 and fat content, 4.31, 0.40 and 4.37 g/100 g in lime, shallot and
ginger respectively. Total fiber of sample was estimated both in the form of soluble and
insoluble fiber. Among all samples valerian showed highest insoluble fiber (77.00%) as it
is the root of the shrub and may have a high cellulose portion. Soluble fiber was highest
in borage (49.9%). Soluble fiber has been specifically related to many health promoting
functions such as lowering of cholesterol (Butt et al., 2007), improving insulin sensitivity
in type I and II diabetic patients (Anderson et al., 1999; Ziai et al., 2005), hypertension
(Wang et al., 2007) and cardiovascular disease (Liu et al., 1999; Merchant et al., 2003).
Vitamin C content was more in lime followed by borage. Ginger showed least vitamin C
content. The ash content of samples was in the range of 9.21 to 3.10 g/100g with the
highest in borage and least seen in shallot. Ash content reflects the mineral content of
samples. Highest calcium content was seen in valerian (829 mg/100g) and least was seen
in shallot (75 mg/100g). Among all analyzed samples, the estimated trace minerals (zinc,
copper, manganese and chromium) were highest in valerian and lime had least trace
minerals. Ginger had highest manganese and chromium after valerian.
As reported by USDA, leaves of borage contain carbohydrates, 3.06 g; protein 1.8 g; fat
0.7 g; calcium, 93 mg; copper, 0.130 mg; iron, 3.30 mg; magnesium, 52 mg; manganese
0.349 mg; zinc 0.20 mg; (USDA, 2011).
1.1. ANTHOCYANIN CONTENT

Anthocyanin could be detected only in borage which had high (104.4mg/100g) content.
Anthocyanins are the largest group of water-soluble pigments in the plant. They belong to
the family of flavonoids. Anthocyanins are responsible for most of the red, blue, and
102
purple colors of fruits, vegetables, flowers, and other plant tissues or products. They are
particularly rich in berries and other fruits with red, blue, or purple color, and in red
wines (Mazza and Miniati, 1993). Anthocyanin exhibits antioxidant property. Mazza et al
(2002) reported that the concentration of anthocyanins in the serum of male subjects that
had consumed 1.2 g anthocyanins from freeze dried blueberries was positively correlated
with the serum antioxidant capacity. Bagchi et al (1998) reported that anthocyanidins and
anthocyanins have exhibited higher antioxidant activity than vitamin C and E. This may
be a reason for its anticarcinogenic activity (Kamei et al., 1995).
Liu et al (2002) reported the anthocyanin content of raspberry to be between 0.17 to 57.0
mg/100g. Other researchers reported 30-750 mg/100g of total anthocyanin in grapes
(Bridle and Timberlake, 1997).
Table 17: Chemical composition of selected herb and spices per (100g dry weight)
Constituents
Moisture (g)
Protein (g)
Fat (g)
Insoluble fiber (%)
Soluble fiber (%)
Carbohydrate (g)
(By difference)
Vitamin C (mg)
Total carotenoids
(mg)
Anthocyanin (mg)
Ash (g)
Calcium (mg)
Phosphorous (mg)
Iron (mg)
Zinc (mg)
Copper (mg)
Manganese (mg)
Chromium (g)
ND: Not detected
Borage
9.900.10
8.100.10
1.350.10
41.100.50
49.90.00
Valerian
7.600.11
4.630.10
1.170.08
77.000.20
7.30.10
Lime
20.060.03
5.240.00
4.310.10
30.270.05
42.50.20
Shallot
11.980.50
7.800.31
0.400.10
6.500.00
33.90.64
Ginger
15.020.04
6.900.09
4.370.03
27.650.06
30.00.04
2.240.02
36.410.00
61.700.33
45.120.1
63.000.50
44.900.40
88.100.41
13.060.07
10.970.08
38.80.8
132.70.1
77.10.2
68.10.1
92.90.2
104.40.25
9.210.40
7820.3
3950.93
75.40.70
2.660.00
1.490.01
1.800.00
33.30.00
ND
8.970.30
8290.8
3281.00
272.00.89
4.800.01
2.690.01
11.470.00
249.00.01
ND
6.260.11
5696.8
1575.70
5.02.10
0.810.07
0.550.00
0.150.00
ND
ND
3.100.06
751.4
2346.30
9.03.00
2.970.03
1.780.04
0.530.31
3.40.35
ND
4.530.61
1040.97
2051.2
9.40.20
1.080.00
0.640.00
10.74001
82.40.00
103
2. IN VITRO ANTIOXIDANT POTENTIAL

2.1. ANTIOXIDANT COMPONENTS OF HERBS AND SPICES
Antioxidant components of all herbs and spices were estimated in 7 different extracts.
Compound which are easier to oxidise are often the best antioxidants. These compounds
are molecules which can donate a free electron or hydrogen atoms to reactive free
radicals (Castaneda-Ovando et al., 2009).
2.1.1. Borage
Antioxidant components, activity and yield of extract of borage were determined in
freshly prepared 7 different extract.
As illustrated in Table 18, in borage, phenolic content was higher in hot water extract
(1.54g/100g) followed by 80% ethanolic extract (1.22 g/100g). Acetonic extract showed
least polyphenol content (0.025 g/100g). Methanolic and ethanolic extract also showed
lesser polyphenol content compared to 80% methanolic and 80% ethanolic extract. In
80% methanolic and 80% ethanolic extracts, values were closer to water extract. It can be
due to high solubility of borage polyphenols in water and also heat stability of borage
polyphenols. Polyphenols are the most abundant antioxidants in the human diet (Scalbert
et al., 2002). Effect of polyphenols on health is well understood. It strongly supports a
role for polyphenols in the prevention of degenerative diseases, particularly
cardiovascular diseases and cancers. The antioxidant properties of polyphenols have been
widely studied by many authors.
Consumption of polyphenols may also have antinutritional effects. The inhibition of
nonheme iron absorption attributable to simultaneous tea consumption is well
understood; high consumption of polyphenols may enhance the risk of iron reduction in
populations of individuals with marginal iron status (Temme and Van Hoydonck, 2002).
Important in this respect is that main sources of polyphenols, such as coffee, tea, and
wine, which are regularly consumed with meals, do not contain vitamin C, which can
enhance absorption of nonheme iron (Zijp et al., 2000). In the case of borage it is a good
source of polyphenol, though measured in dry form, it was still quite good source of
104
vitamin C. It is also interesting that traditionally in Iran borage decoction is used with
addition of dry whole lime. As presented before, borage contained 51.2 and dry lime 70.5
mg of vitamin C/100g of sample. Mehrabani et al (2005a) state that the major phenolic
compound of the ethyl acetate extract of petals of E. amoenum is rosmarinic acid.
Rosmarinic acid has antimicrobial, antiviral, antioxidant and anti-inflammatory
properties, which makes it a precious component for pharmaceutical, food and cosmetic
industries (Petersen and Simmonds, 2003a).
As shown in Table 18, tannins ranged from 165 to 2470 mg. There were more tannin in
hot water followed by 30C water extract. Acetone extract showed the least tannin
content. Tannin content of 80% methanol and ethanolic extract was very close in
concentration to aqueous extract. Tannins are water-soluble polyphenols that are present
in many plant foods; hence there were more tannins in aqueous extract.
Many flavonoids in foods are polymerized into large molecules, either by the plants
themselves or as a result of food processing. These polymers are called tannins (Harborne
and Williams, 2000). There are several subclasses of tannins, three of which are
important to foods and perhaps health (Clifford, 2001). It has been reported by many
authors that tannin decrease iron absorption in the food (Glahn et al., 2002). The
anticarcinogenic and antimutagenic potentials of tannins might be related to their
antioxidative ability, which is important in defending cellular oxidative damage, as well
as lipid peroxidaton. The production of superoxide radicals can be inhibited by tannins
and related compounds (Chung et al., 1998).
Tannin content of several black tea from China, India and Malawi was reported to be in
the range of 0.79-19.4, 7.97-24.4 and 12.1-76.5 g/100g sample respectively
(Smiechowska and Dmowski, 2006). Tannin content of hot water extract of borage was
in the range of what was found in black tea from China. Tinkl (2001) reported that the
tannin contents of hot water extracts of tea, tea bag and herbal tea samples ranged
between 6.20-8.33, 8.03-6.59 and 2.76-6.54 percent (w/w) respectively. The value for
herbal tea was close to borage tannin content.
The highest flavonoid content was observed in hot water extraction followed by 30C
aqueous extract. The least flavonoids was determined in acetonic extract (0.30 g)
followed by ethanolic extract (0.33 g/100g). It was observed that 80% methanolic and 80
105
% ethanolic extract had more flavonoids than pure solvent. It is known that flavonoids
are water soluble compound. Base on this fact we observed more flavonoids in aqueous
extract than solvent extract. Also from results it can be concluded that flavonoids in
borage are not heat sensitive as hot water extraction showed more flavonoids. Flavonoids
are formed in plants from the aromatic amino acids phenylalanine and tyrosine, and
malonate (Cody et al., 1986). Flavonoids are oxidized by radicals, resulting in a more
stable, less-reactive radical. In other words, flavonoids stabilize the reactive oxygen
species by reacting with the reactive compound of the radical. Because of the high
reactivity of the hydroxyl group of the flavonoids, radicals are made inactive, according
to the following equation (Korkina and Afanas' Ev, 1996).
Flavonoid (OH) + R > flavonoid (O) + RH
Echium amoenum was found to be a good source of flavonoids which is supported by
Salehzadeh (1990).
Table 18: Antioxidant components of borage in different extracts (g/100 g)

Constituents
Yield of
Water
Water
(100oC) (30oC)
Methanol Ethanol
Methanol Ethanol
(80%)
(80%)
Acetone
42.5
37.6
35.25
27.53
45.63
51.26
3.03
Total
1.540
1.170
0.450
0.340
1.120
1.220
0.025
polyphenols
0.033
0.028
0.004
0.005
0.018
0.023
0.02
2.470
1.800
1.155
0.945
2.445
2.385
0.165
0.050
0.100
0.084
0.020
0.090
0.097
0.001
4.54
3.92
0.82
0.33
1.10
0.85
0.30
0.042
0.077
0.009
0.004
0.007
0.007
0.000
extract
Tannin
Flavonoids
2.1.2. Valerian
In valerian, 80% ethanolic extract showed highest polyphenols (0.50 g/100g) followed by
80% methanolic extract (0.38 g/100g) with least activity being shown by acetone extract
(0.13 g/100g).
106
Polyphenols are mostly water soluble compounds; however water and solvent extract
showed more. These compounds have phenolic rings with structural elements that bind
these rings to one another. There are differences in number and position of ring and
structural elements which divide them in different groups. Polyphenol compounds are
very big group of phenol containing compound with different physico chemical
properties. Hence, some of the polyphenol compounds of valerian may be soluble in
alcohol. Zheng and Wang (2001) extracted 2g of valerian with 15 ml of phosphate buffer
and reported the total phenolic content of valerian as 1.78 mg of GAE/g of fresh weight.
The reported value is much lesser than the extract with highest polyphenol content
(0.5g/100g) in the present study, which can be due to difference in extracting media and
expression on fresh weight basis.
Tannin content was found to be 135 to 454 mg/100g. It was more in hot water extract
followed by 80% ethanolic and 80% methanolic extract. The least tannin was seen in
acetonic extract. Tannin is water soluble compound and as seen, it was heat stable.
Similar to total polyphenol content, tannin was also extracted more in 80% solvent
extract as both solvent and water extractable compounds can be extracted. As in total
polyphenol, tannin was found to be very less in acetonic extract which shows that acetone
is not appropriate solvent for extracting the tannin compound.
Flavonoids in valerian were more in 30C aqueous extract followed by hot water extract,
indicating higher temperature reduced extractable flavonoid content of valerian. Same as
other compound, 80% ethanolic extract followed by 80% methanolic extract contained
more flavonoids than pure alcoholic extracts.
Total polyphenol content of boiling water extraction of black and green tea is reported to
be 59.3 and 59.8 mg of chlorogenic acid equivalent/g of dry weight (Rababah et al.,
2004). In our study when polyphenols were calculated per g of dry weight, total
polyphenol content of hot water extract of valerian were much lesser than black and
green tea (3.40 mg tannic acid equivalent/g of dry weight).
Yield of the extracts are presented in Table 19.
107
Table 19: Antioxidant components of valerian (g/100 g)

Constituents
Water
o
Water
o
(100 C) (30 C)
Yield of
Methanol
Ethanol
Methanol
Ethanol
(80%)
(80%)
Acetone
5.4
3.25
5.7
5.06
12.2
5.73
0.066
Total
0.315
0.185
0.265
0.250
0.380
0.500
0.130
polyphenols
0.003
0.002
0.003
0.004
0.006
0.002
0.003
0.570
0.430
0.340
0.300
0.442
0.454
0.135
0.087
0.190
0.076
0.025
0.044
0.005
0.014
2.33
3.03
0.60
0.48
0.76
1.05
0.30
0.101
0.123
0.018
0.021
0.004
extract
Tannin
Flavonoids
2.1.3. Ginger
Ginger extract showed highest polyphenol in 100C water extract followed by 30C and
least content was seen in acetonic extract. Ghasemzadeh et al (2010) reported the total
phenolic content of 2 different variety of ginger root in methanol extract, as 10.22 and
13.5 mg gallic acid /g of dry weight (dw). When our value was calculated/g of dw, we
found 6.00 mg tannic acid /g dw in methanolic extract of ginger root. Difference can be
due to the difference in variety. Rababah et al (2004) determined the total phenolic
compound of ginger extract (40 ml acetone:40 ml methanol: 20 ml water: 0.1 ml formic
acid) to be 39.9 mg/g dw. The reported value is much higher than our determination in all
different solvent.
Cai et al (2004) reported total polyphenols of methanolic and aqueous extract of rhizome
of ginger to be 0.74 and 0.38 g/100 dw. In contrast with Cai et al (2004) we found
slightly more polyphenol in aqueous extract compare to methanolic extract.
It was observed that 80% methanolic and 80% ethanolic extract has more polyphenol
than pure methanolic and ethanolic extract. Kaur and Kapoor (2002) reported the total
phenolic compound of 80% ethanolic extract as 221.3 mg/100g which was lesser than our
estimated value (800 mg/100g). Stoilova et al (2007) reported the total phenols of ginger
extract. In alcohol to be 870.1 mg/g dry extract.
108
Tannin content of ginger was found to be in the range of 1.51-0.67 g/100g. The highest
tannin was observed in hot followed by cold water extract. Similar to other samples the
least tannin was seen in acetonic extract. Nwinuka (2005) reported the tannin content of
ginger as 0.01 g/100g.
Flavonoid content was in the range of 2.98-0.249 g quercetin equivalent/100g. The
highest flavonoids were observed in hot water extract (2.98 g/100g) followed by 30C
water extract (1.371 g/100g). The least flavonoids were seen in acetonic extract (0.249
g/100g). Ghasemzadeh et al (2010) reported the flavonoid content of methanolic extract
of rhizome of 2 different variety of ginger as 3.66 and 4.21 mg quercetin/ g of sample. It
is lesser than our value in methanolic extract when calculated per g of dw (8.06 mg/g
dw). Yield of extracts were found to be highest in hot water extract.
Table 20: Antioxidant components of ginger (g/100 g)
Constituents
Yield of
Water
Water
(100oC) (30oC)
Methanol Ethanol
Methanol Ethanol
(80%)
(80%)
Acetone
24.03
22.00
16.04
18.70
23.90
23.30
8.10
Total
0.840
0.838
0.510
0.565
0.780
0.800
0.325
polyphenols
0.002
0.003
0.002
0.004
0.005
0.004
0.002
1.51
1.34
1.12
0.98
1.28
1.15
0.67
0.05
0.08
0.05
0.03
0.01
0.1
0.08
2.98
1.371
0.685
0.278
0.404
0.352
0.249
0.06
0.01
0.005
0.003
0.002
0.002
0.002
extract
Tannin
Flavonoids
2.1.4. Whole Lime

Whole lime highest polyphenols were seen in 100C water extract followed by 80%
ethanolic extract. Methanolic extract of freeze dried lime juice was reported to have
4.20g/100g of lime juice (Patil et al., 2009a). We found phenolic content of 140 mg/100g
in whole dry lime powder.
Cai et al (2004) reported total polyphenols of methanolic and aqueous extract of raw
fruits of citrus aurantifolia to be 5.07 and 4.4 g/100 of sample. Jeong et al (2004)
reported phenolic compound of lime peel in 70% ethanolic extract, aqueous extract (0
109
min and 30 min heat treatment). They found total phenolic compound to be 91.4, 106.9
and 69.1 mol tannic acid equivalent in 50C aqueous, 100C aqueous and 50C 70%
ethanolic extract. Thermal treatment significantly increased polyphenol content of
extract. In our study also hot water extract showed more polyphenol than cold water
extract.
Flavonoid content of whole lime fruit were found to be in the range of 5.72 to 1.7 g/100g.
It was more in hot water extract followed by 80% methanolic and 80% ethanolic extract
(4.6 and 3.2 g/100g respectively). Flavonoid content of hot water extract of whole lime
were found to be more than other tested samples. Flavonoids are ubiquitous compounds
with many medicinal properties. It has been reported that antibacterial properties of
many plants are due to their flavonoid content (Bosio et al., 2000; Cushnie and Lamb,
2005). Okwu and Emenike (2006) reported the flavonoid content of edible pulp portion
of lime (citrus aurantifolia) as 0.29 mg/100g which is much lesser than our finding. The
difference can be due to sample taken for analysis as we analysed whole fruit (with skin).
A high flavonoids in skin and albedo (the white spongy portion) of lime has been
reported (Manach et al., 2004).
Tannin was found to be 0.271 to 0.900 g/100g. The highest tannin was found in hot water
extract followed by 30C aqueous extract (0.900 and 0.856 g/100g respectively).
Acetonic extract exhibited the least tannin content.
The total polyphenol and flavonoids contents per 100 ml of lime juice were reported as
211.70 mg GAE and, 10.67 mg of hesperidine equivalent respectively (Ghafar et al.,
2010). In whole lime water extract (30C & 100C) total polyphenol was 0.420 and 0.480
g/100g sample. From results it can be concluded that lime peel and white spongy portion
have high antioxidant component.
Yield of the extracts are presented in Table 21.
110
Table 21: Antioxidant components of lime (g/100 g)

Constituents
Yield of
extract
Total
polyphenols
Tannin
Flavonoids
Water Water
Methanol Ethanol
Methanol Ethanol
Acetone
(80%)
(80%)
(100C) (30C)
31.5
26.4
17.7
14.8
24.0
27.1
5.3
0.480
0.008
0.900
0.005
5.72
0.6
0.420
0.011
0.856
0.005
2.42
0.07
0.140
0.008
0.601
0.009
2.14
0.10
0.170
0.005
0.531
0.014
1.81
0.09
0.360
0.008
0.795
0.009
4.6
0.50
0.460
0.07
0.731
0.011
3.2
0.01
0.050
0.002
0.271
0.013
1.7
0.02
2.1.5. Shallot
In shallot, polyphenols were more in 100C followed by 30C aqueous extract but were
not detected in methanolic extract. Total phenolic compound in acetonic extract of shallot
was found to be 25 mg which was lesser than reported value by Yang et al (2004). He
reported the phenolic compound of 80% acetonic extract of fresh onion to be 114.7 mg of
gallic acid equivalent/100 g of sample. Difference may be due to extracting media and
freshness of shallot. Brat et al (2006) also reported the polyphenol content of shallot to be
104 mg of GAE/100 g fresh edible portion. Lu et al (2011) reported total phenolic content
of 70% methanolic extract of shallot to be 17.18 mg gallic acid/g fresh weight. In our
study 80% methanolic extract had 0.694 g/100 g of dried sample. Yin et al (2002)
reported that hexane-extract of shallot contained the total phenolics as 5,477.5333.49
GAE mg/kg whereas the total phenolics in water-extract found in shallot were found to
be 4,599.0188.32 GAE mg/kg. In part of crude extract of shallot obtained from crushing
and pressing, total phenolics were 4,086.3784.54 GAE mg/kg.
Shallot flavonoid content was found to be in the range of 2.24 -0.500 g quercetin
equivalent/ 100g of sample acetonic extract had least flavonoid content (0.50 g). Yang et
al (2004) reported 80% acetonic extract of fresh shallot to be 34.4 mg catechin
equivalent/100 g. Ismail et al (2004) reported the total polyphenol content of 70%
ethanolic extract of fresh shallot as 2528 mg/100g. They reported a 13% reduction in
flavonoid content after 1 min blanching in boiling water. Since flavonoids are water
111
soluble compound, it leaches to boiling water so after draining the water, flavonoids loss
is seen. In our study we found more flavonoid in hot water extract.
Tannin content was estimated and found to be 65 to 225 mg/100g. It is more than the
value reported by Nwinuka et al (2005) for tannin content of onion as 10 mg/100g. The
highest tannin content was found in 100C followed by 30C aqueous extract (0.225 and
0.212 g/100g sample). The 80% methanolic and 80% ethanolic extract had more tannin
than pure alcoholic extract.
Yield of the extracts in different medias are provided in Table 22.
Table 22: Antioxidant components of shallot (g/100 g)
Constituents
Yield of
extract
Total
polyphenols
Tannin
Flavonoids
Water Water
Methanol Ethanol
Methanol Ethanol
Acetone
(80%)
(100C) (30C)
(80%)
7.4
6.9
0.100
0.003
0.225
0.008
2.14
0.010
0.099
0.004
0.212
0.004
1.80
0.003
2.01
ND
0.104
0.004
0.512
0.004
2.2
4.6
4.9
3.05
0.020
0.0021
0.089
0.003
0.478
0.004
0.090
0.003
0.195
0.006
0.694
0.006
0.060
0.004
0.174
0.003
0.717
0.003
0.025
0.003
0.065
0.001
0.553
0.030
Borage exhibited the highest phenolic (1.54 g/100g) content compared to other samples.
In all samples acetonic extract showed least polyphenols content except shallot and the
hot aqueous extract showed highest polyphenol except valerian.
The polyphenol content of samples followed the order: borage> ginger> valerian> lime>
shallot. As results indicate, 80% methanolic and 80% ethanolic extract showed higher
content than the pure methanolic and ethanolic extract. It can be explained on the basis
that in 80% alcoholic extract both water and alcohol soluble compound can be extracted
so the content will be more than pure alcoholic extract. Antioxidant activity of plant
extract is usually linked to their phenolic content. Hydrogen donating characteristics of
the phenolic compounds is responsible for the inhibition of free radical induced lipid
peroxidation (Yen et al., 1993). Phenolic compounds are also known as high level
antioxidants because of their ability to scavenge free radicals and give oxygen species
such as singlet oxygen, superoxide free radicals and hydroxyl radicals (Hall, 1997),
112
though, it is well accepted that non phenolic antioxidants might also contribute to the
antioxidant activity of plant extract (Hassimotto et al., 2005; Harish and Shivanandappa,
2006). In a study, researchers estimated total polyphenol content of 35 different herbs and
medicinal plants in 80% methanolic extract. The range of polyphenols seen was between
0.8-42.1mg GAE/g dry weight (dw) and for white onion it was reported to be 2.5 mg of
GAE/g (Kahkonen et al., 1999). In our study when total polyphenols content as TAE in
80% methanolic extract /g of dw was calculated, we found the values to be 12.43, 4.11,
9.17, 4.5 and 1.02 for borage, valerian, ginger, lime and shallot.
Turkmen et al (2006) reported polyphenol content (mg/g) of mate and black tea in
different solvent extracts, the values being 30.5 and 64.2 in water extract, 1.8 and 2.6 in
acetonic extract, 53.7 and 83.5 in 80% ethanolic extract, 2.1 and 4.8 in ethanolic extract,
56.0 and 85 in 80% methanolic extract and 13.5 and 35.5 in absolute methanolic extract
in black tea and mate respectively. As data showed polyphenol content of black tea and
mate varied in different extraction solvent and the order of polyphenol content was 80%
methanolic extract> 80% ethanolic extract> water > methanol> ethanol> acetone for
both black tea and mate. But in our study order of polyphenol content of borage and
valerian (traditionally used as a tea) in different solvent was as follows, hot water > 80%
ethanol> 30C water > 80% methanol > methanol > ethanol > acetone and 80% ethanolic
> 80% methanolic> 100C water> methanol> ethanol> 30C water> acetone. It can be
due to different polyphenol components in our samples in comparison with what is found
in black tea and mate. Kaur and Kapoor (2002) reported phenolic compound of 80%
ethanolic extract of ginger as 221.3 mg/100g but we found 800 mg/100g.
Among all the samples aqueous extract exhibited the highest flavonoids and acetonic
extract showed the least flavonoids compared to other extracting media. Whole dry lime
followed by borage showed the highest flavonoids and shallot showed the least flavonoid
content. Between five tested samples analyzed tannin content of borage was more than
other samples in all different extracts. Except valerian all other extracts showed the
highest tannin in 100C aqueous extract and in all samples the least tannin was seen in
acetonic extract.
113
2.2. ANTIOXIDANT ACTIVITY

The antioxidant activity of all the selected samples was estimated using three different
assays. Since the antioxidant potential of any substance is due to the presence of different
components, the antioxidant activity is best measured using multiple assays rather than
depending on any one assay, hence total antioxidant activity, reducing power and free
radical scavenging activity was measured and results are presented below.
2.2.1. Total antioxidant activity

Borage showed the highest activity in 100C followed by 30C aqueous extract. The least
activity was observed in acetonic extract. Among all samples, after shallot, borage
showed the highest antioxidant activity.
Valerian had highest activity in 80% methanolic extract followed by 100% methanolic
extract. The least activity was observed in acetonic extract.
Ginger showed the highest activity in methanolic extract followed by ethanolic extract
and least was seen in acetonic extract.
In lime total antioxidant activity was more in hot water extract followed by 30C water
extract. Similar to all other samples lime also showed the least activity in acetonic
extract.
Shallot showed the highest antioxidant activity compared to all other samples. The
highest activity was seen in 100C aqueous extract followed by 80% methanolic extract.
Ethanolic extract did not show any antioxidant activity.
Except valerian which showed the highest activity in 80% methanolic extract, all other
samples showed highest total antioxidant activity in 100C aqueous extract.
Yang et al (2004) reported that total antioxidant activity of shallot was significantly
higher than 10 different tested onions.
114
Table 23: Total antioxidant activity of samples in different extracting media (mol
ascorbic acid equivalent /g of sample)
Extracting media
Water (100C)
Water (30C)
Methanol
Ethanol
80% Methanol
80% Ethanol
Acetone
Borage
2,38,235
111
2,29,706
105
146,325
83
89,706
67
108,639
38
70,000
62
10,698
44
Valerian
31,985
82
22,800
102
64,117
139
61,029
99
98970
87
32,941
55
32,463
64
Ginger
73,529
121
79,400
88
98,822
74
91,176
66
85,294
47
8,000
38
32,056
27
Lime
90,588
25
88,225
94
73,235
33
51,410
28
64,411
45
39,110
32
1,753
19
shallot
7,72,059
42
6,48,525
56
64,705
38
0
1,90,073
69
58,829
61
3,250
29
2.2.2. Antioxidant activity by reducing power assay

a) Borage
The reducing power of bioactive compounds is said to be associated with antioxidant
activity (Yen et al., 1993; Siddhuraju et al., 2002). Hence it is essential to determine the
reducing power of phenolic constituents to explain the relationship between their
antioxidant effect and their reducing power. The reducing power of different solvent
extracts of borage is shown in Figure 11. As illustrated, borage showed highest reducing
power properties in hot water and 30C water extract followed by 80% ethanolic and 80%
methanolic extract. The least reducing power properties were exhibited by acetone
extract. Borage showed high antioxidant components and the high reducing power ability
can be attributed to their presence
.
115
Figure 11: Reducing power of borage in different extracting media
Optical Density (nm)
2.5
2
1.5
A
B
0.5
0
Water
(100C)
Water
(30C)
Methanol
Ethanol
Methanol
(80%)
Ethanol
(80% )
Acetone
[Concentration, A: 2000 ppm, B: 4000 ppm, C: 6000 ppm, D: 8000 ppm]

b) Valerian
Valerian showed highest reducing power properties in 80% methanolic and 80%
ethanolic extract. Acetone extract showed least activity. Among all samples,
comparatively valerian had lesser reducing power ability. The ability to reduce Fe (III)
may be attributed to hydrogen donation from phenolic compounds (Shimada et al., 1992),
which is also related to the presence of reductant agent. In addition, the number and
position of hydroxyl group of phenolic compounds also rule their antioxidant activity
(Rice-evans et al., 1995).
Figure 12: Reducing power of valerian in different extracting media
1.4
1.2
1
A
0.8
0.6
0.4
0.2
0
Water
(100C)
Water
(30C)
Methanol
Ethanol
Methanol
(80%)
Ethanol
(80%)
Acetone
116
c) Ginger
Ginger exhibited highest activity in 80% methanolic and 80% ethanolic extract
respectively. The least activity was observed in acetonic extract. El-Ghorab et al (2010)
reported ferric reducing antioxidant power of fresh and dried ginger in methanolic and
hexane extracts. It was observed that, the methanol extracts has higher potential than
hexane extracts to reduce the ferric ions to ferrous ions. The maximum absorbance value
was reported to be 1.138 for fresh ginger methanol extract followed by dried ginger with
a value of 0.847 at a concentration of 240 ppm. Chen et al (2008a) determined the
antioxidant activity of 18 Taiwan endemic species of ginger and reported that reducing
power of methanolic extract of samples were in the range of 0.47-1.6 nm at the
concentration of 100mg/ml (100000 ppm).
Figure 13: Reducing power of ginger in different extracting media

1
Optical Density
0.8
0.6
A
B
0.4
C
D
0.2
0
Water
(100C)
Water
(30C)
Methanol
Ethanol
Methanol
(80%)
Ethanol
(80%)
Acetone

d) Lime
In lime higher activity was seen in 80% methanolic extract followed by 100C aqueous
extract and least activity was seen in acetonic extract. The power of certain antioxidants
is associated with their reducing power (Jayaprakasha et al., 2001), which is related to the
presence of reductanes (Duh, 1998). The reducing power of 70% ethanolic and aqueous
extract was reported to increase significantly by heat treatment (Jeong et al., 2004) . In
117
our study hot water extract showed significantly higher reducing power properties
(P=0.000).
Figure 14: Reducing power of lime in different extracting media
0.6
0.5
0.4
0.3
B
C
0.2
D
0.1
0
Water
(100C)
Water
(30C)
Methanol
Ethanol
Methanol
(80%)
Ethanol
(80%)
Acetone

e) Shallot
Shallot showed the highest activity in 100C and 30C aqueous extract but in alcoholic
extract activity was not observed. The least activity was seen in acetonic extract. It is
stated that extracts obtained by high polarity solvents are significantly more effective in
reducing power and radical scavengers than those obtained by less polarity solvents. This
specifies that antioxidant or active compounds of different polarity might be present in
samples. Change in solvent polarity varies its ability to dissolve a selected group of
antioxidant compound and persuade the antioxidant activity determination (Zhou and Yu,
2004). The order of reducing power ability was seen as follows: Borage> valerian>
ginger> lime> shallot.
118
Figure 15: Reducing power of shallot in different extracting media
0.12
0.1
0.08
A
0.06
0.04
0.02
0
Water
(100C)
Water
(30C)
Methanol
Ethanol
Methanol
(80%)
Ethanol
(80%)
Acetone

2.2.3. Free radical scavenging activity by DPPH
DPPH is a stable free radical in methanol or aqueous solution and accepts an electron or
hydrogen radical to turn into stable diamagnetic molecule. It is usually used as a substrate
to evaluate the antioxidative activity of antioxidants (Duh et al., 1999), thus we estimated
the antioxidant activity through free radical scavenging of samples, and results are
presented in Figure 16-20.
a) Borage
Among all the samples borage showed highest activity compared to others. As illustrated
in figure 12 at 0.4-1.0 mg, the FRSA was highest in water the value being 94% for 1.0
mg concentration. The activity was slightly reduced when room temperature water was
used. However, in solvents, comparatively, the activity was lesser indicating that the
antioxidant activity was prompted by water soluble components. The results were similar
to what were observed in two other assays, namely total antioxidant activity and reducing
power.
119
Figure 16: Free radical scavenging activity of borage in different extracting media
Concentration- Acetone extract, A: 5.0, B: 10.0, C: 15.0 and D: 20.0 mg of sample, all
other extracts, A: 0.4, B: 0.6, C: 0.8 and D: 1.0 mg of sample
b) Valerian
Valerian in 80% methanolic extract exhibited highest activity, compared to other
extracting media. In valerian, a slightly different profile was observed for FRSA (Figure
17), while the 80% methanolic extract had highest activity, at 70% for 1.0 mg sample, for
hot water extract the equivalent was only 9%. However, activities were seen for other
extracts albeit at higher concentrations. Acetone and ethanolic extracts had lower FRSA.
The antioxidant activity of valerian followed a different profile in all three assays.
120
Figure 17: Free radical scavenging activity of valerian in different extracting media
Concentration- 80% methanol, A: 0.4, B: 0.6, C: 0.8 and D: 1.0 mg of sample, all other
extracts, A: 2.0 mg, B: 4.0 mg, C: 6.0 mg, D: 8.0 mg of sample
c) Ginger
Ginger in 80% methanolic extract exhibited highest activity, compared to other extracting
media. The activity shown in the figure is for a lower concentration of ginger for 80%
methanolic extract than other extracts. As due to high antioxidant activity, the activities
were determined at a lower concentration. Ghasemzadeh et al (2010) reported the free
radical scavenging activity of two varieties of Malaysian ginger as 51.41 and 58.22% in
concentration of 0.04 mg. The reported value is much more than our observation. We
found that 2.5 and 5mg of sample exhibited 39.6 and 64.7% free radical scavenging
activity. In present study, difference in values can be due to nature of sample as for our
study, commercial sample was purchased, whereas the authors cultivated ginger in
greenhouse under controlled conditions and used for determination of antioxidant
activity.
121
Figure 18: Free radical scavenging activity of ginger in different extracting media
Concentration- 80% methanol, A: 0.4, B: 0.6, C: 0.8 and D: 1.0 mg of sample, all other
extracts, A: 2.5, B: 5.0, C: 7.5 and D: 10 mg of sample
d) Lime
Lime showed highest activity in 80% methanolic followed by 100C aqueous extract in
2.5 to 10 mg of sample and the least activity were seen in acetonic extract. Bocco et al
(1998) demonstrated that citrus peel and seed extracts have high levels of phenolics,
which have strong antioxidant capability. Jeong et al (2004) reported the radical
scavenging activity of lime peel in 50C, 70% ethanolic extract as 30.6%, 50C water as
18.30 % and 100C water extract as 24.29 %.
122
Figure 19: Free radical scavenging activity of lime in different extracting media
100
% Activity
80
A
60
B
40
C
D
20
0
Water
(100 C)
Water
(30C)
Methanol Ethanol Methanol Ethanol

(80%)
(80%)
Acetone
Concentration- All extracts, A: 2.5, B: 5.0, C: 7.5 and D: 10 mg of sample
e) Shallot
Shallot showed the optimum level of activity in hot and 30C water extract and least
activity was observed in acetonic extract. Methanol and ethanol extract did not show any
activity as observed in reducing power assay. Lu et al (2011) reported the shallot radical
scavenging activity as 5.71 mol Trolox/g fresh weight. The free radical scavenging
activity of samples was found to be as follows:
Borage> valerian= ginger> lime> shallot
It is well understood that both agronomic or environmental factors and genetics play
important roles in the phenolic composition and accordingly nutritional quality of crop
(TomsBarbern and Espn, 2001), so differences between studies can be correlated to
differences in environmental factors and genetics of plant.
123
Figure 20: Free radical scavenging activity of shallot in different extracting media
60
% Activity
50
40
A
30
20
C
D
10
0
Water
(100C)
Water
(30C)
Methanol
Ehanol
Methanol
(80%)
Ethanol
(80%)
Acetone
Concentration- All extracts, A: 5, B: 10, C: 15 and D: 20 mg of sample

2.2.4. Correlation between antioxidant component and antioxidant activity
Antioxidant components and activity are highly dependent on extracting solvent and
concentration of solvent (Turkmen et al., 2006) but also vary within the samples. Many
researchers have reported about the relationship between phenolic content and
antioxidant activity. In some studies they found a correlation between the phenolic
content and antioxidant activity (Velioglu et al., 1998; Liu et al., 2002; Sellappan and
Akoh, 2002; Sun et al., 2002; Apak et al., 2006) whereas others found no relationship
(Kahkonen et al., 1999).
The antioxidant components of all samples extracted in water and in solvents were
correlated with their antioxidant activity as determined by their different assays and
results are presented in Table 24. Analysis was done for results of water extracts (2 sets
of value, and solvent extracts (4 sets of values, acetonic extract were not included
because of very low activity) were treated individually.
In our study except for valerian and ginger, all samples showed high correlation between
antioxidant components and all three method of antioxidant activity in water extracts at
two temperature (R2=1). In valerian only flavonoids showed negative correlation with
antioxidant activity and ginger showed positive correlation only between polyphenol and
antioxidant activity. In valerian, it can be explained due to the fact that though
124
antioxidant activity was more in 100C water than 30C, total flavonoids were lesser in
100C than 30C aqueous extract which can be due to heat sensitivity of valerian
flavonoids.
In solvent extracts, except valerian and ginger, all samples showed correlation with
DPPH and antioxidant components. Polyphenol and total tannin showed correlation with
reducing power in all samples except shallot. In valerian only the tannin content had
correlation with reducing power and total antioxidant activity (R2= 0.696) and (R2=0.713)
respectively. In borage and lime correlation was seen with reducing power and all three
antioxidant components, whereas shallot showed negative correlation. In solvent extract
of ginger, only flavonoids did not show correlation with DPPH method and all other
compound showed correlation with antioxidant activity with 3 different methods.
In solvent extracts, for all samples antioxidant components showed positive association
with free radical scavenging activity, though the extent of association varied. Borage
showed high correlation between polyphenols and tannins (R2 =0.94-0.96), and a slightly
lesser correlation with flavonoids; for valerian, it was lesser for all components (0.4160.54); for ginger, it was low for flavonoids but very high for polyphenols and tannins; for
lime and shallot, good correlation was seen.
For reducing power assay, correlations were high for borage and lime, for all components
(R2= 0.836-0.954). Ginger showed high value for polyphenol and tannin but low for
flavonoids whereas for valerian it was low for all. Shallot did not exhibit any association
between antioxidant components and reducing power.
For total antioxidant, results were different for all samples. For borage, there was a week
correlation for flavonoids, but others were negative, for valerian, tannins were better but
others were low, for ginger, tannins showed good correlation, others were low, lime and
shallot followed the same trend. It can be said that, the antioxidant activity of all herbs
and spices could be correlated with their antioxidant components very well.
125
Table 24: Correlation coefficient between antioxidant compound and antioxidant

activity of samples
Water Extract
Correlation
Coefficient
Solvent Extract
DPPH
Reducing
Power
Total
Antioxidant
DPPH
Reducing
Power
Total
Antioxidant
Flavonoids
0.74
0.95
0.243
Polyphenols
0.94
0.93
-0.39
Total tannin
0.96
0.92
-0.4
Flavonoids
-1
-1
-1
0.54
0.40
0.32
Polyphenols
0.45
0.52
0.38
Total tannin
0.416
0.696
0.713
Flavonoids
-1
-1
-1
0.493
0.505
0.613
Polyphenols
0.901
0.847
0.579
Total tannin
-1
-1
-1
0.985
0.887
0.885
Flavonoids
0.843
0.836
0.408
Polyphenols
0.746
0.851
0.318
Total tannin
0.931
0.954
0.740
Flavonoids
0.860
-0.195
0.654
Polyphenols
0.970
-0.218
0.758
Total tannin
0.860
-0.600
0.838
[R Values]
Borage
Valerian
Ginger
Lime
Shallot
126
3. ANTIBACTERIAL ACTIVITY
With the realization that the effective life span of antibiotic medicines are limited and
over prescription and misuse of traditional antibiotics may cause microbial resistance, the
use of plant extract for medicinal treatment has become popular (Alam et al., 2009).
Nowdays, nearly 30% or more of the modern pharmacological drugs are derived directly
or indirectly from plants and their extracts dominate in homeopathic or ayurvedic
medicines (Banso, 2009; Jabeen et al., 2009; Ahameethunisa and Hopper, 2010;
Murugesan et al., 2011).
Authentic cultures of human pathogenic bacteria viz., Escherichia coli (MTCC 7410),
Klebsiella pneumonia (MTCC 7407), Bacillus subtilis (MTCC 121), Bacillus cereus
(MTCC 1272), Salmonella typhi (MTCC 733), Ps. aeruginosa (MTCC 424) and
Staphylococcus aureus (MTCC 7443) were obtained from Microbial Type Culture
Collection, Chandigarh, India and they served as test bacteria.
Antibacterial activity of all samples was examined in aqueous and methanolic extract at
50 l/well. In all extracts, 2 g of sample in 200 ml of extracting media were used for
extraction of aqueous and methanolic extract. Dried extract was reconstituted with the
ratio of 1:3 and subjected for antibacterial activity. MIC of samples was determined in
both extracts.
3.1. ANTIBACTERIAL ACTIVITY OF AQUEOUS EXTRACT

Shallot and lime showed significant activity against almost all 6 gram positive and gram
negative bacteria. Results are presented in Table 25 and Figure 21-34.
As presented in Table, shallot and lime showed highly significant antibacterial activity
against all bacteria. Against E. coli, shallot showed more activity than lime and as
presented in Table, diameter of zone of inhibition was 26.5 mm whereas lime had 19.0
mm zone of inhibition. Minimum inhibitory concentration (MIC) were measured at 05,
10, 25 and 50 l. Inhibition was in the range of 14 to 27 mm in shallot and 12 to 19 mm
in lime. Borage, valerian and ginger did not show any activity against E. coli.
Observation for ginger was in agreement with other studies. Onyeagba et al (2004)
evaluated aqueous extract of dried ginger. They extracted 20 g dried ginger with 100 ml
water and added the sample to the disk with 5mm diameter and observed that it did not
127
inhibit E. coli. They also reported that at same concentration fresh lime juice inhibited
the growth of E. coli with diameter zone of 11 mm.
Indu et al (2006) determined the antibacterial properties of 100% fresh ginger juice, and
75%, 50% and 25% diluted ginger juice against 20 serogroups of E. coli (both pathogenic
and non-pathogenic). The pathogenic serogroups included enterotoxigenic E. coli and
enterohemorrhagic E. coli. They observed that100% ginger showed activity only against
2 pathogenic strain of E. coli and it did not show any activity in remaining 18 strains.
Inhibition was reported as 10 and 18 mm in enterotoxigenic E and enterohemorrhagic E.
coli respectively. Diluted juice did not show activity in any strain and any concentrations
except 75% ginger which showed activity of 13mm against enterohemorrhagic E. coli.
In the other study Ekwenye & Elegalam (2005) reported the antibacterial potential of
aqueous extract of ginger against E. coli. They extracted 5g of ginger in 200 ml of
distilled water. The extract was dried and reconstituted by the ratio of 1:2. After
sterilizing the extract the inhibition of ginger extract was evaluated. The aqueous extract
of ginger did not show activity against E. coli at concentration of 1g of sample.
Fresh ginger and commercial ginger paste were inoculated with a three strain cocktail of
overnight cultures of E. coli O157: H7 and stored at 4C and 8C for 2 weeks. Each paste
exhibited different antimicrobial effects alone and in ground beef or buffered peptone
water at 4C and 8C for 2 weeks. Commercial ginger paste showed strong antimicrobial
properties with complete inactivation of E. coli O157:H7 in the paste at 3 days at 4C and
8C. However, fresh ginger paste showed antimicrobial activity only at 8C. Only
commercial ginger paste had antimicrobial activity in buffered peptone water at 4C for 2
weeks. However, commercial ginger paste showed antimicrobial activity in ground beef
at 3 days and after (about 12 log CFU/g) compared to control samples at 8C for 2
weeks (Gupta and Ravishankar, 2005).
128
Figure 21: Antibacterial activity of aqueous extract of lime and shallot against E. coli
Sh: Shallot, L: Lime, V: valerian, Bo: Borage, G: Ginger, -C: negative control
Figure 22: Minimum inhibitory concentration of aqueous extract of lime and shallot against E.
coli
Lime
Shallot
+C: positive control, -C: negative control, A: 5 l, B: 10 l, C:25 l and D: 50l
Aqueous extract of both shallot and limes exhibited a remarkable inhibition in growth of
Staph. aureus. In comparison shallot showed more inhibition (33.5 mm) than lime (23
mm). Other samples did not exhibit antibacterial properties against Staph. aureus. When
minimum inhibitory concentration was examined at 50, 25, 10 and 5 l it was observed
129
that shallot and lime showed activity in the range of 33-12 and 22-0 mm respectively. At
same condition inhibition was observed with gentamycin antibiotic which interestingly
was 1mm lesser than shallot at the same concentration. Other samples did not show
inhibition. Onyeagba et al (2004) reported that aqueous extract of dried ginger does not
have antibacterial activity against Staph. aureus, whereas fresh lime juice could inhibit
with zone of 17 mm.
Figure 23: Antibacterial activity of aqueous extract of lime and shallot against Staph. aureus
Sh: Shallot, L: Lime, +C: positive control, -C: negative control

Figure 24: Minimum inhibitory concentration of aqueous extract of lime and shallot against
Staph. aureus
Lime
Shallot
130
Bacillus subtilis was inhibited only with shallot and lime. Diameter zone of inhibition by
shallot was 29 and lime 28.8 mm. Minimum inhibitory concentration was done and zone
of inhibition was found to be in the range of 29-18 and 28-20 in shallot and lime
respectively.
Figure 25: Antibacterial activity of aqueous extract of lime and shallot against B. subtilis

Figure 26: Minimum inhibitory concentration of aqueous extract of lime and shallot against B.
subtilis
Lime
Shallot
+C: positive control, -C: negative control, A: 5 l, B: 10 l, C: 25 l and D: 50l
131
Typhoid fever causes an estimated 16.6 million cases and 600,000 deaths worldwide each
year (Perilla and Diseases, 2003), so it may be useful to find a medicinal plant which has
property to inhibit these bacteria.
Shallot showed a zone of 32.5 and lime showed 30.5 mm. MIC for shallot and lime was
in the range of 32-15 and 29-12 mm respectively.
Ekwenye & Elegalam (2005) studied the antibacterial potential of aqueous extract of
ginger against Sal. typhi and reported that salmonella typhi is sensitive to aqueous extract
of fresh ginger and it showed a zone of 8 mm at 1000 mg/ml of sample. But since we
used dry ginger, activity was not seen. It may be due to sensitivity of active compound to
storage and drying. This finding is supports the study conducted by Onyeagba et al
(2004). They found out dried ginger did not exhibit antibacterial potential against
salmonella spp and lime juice showed diameter inhibition of zone by 13mm.
Figure 27: Antibacterial activity of aqueous extract of lime and shallot against Salmonella
typhi
132
Salmonella typhi
Lime
Shallot
Shallot showed a remarkable activity against kleb. pneumonia (29.5mm). Inhibition was
3.5 mm more than gentamicin antibiotic. Zone of inhibition was measured as 18.5 mm in
lime. Activity was observed in 4 different concentration and found to be 29-19 and 21-0
mm in shallot and lime respectively.
Figure 29: Antibacterial activity of aqueous extract of lime and shallot against kleb.
pneumonia
133
Kleb. Pneumonia.
Lime
Shallot
Lime had slightly more inhibition than shallot, against ps. aeruginasa. At concentration
of 50 l, lime showed zone of 23.5 mm and shallot had 21.5 mm inhibition. MIC was in
the range of 20-13 and 21-11 mm in shallot and lime respectively. Gentamicin showed
inhibition of 27 mm.
Figure 31: Antibacterial activity of aqueous extract of lime and shallot against Ps. aeruginosa
L: Lime, G:ginger, Bo: Borage Sh: Shallot, V: Valerian, -C: Negative control
134
Ps. aeruginosa
Lime
Shallot
Both shallot and lime showed antibacterial activity against B. cereus. Shallot showed
slightly more activity than lime. As presented in Table 25 a zone of 23.5 and 18.5 mm
was observed in shallot and lime respectively.
Figure 33: Antibacterial activity of aqueous extract of lime and shallot against B.cereus
L: lime, G:ginger, Sh: shallot, Bo: borage, V: valerian, +C: positive control
135
Figure 34: Minimum inhibitory concentration of aqueous extract of lime and shallot against B.
cereus
Lime
Shallot
+C: Positive control, -C: Negative control, A: 5 l, B:10 l, C:25 l and D: 50l
Table25: Antibacterial activity of aqueous

Samples
E. coli
Bacillus
Staph.
Ps.
aureus
aeruginosa subtilis
Kleb.
Bacillus
Sal.
pneumonia
cereus
typhi
26.5
33.5
21.5
29.0
29.5
23.5
32.5
0.71
0.71
0.71
1.41
0.71
0.71
0.71
Borage
Valerian
19.0
23
23.5
28.5
18.5
18.5
30.5
1.41
0.71
1.41
0.71
0.71
0.71
2.12
Shallot
Lime
Ginger
Concentration: 50l/ well.

Zone of inhibition is in mm
.
136
Table 26: MIC of aqueous extracts against gram positive bacteria

Bacillus cereus
Spice
Staph. aureus
Bacillus subtilis
50
25
10
05
50
25
10
05
50
25
10
05
Shallot
24
22
20
18
33
31
29
21
29
24
20
18
Lime
18
16
15
14
22
20
15
28
25
21
20
Gentamicin
31
32
34
Water
Table 27: MIC of aqueous extracts against gram negative bacteria

Sal. Typhi
Ps. aeruginosa
50 25 10 05
50 25 10
05 50
25 10 05 50 25 10 05
Shallot
32 27 24 15
20 18 15
13 29
26 23 19 27 25 23 14
Lime
29 25 23 12
21 17 15
11 21
18 12 -
19 17 15 12
Samples
Kleb. pneumonia
E. coli
Gentamicin 29 -
26
25 -
Water
3.2. ANTIBACTERIAL ACTIVITY OF METHANOLIC EXTRACT

Antibacterial activity of all samples in methanolic extract was studied and similar to
aqueous extract, ginger, valerian and borage did not show any inhibition in selected
bacteria.
Diameter of inhibition zone (25 mm) was shown by shallot and lime against E. coli. MIC
found to be in the range of 25-18 and 26-12 mm in shallot and lime respectively. But E.
amoenum, ginger and valerian did not show activity. Results are in agreement with other
studies. Mansouri et al. (2005) also examined antibacterial properties of methanolic
extract of Echium amoenum against E. coli and reported that petals of E. amoenum did
not show antibacterial properties against E. coli. Onyeagba et al (2004) reported the
ethanolic extract of dried ginger does not have antibacterial potential against E. coli.
137
Figure 35: Antibacterial activity of methanolic extract of lime and shallot against E. coli
Sh: Shallot, L: Lime, G: Ginger +C: Positive control, -C: Negative control
Figure 36: Minimum Inhibitory concentration of methanolic extract of lime and shallot against
E. coli
Lime
Shallot
Lime and shallot showed similar activity against Staphylococcus aureus. As presented in
Table 28 diameter of inhibition zone were 25 mm in both lime and shallot.
Mansouri (1999) tested ethanolic extracts of E. amoenum against Staphylococcus aureus
(489 samples) and observed that it did not inhibit bacterial growth.
138
Figure 37: Antibacterial activity of methanolic extract of lime and shallot against Staph. aureus

Staph. aureus
Lime
Shallot
Ps. aeruginosa was inhibited with methanolic extract of shallot and lime. Zone of
inhibition were found to be 22.5 and 23.5 mm in shallot and lime respectively. At
different concentration zone of inhibition were in the range of 22-15 and 23-15 mm
respectively. Inhibition was found to be almost same as gentamicin.
139
Figure 39: Antibacterial activity of methanolic extract of lime and shallot against
Ps.aeruginosa
Sh: Shallot, L: Lime, G: Ginger, Bo: Borage, V: Valerian -C: Negative control
Ps.aeruginosa
Lime
Shallot
+C: Positive control, -C: Negative control, A: 5 l, B: 10 l, C: 25 l and D: 50l
Diameter of inhibition zone was 32 mm in shallot extract against bacillus subtitles and
lime showed inhibition zone almost same as shallot (31 mm). Minimum inhibitory
concentration of shallot was found to be in the range of 31-11 mm and lime ranged
between 30-21 mm. In this experiment gentamicin antibiotic showed a zone of 38 mm.
140
Figure 41: Antibacterial activity of methanolic extract of lime and shallot against B. subtilis
B. subtilis
Lime
Shallot
Shallot and lime showed 24 and 25 mm zone respectively. The MIC of shallot and lime
were in the range of 24-18 and 25-17 mm respectively. Gentamicin was found to inhibit
the growth with the diameter of 24mm. Shahidi Bonjar (2004) examined the antibacterial
properties of ethanolic extract of E. amoenum and ginger against kleb. Pneumonia and
141
observed that both ginger and borage did not show antibacterial properties in ethanolic
extract against kleb. pneumonia.
Figure 43: Antibacterial activity of methanolic extract of lime and shallot against Kleb.
Pneumoniae

Kleb. Pneumoniae
Lime
Shallot
Against B. cereus, lime exhibited more diameter of inhibition zone than shallot. Shallot
had diameter of 25 mm and lime showed 27 mm inhibition zone. The activity of lime
were found to be very close to the inhibition by gentamicin, 28 mm. when activity was
142
evaluated in different concentration, a range of 25-18 mm and 26-16 mm were observed

in shallot and lime respectively.
Figure 45: Antibacterial activity of methanolic extract of lime and shallot against B. cereus
Sh: Shallot, L: Lime, G: Ginger, Bo: borage, +C: Positive control, -C: Negative control
B. cereus
Lime
Shallot
Shallot and lime had 25 and 24 mm diameter of inhibition zone against salmonella typhi.
Inhibition was quite lesser than gentamicin (30 mm). Inhibition at different concentration
143
was in the range of 24-10 mm in shallot and 24-16 mm in lime. Onyeagba et al (2004)
reported that salmonella spp does not inhibit with ethanolic extract of dried ginger. Azu
et al (2007) studied the antimicrobial properties of various extracts of fresh Allium cepa
(onions) and fresh Zingiber officinale (ginger) against Escherichia coli, Salmonella typhi
and Bacillus subtilis. Sensitivity pattern of staph. aureus and Ps. aeruginosa to coldwater extract of ginger was reported to be in the range of 13-19 mm against salmonella
typhi and 12-17 mm against ps. aeruginosa in the concentration of 0.1-0.8 gml-1.
Inhibition was not seen in the hot water extract against Salmonella typhi. It can be
explained by sensitivity of antibacterial component of ginger to thermal treatment. But
activity was reported in hot water extract against ps. aeruginosa. When the inhibition was
compared with cold water extract, inhibition was found to be lesser in hot water extract.
In this study inhibition was very less in most of the concentrations and extracts, inhibition
was not seen. Although onion and shallot are in a same botanical family but activity of
shallot was found to be much more than onion (Allium cepa). In opposite, in aqueous and
methanolic extract of ginger, inhibition was not seen in any of the bacteria. If can be due
to the variety and also in present study we have used dried ginger but Azu et al (2007)
used fresh ginger for the study.
It was clear from this work that the solvent of extraction affected the degree of
antibacterial activity of the extracts.
Figure 47: Antibacterial activity of methanolic extract of lime and shallot against Sal. typhi
144
Sal. typhi
Shallot
Lime
Table 28: Antibacterial activity of methanol extract

Staph. Ps.
Bacillus
Kleb.
Bacillus
Samples E. coli
aureus aeruginosa subtilis
pneumonia cereus
25.5
25.0
32.0
24
25.0
Shallot
22.5
0.71
1.41
1.41
0.71
1.41
Borage
Valerian
25.0
25.0
23.5
31
25
27.0
Lime
1.41
0.71
0.71
2.12
0.71
0.71
Ginger
Zone of inhibition is in mm
Sal.
typhi
25.0
1.41
24.0
0.00
-
Table 29: MIC of methanolic extracts against gram negative bacteria

Samples
E. coli
Sal. typhi
Ps. Aeruginosa
Kleb. pneumonia
50 25 10 05 50 25 10 05 50 25 10 05 50 25 10 05
Shallot
25 23 21 18 24 20 14 10 22 19 18 15 24 22 20 18
Lime
26 19 17 12 24 19 18 16 23 20 17 15 25 22 19 17
Gentamicin 29 -
30 -
24 -
24 -
Methanol

Zone of inhibition is in mm.
145
Table 30: MIC of methanolic extracts against gram positive bacteria

Samples
Bacillus cereus
Staph. aureus
Bacillus subtilis
50
25
10
05
50
25
10
05
50
25
10
05
Shallot
25
24
21
18
24
19
16
14
31
29
25
11
Lime
26
24
21
16
25
22
20
13
30
26
24
21
Gentamicin
30
34
38
Methanol

Zone of inhibition is in mm.
In aqueous extract of C. aurantifolia and allium scallion, difference in activity was not
significant between gram positive and gram negative tested bacteria.
But between shallot and lime, aqueous extract of shallot showed more activity in both
gram positive and gram negative bacteria. In methanol extract lime showed slightly more
inhibition compared to shallot. Diameter zone of inhibition in shallot and lime did not
show significant difference between gram positive and gram negative tested bacteria.
It can be concluded that among samples shallot and lime showed antibacterial potential
against 7 tested bacteria. Aqueous extract exhibited slightly higher activity than
methanolic extract.
4. IN VIVO ANTIOXIDANT POTENTIAL OF BORAGE AND

SHALLOT
4.1. EFFECT OF BORAGE SUPPLEMENTATION ON ANTIOXIDANT STATUS
OF DIABETIC PATIENTS
It is well known that oxidative stress is more in diabetic patients and cause lipid
peroxidation and production of free radicals. Oxidative stress and free radicals are
associated with increase in diabetic complications and also chance of cancer. To evaluate
in vivo antioxidant potential, effect on lipid profile and safety of borage, 60 NIDDM
subjects without complications were divided to 2 groups of 30. Thirty subjects received
500 mg petals of borage/day for 30 days and other group was received placebo capsules
/day for 30 days. Twenty four hours food recall was taken before and after the study.
146
Blood was analyzed for antioxidant activity, lipid peroxidation and biochemical
parameters. Results are presented in figure 13-15.
4.1.1. Total Thiol

Total thiol protein was determined before and after the study. Protein thiols along with
bilirubin and uric acid are the major endogenous antioxidants (Kampa et al., 2002). In the
cell membranes; however, thiol compounds such as glutation prevent ethanol-induced
liver injury through the elimination of acetaldehyde (Hirayama et al., 1983). Thiols are a
class of organic sulfur derivatives which are distinguished by the presence of sulfhydryl
residues (-SH) at their active site. One of the characteristic features of most thiols is their
capability to act as reducing agents. Therefore, in the case of an oxidant-thiol interaction,
the oxidant is neutralized to a relatively less toxic byproduct at the expense of the
reducing power of thiol, which itself gets oxidized to a disulfide (Sen and Packer, 2000).
Since in degenerative disease like diabetes, oxidative stress is high, thiol molecule
interact with oxidant so the concentration of thiol molecule will reduce in blood.
Diabetes mellitus is increasing worldwide, resulting from the obesity, inflammation and
hyperglycemia. Both type I and type II diabetes are powerful and independent risk factors
for coronary artery disease, stroke and peripheral arterial disease (Wattanakit et al., 2005;
Ziegler, 2005). It is proved that oxidative stress is a major contributor to diabetic
complications so antioxidant supplementation can reduce free radicals and prevents
complications (Vivekananthan et al., 2003; Eidelman et al., 2004; Sesso et al., 2008).
A decrease in plasma thiol levels has been reported in diabetic patients (Semenkovich
and Heinecke, 1997; Pasaoglu et al., 2004). An increase of the antioxidant capacity of
plasma indicates absorption of antioxidants and an improved in vivo antioxidant status
(Cao et al., 1998).
Figure 49 presents the total thiol of study group and control group before and after the
study. As shown in figure, in experimental subjects, total thiol of samples significantly
increased at the end of the study whereas in control group, which had received placebo,
there were no significant changes in serum total thiol molecule before and after the study.
Initially total thiol content of intervention group was found to be 0.140 mol/ml which
increased to 0.243 mol/ml after 30 days of borage supplementation. Whereas total thiol
147
in control group initially was 0.157 mol/ml and after 30 days of taking placebo it was
reported to be 0.164 mol/ml which was statistically not significant. When increase was
calculated as percent increase over control, a 73% increase was seen in study group but in
control group, total thiol increased by 4%. Ranjbar et al (2006) report a similar study
where the total thiol content of Iranian healthy adults was 0.49 mol/ml and after giving
borage decoction two times in a day for duration of 14 days, total thiols were elevated to
0.56 mol/ml.
Hence, researchers support the fact that when oxidative stress is more, total thiol reduces,
due to antioxidant potential and neutralizing free radicals.
Figure 49: Effect of borage supplementation on Serum total thiol molecule of
diabetic patients before and after the study (mol/ml)
0.3
Total thiol mol/ml
0.25
0.2
0.15
Before
After
0.1
0.05
0
Study group
Placebo
4.1.2. Serum lipid peroxide level

Serum lipid peroxide level of patients was analyzed before borage supplementation and at
the end of the study. As presented in Figure 50, serum lipid peroxides were 4.9 nmol/ml
before supplementation and after a month of supplementation the levels decreased
drastically (4.3 nmol/ml) and when analyzed statistically, there was a significant
difference in serum lipid peroxide, before and after the study in the study group. In the
control group which had taken placebo, serum lipid peroxide was found to be 4.82 and
4.71 nmol/ml before and after the study respectively and after statistical analysis showed
148
no significant difference. Lipid peroxidation, glutathione levels, glutathione peroxidase

and glutathione reductase, catalase, superoxide dismutase are biomarkers of oxidative
stress (Maritim et al., 2003). Many researchers suggest the possibility of antioxidant
supplementation on reduction of lipid peroxidation to reduce the oxidative stress (Park
and Choi, 2002). Since oxidative stress in diabetic patients plays an important role in
incidence of diabetic complementation it is very important to reduce oxidative stress in
diabetic patients. The level of serum lipid peroxides in diabetics has been reported by
many authors. Freitas et al (1997) reported the LPO level of diabetics as 4.51 nmol/ml.
Kei (1978) found the LPO level in NIDDM without complication is 4.73 nmol/ml and
Kumawat et al (2009) reported LPO level to be 5.64 nmol/ml in diabetic retinopathy,
7.86 nmol/ml in diabetic neuropathy and 7.66 in diabetic nephropathy. Samuel and
Johncy (2010) reported the LPO level of NIDDM without complications as 4.73 nmol/ml,
with retinopathy 5.65, with pre-neuropathy 5.60, with nephropathy 5.73, and with
retinopathy and pre-neuropathy 5.59. As shown here, there is a positive correlation
between LPO level and diabetic complication. Ranjbar et al (2006) studied the effect of
borage decoction, 2 times a day for duration of 14 days, on lipid peroxidation level of
healthy young (18-25 years old). They reported a significant reduction in lipid
peroxidation level after 14 days supplementation.
Figure 50: Effect of borage supplementation on Serum lipid peroxidation of diabetic
patients before and after the study (nmol/ml)
Lipid peroxidation nmol/ml
5.2
4.8
Before
After
4.4
4
Study Group
Placebo
149
4.1.3. Total antioxidant capacity (TAC) by FRAP assay

Serum total antioxidant capacity of patients in both study and control group were
measured before and after supplementation. Total antioxidant capacity was determined
by FRAP assay. As illustrated in figure, after 30 days supplementation of borage the total
antioxidant capacity was significantly increased, whereas in control group which had
received placebo, changes in antioxidant capacity were not significant. In intervention
group, TAC increased from 707 to 1002 mol/ml but in control group it was 733
mol/ml before giving placebo and 731 mol/ml after study. Present results are in
agreement with previous results. Ranjbar et al (2006) studied the effect of borage
decoction on TAC in healthy adults. They observed after 14 days consumption of 2 cup
borage decoction, TAC significantly increased in healthy subjects. They reported an
increase from 1450 to 1700 mol/ml.
Figure 51: Effect of borage supplementation on Serum total antioxidant activity of

diabetic patients before and after the study (mol/ml)
1200
TAC mol/ml
1000
800
Before
600
After
400
200
0
Study group
Placebo
4.1.4. Food intake

Since the antioxidant levels in blood may also be influenced by dietary intake, the
nutrient intake of subjects was determined using 24 hours recall dietary survey twice
during the study, before and at the end of study. Results are given in Table 31 and 32.
150
As shown in Table 31, male subjects in experimental and control group were taking
energy, protein, iron, carotenoids, riboflavin and niacin lesser than RDA whereas intake
of fat and calcium were found to be more than RDA. Thiamin and vitamin C intake were
slightly lesser than RDA.
Female subjects were found to take lesser energy, iron and carotenoids than RDA for
reference Indian woman. In both experimental and control group. Protein, Vitamin C and
thiamin were found to be meet requirements and niacin and riboflavin intake were found
to be very close to RDA value.
Since RDA for Indian, does not provide recommendation for dietary fiber, it was
compared with RDA for Americans. Dietary fiber in both male and female were found to
be lesser than RDA for Americans.
Dietary intake was calculated as percent adequacy of nutrient and data are presented in
Figure 52 and 53.
Table 31: Average nutrient intake of male diabetic patients before and after borage
supplementation
Experimental group
Control group
Before
After
Before
After
Energy (kcal)
2320
2270
2100
2150
Protein (g)
50.7
51.1
52.0
51.2
Fat (g)
40.8
45.1
42.3
44.0
Fiber (g)
26.8
25.2
27.5
25.5
Calcium (mg)
590
550
530
542
Iron (mg)
12.0
11.1
13
12.5
Carotenoids (g)
1121
1032
1281
1242
Thiamin (mg)
1.08
1.10
1.10
1.05
Riboflavin (mg)
0.74
0.79
0.77
0.80
Niacin (mg)
9.21
10.21
9.70
9.90
Vitamin C
30.30
32.1
35.6
34.2
RDA: Recommended Dietary Allowance for Indian male
*: WHO recommendation (WHO, 2003)
Macronutrient
RDA
2425
60
20
30*
400
28
2400
1.2
1.4
16
40
151
Table 32: Average nutrient intake of female diabetic patients before and after
borage supplementation
Experimental group
Control group
Before
After
Before
After
Energy (kcal)
1864
1971
1880
1990
Protein (g)
50.0
50.4
48.4
50.1
Fat (g)
50
51
54
53.5
Fiber (g)
12.17
17.3
14.0
17.8
Calcium (mg)
634
580
552
490
Iron (mg)
10.8
11.8
10.2
9.9
Carotenoids (g)
1018
1022
1032
1029
Thiamin (mg)
0.98
1.0
0.97
0.98
Riboflavin (mg)
0.92
1.01
0.98
1.00
Niacin (mg)
8.39
9.9
10.4
10.9
Vitamin C (mg)
43
41
45
42
RDA: Recommended Dietary Allowance for Indian female
*: WHO recommendation
Macronutrient
RDA
1875
50
20
30*
400
30
2400
0.9
1.1
12
40
Figure 52: Percent adequacy of nutrient intake of male diabetic patients before and
after borage supplementation
250
200
150
100
Before-E
After-E
50
0
Before-C
After-C
E: experimental group, C: Control group
152
Figure 53: Percent adequacy of nutrient intake of female diabetic patients before
and after borage supplementation
300
250
200
150
100
50
Before-E
After-E
Before-C
After-C
E: experimental group, C: Control group

4.1.5. Blood glucose estimation
As presented in Figure 54, initially fasting blood glucose of patients in study group was
110 mg % and after supplementation it was found to be 113.5 mg%. Post prandial blood
glucose was measured 2 hours after breakfast. Initially it was 167 mg% and after study
the mean value for post prandial blood glucose was 160 mg%. HbA1C were 7.34 and 7.01
% before and after the study. When data was statistically analyzed, no significant
difference in both FBG and PPG level before and after the study was observed.
Other studies also support our finding. Park and Choi (2002) studied the effect of tocopherol supplementation on oxidative stress in type II diabetes mellitus, and found
that there is not a significant difference in blood glucose before and after
supplementation. Czernichow et al (2006) also reported that antioxidants does not affect
blood glucose.
153
Figure 54: Effect of borage supplementation on Serum Blood glucose of diabetic

patients before and after the study (mg%)
250
200
mg %
150
Before
100
After
50
0
FBG-S
PPBG-S Hba1c-S
FBG- C
PPBG- C Hba1c-C
FBG: Fasting blood glucose in study group

PPBG: post prandial blood glucose in study group
S: Study group
C: Control group
4.1.6. Lipid profile test
Lipid profile of all patients- including control group- before and after the study was
determined. As presented in Figure 55, cholesterol level in study group was 195 mg %
before study and after one month supplementation; it reduced to 166 mg%. Triglyceride
reduced from 159 to 131 mg%, LDL reduced from 123 to 109 mg% and VLDL from 33
to 27mg%. When data were analyzed statistically a highly significant reduction was seen
in cholesterol level, triglyceride, VLDL and LDL of study group patients after 30 days
supplementation and P value was found to be 0.001, 0.0008, 0.002 and 0.025
respectively. When the reduction was calculated as percent of value before treatment,
15% reduction was seen in total cholesterol and 18% and 18.2% reduction were seen in
triglyceride and VLDL level respectively, whereas in control group, after the completion
of study, differences was not seen in plasma lipid.
Heart disease is a major disease all over the world. Hypercholesterolemia is an important
risk factor for atherosclerosis (Real et al., 2001). Some of cholesterol- lowering
154
nutraceuticals and functional foods are dietary fiber, polyphenols, phytoestrogens, tea
catechins and garlic (Chen et al., 2008b).
As reported earlier, the soluble fiber of borage was found to be 45% of dry petal. It is
well known that soluble fiber can reduce plasma lipid concentration (Brown et al., 1999;
Chandalia et al., 2000). Aller et al (2004) administered subjects with 1.97 g soluble fiber
and observed that a modest increase in soluble fiber intake in healthy subjects improved
LDL cholesterol and glucose levels. Chandalia et al (2000) studied the effect of high fiber
diet on 13 patients with type 2 diabetes mellitus. The high-fiber diet (total, 50 g; 25 g of
soluble fiber and 25 g of insoluble fiber containing foods not fortified with fiber)
reduced plasma total cholesterol concentrations by 6.7% (P=0.02), triglyceride
concentrations by 10.2% (P=0.02), and very-low-density lipoprotein cholesterol
concentrations by 12.5 % (P=0.01).
Though each capsule provided 225 mg of soluble fiber, but still significant reduction was
seen in study group.
In addition, each capsule also provided 7.7 g polyphenols, 12.35g tannin and 22.7g
flavonoids. It has been reported that polyphenols can reduce blood lipid concentration
(Bradamante et al., 2004; Vita, 2005). Osada reported polyphenols from unripe apple has
effect on lowering serum lipid profile in rats (Osada et al., 2006). Nagasako-Akazome et
al (2005) studied the effect of unripe apple polyphenol on lowering blood lipid profile.
They gave apple polyphenol in the form of tablet at 3 different doses (300, 600 and 1500
mg), it was observed that the serum total cholesterol at week 0 and week 4 were 226.5
and 224.1 g/dl in placebo group, 226.9 and 222.7 mg/dl in low dose group, 231.2 and
224.7 mg/dl in medium dose group and 226.6 and 216 mg/dl in high dose group. They
observed a significant difference in cholesterol level of high dose group. Also it has been
suggested that the daily consumption of 2 g of phytosterols can effectively lower the
cholesterol by 9-14% in humans with little or no effect on HDL-C and TG levels (Law,
2000)
Erba et al (2005) investigated the effect of the addition of two cups of green tea
(containing approximately 250 mg of total catechins) to a controlled diet in a group of
healthy volunteers with respect to a group following the same controlled diet but not
consuming green tea. After 42 days, use of green tea caused a significant increase in
155
plasma total antioxidant activity [from 1.79 to 1.98 mol Trolox equivalent (TE)/ml,
P<.001], significant decreases in plasma peroxides level (from 412 to 288 Carr U, P<.05)
and induced DNA oxidative damage in lymphocytes (from 14.2% to 10.1% of DNA in
tail, P<.05), a moderate although significant decrease in LDL cholesterol (from 119.9 to
106.6 mg/dL, P<0.05) with respect to control. The present study suggests the ability of
green tea, consumed within a balanced controlled diet, to improve overall the
antioxidative status and to protect against oxidative damage in humans.
Figure 55: Effect of borage supplementation on Lipid profile of diabetic patients

before and after the study (mg/dl)
250
mg/dl
200
150
100
Before
After
50
0
-S: Study group

-C: control group
156
Table 33: Effect of borage supplementation on Lipid profile of diabetic patients

before and after the study (mg/dl)
Lipid profile
Total Cholesterol
Triglycerides
HDL
LDL
VLDL
Chol/HDL
LDL/HDL
TG/HDL
Non HDl Chol
Intervention group
Before
After
202
164
165
130
40.18
41.18
123.2
108.8
33.0
27.3
4.88
4.04
3.1
2.66
3.98
3.2
154
124
Placebo group
Before
After
170.5
174.1
153.4
163.2
37.4
39.3
97.6
97.7
30.5
32.5
4.59
4.45
2.64
2.5
4.1
4.17
133.1
134.9
4.1.7. Liver function test

At week 0 and week 4, liver function test was done to observe if the supplementation can
affect the liver function. As illustrated in Table 34, total bilirubin was 0.75 at the
beginning and after study it was 0.7 mg%. Direct and indirect bilirubin, total protein,
albumin and globulin at week 0 were found to be 0.36, 0.38, 7.02, 3.52 and 3.49
respectively and at week 4, it was 0.36, 0.34, 7.06, 3.50 and 3.56 respectively. Liver
enzymes were also estimated at week 0 and week 4. Before study SGPT, SGOT and
alkalinephosphatase were 27.47, 25.76 and 164.3 u/l and after 30 days supplementation it
was found to be 27.7, 28.07 and 171.7 u/l respectively.
Pyrrolizidine alkaloids are hepatotoxin compound which are present in some species of
Echium (Mehrabani et al., 2006). The total alkaloid content of dried petals of E.
amoenum was reported to be 0.01%. In petals of this plant, structures of four
pyrrolizidine alkaloids namely: echimidine I, echimidine isomer II, 7-angeloyl
retronecine III and 7-tigloyl retronecine IV were identified (Mehrabani et al., 2006).
In the other study conducted by Mehrabani et al (2007), three doses of 40 mg/kg,
400mg/kg and 800mg/kg of the dried extract of decoct of E. amoenum (according to the
consumed doses by human) were administrated by oral gavages for duration of 28 days in
157
rats. Water as solvent was given to the control group. Each group contained five female
and five male rats. On the 29th day serum samples were collected for liver function tests
(AST, ALT, total bilirubin and alkaline phosphates) and liver specimens were isolated for
histopathologic study. They reported that difference between experimental and control
groups in all tests were not significant (P>0.05) and histopathologic studies of livers
showed no evidence of hepatotoxicity.
In the present study, at lower dose (500 mg/day) of dried petals of Echium amoenum we
did not observe any significant changes in any liver function test.
Table 34: Effect of borage supplementation on Liver function test of diabetic

patients before and after the study
Blood Parameters
Bilirubin Total (mg%)
Direct (mg%)
Indirect (mg%)
Total Protein (g%)
Albumin (g%)
Globulin (g%)
A/G Ratio
SGOT (u/l)
SGPT (u/l)
Alkaline Phosphatase
(u/l)
Intervention group
Before
After
0.75
0.70
0.36
0.36
0.38
0.34
7.02
7.06
3.52
3.50
3.49
3.56
1.01
0.99
25.76
28.07
27.47
27.7
164.3
171.7
Control group
Before
After
0.76
0.65
0.36
0.3
0.403
0.301
7.15
7.17
3.38
3.5
3.77
3.66
0.89
0.96
24.1
21.5
27.3
18.5
180.2
154.6
4.1.8. Renal profile

Renal function test including urea, creatinine and uric acid were evaluated at the
beginning and at the end of the intervention. As shown in Table 35, urea was 26.12 and
30.55 mg% in study group and control group and after supplementation it was 26.05 and
28.98 mg% respectively. Creatinine and uric acid were also in similar ranges and did not
differ significantly before and after supplementation either in control or experimental
group. (study group: 0.91 and 4.47 in the beginning and 0.88 and 4.52 mg % at the end.
Control group: 0.97 and 5.65 before the study and 0.97 and 5.30 mg% after the fourth
158
week). When data was analyzed statistically, there was no significant difference in renal
profile of study group as well as control group before and after the study.
Zahedi et al (2004) studied the effect of Echium Amoenum on the renal function tests in
rats. They administrated the percolated extract of this plant with doses of 100 and 200
mg/kg to 5 groups of rats via orogastric tube for 7 days. It was reported that renal
function tests including Blood Urea Nitrogen and creatinine did not change significantly
after oral administration of extract.
Table 35: Effect of borage supplementation on renal profile of diabetic patients

before and after the study (mg%)
Renal
profile
Urea
Creatinine
Uric acid
Study group
Before
After
26.12
26.05
0.91
0.88
4.47
4.52
Control group
Before
After
30.55
28.98
0.97
0.97
5.65
5.30
In present study it was observed that petals of borage showed antioxidant potential and
significantly reduced lipid peroxidation in diabetic patients. It was also observed that it
reduced serum total cholesterol, triglyceride, LDL and VLDL. Liver function test and
renal test showed that 500 mg borage per day for 30 days did not affect any parameter.
4.2. EFFECT OF SHALLOT SUPPLEMENTATION ON ANTIOXIDANT

STATUS OF CIGARETTE SMOKERS
Cigarette smoke, as a pollutant, has been established to include a variety of xenobiotics,
some of which are known to be oxidant or free radicals that can directly and indirectly
initiate and propagate the process of lipid peroxidation (Hoshino et al., 1990).
The enhanced susceptibility of erythrocytes of smokers to peroxidation may reflect the
lower activities of glucose-6-phosphate dehydrogenase and glutathione peroxidase.
Decreased activity of glucose-6-phosphate dehydrogenase can be caused by extracellular
or intracellular lipid hydroperoxides (Khandwala and Gee, 1973). Differences in
159
glutathione peroxidase activity between smokers and nonsmokers have been reported
previously and may be associated with decreased selenium status (Ellis et al., 1984).
Thus, it is believed that smokers encounter a sustained free radical load. It has been
shown that cigarette smoking caused an increase in blood and serum malonaldehyde
content. Many studies have shown an increased lipid peroxidation due to cigarette
smoking. Allium families are reported with high antioxidant potential. In this study to
evaluate antioxidant potential of shallot in cigarette smokers, 40 healthy subjects were
selected for study. Subjects, who smoke minimum 5 cigarettes per day since last 3 years
in the age group between 25-40 years, were included for the study. Subjects were
assigned to 2 groups of 20 each (study group and control group). Control group received
placebo (rice flour) whereas study group received 500 mg shallot capsule twice a day.
Antioxidant activity, lipid peroxidation and total thiol was measured in serum of subjects
before and after supplementation and presented in Figure 56-58.
4.2.1. Total thiol

As illustrated in Figure 56, total thiol protein of smokers increased significantly
after 30 days of shallot supplementation. Subjects were examined for serum total
thiol before supplementation and it was found to be 0.146 mol/ml in study group
and 0.157 mol/ml in placebo group. When shallot capsules were given to study
group and placebo to control group, it was observed that total thiol was 0.243 and
0.158 mol/ml in study and control group respectively. When data were analyzed
statistically, a significant difference was observed in study group before and after
the study (P= 0.000) but difference was not significant (P= 0.439) in control group
which had received placebo capsules. Negative correlation was observed between
No. of cigarettes per day and serum total thiol, indicating that when the number of
cigarettes smoked per day increases, serum total thiol decreases. It can be due to the
increase in oxidation of thiol molecule.
160
Figure 56: Effect of 30 days shallot supplementation on serum total thiol molecule of
cigarette smokers (mol/ml)
0.300
0.250
mol/ml
0.200
Before
0.150
After
0.100
0.050
0.000
Study group
Placebo
Shallot (Allium ascalonicum L.), belonging to the Family Alliaceae, is one of the
promising plants which demonstrates significant antioxidant as well as anti-inflammatory
properties, useful in the protection of various diseases such as respiratory and nervous
diseases. The important activity compounds in Alliaceae family such as onion, garlic or
shallot are different but they mainly contain total phenolic compound that have the OH
group (Cao et al., 1997). In addition, most of phenolic compounds such as furostane
saponins and high level of quercetin, isorhametin and other glycosides are present in
shallot (Fattorusso et al., 2002).
The major cause of chronic oxidative stress in humans is exposure to free radicals in
cigarette smoke. Cigarette smoke free radicals are considered an important cause of
atherosclerosis and cancer (Pryor, 1997; Pittilo, 2000). Thiols are powerful reducing
agents that are capable of acting as antioxidants in vivo. Thiols exist in three forms: freethiol and two types of disulfides, namely, homodisulfides and heterodisulfides. Several
aminothiols, e.g., cysteine, homocysteine (Hcy), and GSH, and disulfides (e.g., cystine,
homocystine, and oxidized glutathione), interact by means of redox and disulfide
exchange (Iciek et al., 2004). This dynamic system (with respect to thiol status) is
important for normal physiologic function (Morris, 2002). Changes in the redox thiol
161
status lead to the induction of oxidative stress and apoptosis. As both an intracellular and
extracellular redox buffer, t-SH plays important roles in the in vivo prevention of
atherosclerosis (Ueland et al., 1996). t-SH plays a prominent role in antioxidant reactions,
and in catalysis, regulation, and electron-transport reactions, and in reactions that
preserve the correct structure of proteins. Mixed disulfides with proteins are formed by
reaction of S-thiolation, in which protein thiols conjugate with non-protein thiols (Klatt
and Lamas, 2000). This process plays a regulatory and an antioxidant role, since it
protects protein SH groups against irreversible oxidation from SO2H and SO3H;
moreover, it participates in signal transduction (Padgett and Whorton, 1998).
Maintaining the intracellular thiols, such as GSH, in their reduced form, may allow for
the maintenance of plasma homocysteine and other intracellular thiols in redox states
(Moriarty et al., 2003). Since the plasma GSH concentration reflects its levels in various
tissues, a reduced plasma concentration of GSH may be a diagnostic indicator of a
pathological state (Ashfaq et al., 2006).
GSH is the most important endogenous antioxidant in humans. It is often accompanying
by other endogenous thiols, such as cysteine, cysteinylglycine and even Hcy (in low
concentration). These thiols scavenge ROS and are involved in preserving the prooxidantantioxidant balance in human tissues (Zinellu et al., 2006).
GSH is an abundant tripeptide that protects against oxidative stress and damage in nearly
all cells and tissues (Via, 1990; Cotgreave and Gerdes, 1998). It is the major
intracellular antioxidant and functions by scavenging free radicals, detoxifying lipid
peroxides via glutathione peroxidase, and conjugating reactive electrophilic toxicants and
carcinogens. In addition, GSH is involved in numerous other cellular pathways including
protein and DNA synthesis, DNA repair, and immune surveillance. GSH is oxidized to its
disulfide form (GSSG), but is subsequently reduced back to GSH by GSH reductase. An
alternative pathway for GSSG metabolism is protein glutathiolation (also referred to as
glutathionylation) where thioldisulfide exchange occurs with cysteine (Cys) residues in
proteins to form GSSP. The formation of GSSP within cells can be substantial and reach
200 M in certain tissues (Kleinman et al., 2003). There is considerable evidence that
glutathiolation represents an important redox-sensitive regulator of cellular activities
(Sies et al., 1987; Klatt and Lamas, 2000). An induction of GSH synthesis may
162
accompany a smoking-related increase in GSH utilization through oxidation to GSSG

and GSSP, resulting in increases in both GSH and GSSP. This would suggest that GSSP:
GSH ratios are less sensitive to oxidative stress than GSSP alone. The amount of protein
that is glutathiolated in the blood of smokers is reported to be high (ranging from 0.05 to
0.38 mmol/l), 34 to 43% higher than those observed in nonsmokers. A doseresponse
relationship is reported to be apparent between GSSP levels and tobacco smoke
measurements such as cigarettes smoked per day, blood cotinine (an alkaloid found in
tobacco and also a metabolite of nicotine) and blood thiocyanate. A similar but smaller
increase in plasma GSSP levels was also found in smokers compared with nonsmokers
(Muscat et al., 2004). These findings provide compelling evidence that blood GSSP is an
indicator of oxidative stress, and those abundant free radicals in cigarette smoke cause
increases in blood GSSP concentrations. As shown in Figure 57 interestingly a negative
correlation was observed between total thiol and No. of cigarette smoked.
Figure 57: Correlation between total thiol and no of cigarette
No. of cigarrete
25
20
15
10
5
0
0
0.05
0.1
0.15
0.2
0.25
0.3
Total thiol
4.2.2. Serum lipid peroxidation

As explained earlier, smoking also produces xenobiotics and toxic compounds in the
body. Many researchers have also reported lower serum vitamin C content in smokers
compared with nonsmokers. Thus lipid peroxidation increases in smokers.
Serum lipid peroxidation was found to be 4.9 and 4.79 at the beginning of the study in
study and control group respectively. After 30 days supplementation, estimation was
repeated again and found to be 4.3 and 4.71 in study and control group respectively.
Statistical analysis showed a significant reduction in the serum lipid peroxidation level of
163
subject after shallot supplementation (P= 0.000), whereas in control, difference was not
significant (P= 0.05). A negative correlation was observed between total thiol and lipid
peroxidation, which shows that, when oxidative stress is more the total thiol molecule
reduces due to the oxidation (R2 = -0.369). Also a positive correlation (R2 = 0.267) was
observed between serum lipid peroxidation and number of cigarettes.
Figure 58: Effect of 30 days shallot supplementation on serum lipid peroxidation of
cigarette smokers (nmol/ml)
5
nmol/ml
4.8
4.6
Before
After
4.4
4.2
4
Study group
Placebo
Figure 59: Correlation between serum

lipid peroxidation and No. of cigarette
per day
Figure 60: Correlation between

serum lipid peroxidation and total
thiol
0.3
0.25
5
Total thiol
lipid peroxidation level
4
3
2
0.2
0.15
0.1
0.05
0
0
10
20
No. of cigarrette
30
Lipid peroxidation level
164
Liu and Wei (1999) reported that, the level of total blood glutathione is negatively
correlated with the level of plasma lipid peroxides (r=0.305,P=0.002) and was
positively correlated with the smoking index (r=0.307, P=0.019) of all the study subjects.
These results indicate that the activities of glutathione peroxidase and glutathioneStransferase reduced to a great extent under smoking-mediated oxidative stress in the
blood of both young and aging smokers. Moreover, the compensatory generation of total
blood glutathione may effectively prevent plasma lipids from peroxidation in young
smokers, although the activities of glutathione peroxidase and glutathioneS-transferase in
plasma were decreased. By contrast, total blood glutathione was inadequate for such
protection in the aging smokers. We suggest that supplementation of thiol-group-related
agents may be considered for the prevention or alleviation of oxidative stress in aging
smokers, whose capability and capacity for the disposal of smoking-mediated free
radicals and reactive oxygen species are compromised
Leelarungrayub et al (2004) evaluated the antioxidant potential of Thai shallot. It is
reported that Thai shallot protect human erythrocytes from possible damage from
external or internal radicals such as H2O2 or peroxyl radical such as 2,2-Azobis (2amidino-propane) dihydrochloride. They reported that thai shallot is able to inhibit lipid
peroxidation and glutathione depletion in erythrocytes and suggested that the Thai shallot
extracts have protective effect on the GSH deterioration in vitro from protein
hydroperoxide (PrOOH) or hydroxyl radical from gamma irradiation (Leelarungrauyub et
al., 2004). Thai shallot extracts can also protect and scavenge the protein and lipid
hydroperoxide (LOOH) formation in vitro study (Leelarungrayub et al., 2004).
4.2.3. Total antioxidant by FRAP assay

It has been reported that the smokers, because of their increased oxidative stress, had
12% lower FRAP than did the nonsmokers. These data suggest that smokers have less
plasma antioxidant potential, which would be consistent with their greater plasma
isoprostane concentrations (Bruno et al., 2005). Plasma uric acid is the greatest predictor
of FRAP and accounts for 60% of the total predicted FRAP, whereas ascorbic acid
contributes to 15% of the value (Benzie and Strain, 1996).
165
Results revealed that significant increase was there in experimental group whereas
changes in control group were not significant. Slightly positive correlation was observed
between serum total antioxidant capacity and total thiol molecule. A negative correlation
was seen between serum total antioxidant capacity and No. of cigarettes smoked per day,
whereas correlation between serum total antioxidant capacity and lipid peroxidation level
and between TAC and total thiol molecule was found to be positive.
Figure 61: Effect of 30 days shallot supplementation on serum total antioxidant
capacity of smokers (mlo/ml)
690
mol/ml
680
670
Before
After
660
650
640
Study group
Placebo
690
680
670
660

serum total antioxidant capacity
and No. of cigarettes per day
25
No. of cigarettes
TAC

serum total antioxidant capacity and
total thiol
650
640
630
620
20
15
10
5
0
0.1
0.2
Total thiol
0.3
620
640
660
680
700
TAC
166
Fifure 64: Correlation between serum total antioxidant capacity and lipid
peroxidation level
Lipid peroxidation level
6
5
4
3
2
1
0
620
640
660
680
700
TAC
4.2.4. Food intake

Food intake was evaluated using 24 hours food recall in both control and study group
before and after the study. It was observed that, except protein, iron, and vitamin A all
evaluated nutrients were lesser that RDA. It is well known that vitamin C requirement is
more in cigarette smokers, however, consumption was lesser than recommendations for
healthy adult male. This can cause more oxidative stress and more lipid peroxidation.
Table 36: Average nutrient intake of cigarette smokers before and after shallot
supplementation
Experimental group
Control group
Macronutrient
RDA
Before
After
Before
After
Energy (kcal)
2530
2670
2715
2600
2800
Protein (g)
66.09
70.5
68.4
72.2
70
Fat (g)
27
31
32
30
28
Fiber (g)
24
22
25
26
38*
Calcium (mg)
675
712
650
669
1000
Iron (mg)
17
16
14
13
8
vitamin A (g)
893
910
800
887
900
Thiamin (mg)
0.9
1.0
1.1
0.97
1.2
Riboflavin (mg)
1.07
1.01
0.99
1.03
1.3
Niacin (mg)
14.1
13.6
14.3
14.0
16
Vitamin C (mg)
46
52
48
54
90
RDA: recommended daily allowance for American.
167
It can be stated that smoking introduces lipid peroxidation in human body. Daily shallot
supplementation exhibited significant improvement in serum total antioxidant activity. It
was observed that it reduced lipid peroxidation and increased total thiol significantly.
5. NUTRITIONAL AND MEDICINAL PROPERTIES OF SAMPLES

5.1. INFLUENCE OF PETALS OF BORAGE ON EIGHT DIFFERENT MOOD
STATES IN ADULT HUMAN SUBJECTS
World Health Organization includes the neurological and psychiatric disorders as an
important and growing cause of morbidity (WHO, 2001). Keshari et al (2011) reported
that beyond 25% of people are affected by mental and behavioral diseases at some point
during their lives.
Traditionally decoction of petals of borage is used in Iran to reduce anxiety, stress,
depression and as a mood enhancer. Anxiolytic effect of borage has been studied on mice
by authors (Rabbani et al., 2004; Gholamzadeh et al., 2009). Since mental and behavioral
disorders are common and can affect on individual and his family life, and lack of studies
on anxiolytic effect of borage on human, present study was planned to investigate the
effect of borage on different mood state in human subject.
Fifty subjects aged 25-35 years were asked to answer questionnaire at day 0 and 30. All
subjects were given one borage capsule containing borage petal (500 mg) per day for 30
days. Subjects with at least 5 mood states in high score were considered as experimental
group and subject with at least 5 stated in average score, were considered in control
group. Raw data were converted to sten score as given in the manual of eight state
questionnaire (8SQ). Score of 1-4 were described as low, 5-6 average and 7-10 high sten
score. The results are presented in Table 37- 38 and Figure 65A and B.
5.1.1. Stress and anxiety
As per manual, higher sten score for anxiety is described by worried, easily rattled tense,
emotionally upset, easily angered, high strung, easily annoyed mood states.
Experimental group showed a sten score of 7.55 for anxiety at the day 0. The score was
reduced to 6.75 at day 30. Statistical analysis showed highly significant difference
168
between score of day 0 and 30 (P=0.000). In control group, score of 5.47 was slightly
reduced to 5.24 at day 30. Reduction was found to be marginally significant in control
group (P=0.019). In study group, score of stress was found to be 7.22 at the beginning of
the study. Sten score was reduced to 6.24 after 30 days supplementation, whereas in
control group the score were 5.16 and 4.75 before and after the intervention. When scores
were statistically analyzed, highly significant difference was observed in the score of
before and after intervention in both experimental and control group (P=0.000).
Stress and anxiety are common psychiatric symptoms of the modern lifestyles. In small
quantities, stress and anxiety are beneficial; they can motivate and help one be more
productive. But, intensive stress, or a strong reaction to stress, is dangerous. It can cause
general poor health in addition to specific physical or psychological illnesses like
infection, heart disease, or depression. Persistent and insistent stress often initiates
anxiety and unhealthy behaviors like over eating.
Anxiety is known as Central Nervous System disorder (Weinberger, 2001; Kjernisted and
Bleau, 2004). Clment et al (2002) stated that anxiety is a common emotional incidence
in humans. Anxiety is an emotional state, unpleasant in nature and is related with
uneasiness, worry and concern or fear about some defined or undefined future threat
(Gupta et al., 2010). It is a normal reaction to stress and is distinguished by heart
palpitations, fatigue, nausea and shortness of breath.
In physiological point of view, anxiety is caused by unbalanced neurotransmitters in the
brain. Brain synthesizes numerous neurotransmitters like acetylcholine, adrenaline,
dopamine, endorphins, serotonin, gamma amino butyric acid, glutamate etc. Most
information has come from studying the action of anxiety-reducing or anxiolytic drugs.
The outcome of studies suggest that anxiety can be attributed dysfunction of one or more
neurotransmitters and their receptors (Khanum and Razack, 2010). The anxiolytic effect
of Echium amoenum was reported by (Shafaghi et al., 2002). They investigated the
putative activity of hydroalcoholic and aqueous infusion extracts of Echium amoenum L.
in mice with using two methods (the rotarod model of motor coordination and the
elevated plus maze model of anxiety). Once, one hour before performing the tests the
extracts were administered intraperitonealy. They reported that the hydro alcoholic
extract of Echium amoenum in the dose of 125, 250 and 500 mg/kg did not show
169
significant effect on motor coordination whereas the aqueous extract at dose of 62.5, 125,
250 and 500 mg/kg interrupted motor coordination significantly. Also it is reported that at
the dose of 5, 10, 20, 30, 62.5, 80 and 125 mg/kg, intraperitoneal injection of aqueous
extract demonstrated a significant dose-dependent increase in time spent in open arm
whereas significant change was not observed in open arm entries, closed arm entries and
total arm entries. It was observed that in 125 mg/kg group the anxiolytic effect was most
evident. It is almost manifest that the extract produces its anxiolytic effect in the doses in
which no change in motor activity is apparent. The effect was compared with
conventional anxiety medicine (diazepam) at the dose of 0.25, 0.5, 1.0 and 2.0 mg/kg in
the same setting. It was reported that the maximal efficiency of the extract is significantly
lower than diazepam. They concluded that single administration of aqueous extract of
Echium amoenum L. shows a significant but mild to moderate anxiolytic effect in mice.
In the other study, Rabbani et al (2004) showed the ethanolic extract of Echium amoenum
flowers at the dose of 50 mg/kg can increase the percentage of time-spent and the
percentage of arm entries in the open arms of the elevated plus-maze and decreased the
percentage of time-spent in the closed arms of elevated plus-maze. Also they reported
that Echium amoenum prolonged the ketamine-induced latency to sleep but did not show
significant effects on total sleeping time induced by ketamine. In these animals, the
locomotor activity was affected but not to the same extent as seen for diazepam. Like
other study, they also suggested that the extract of E. amoenum seems to possess
anxiolytic effect but the effect is lesser than diazepam.
5.1.2. Depression
The score of depression were found to reduce in experimental group from 6.71 to 6.45
whereas very small difference was observed in control group (5.1 to 5.0). The statistical
analysis showed a significant reduction in experimental group (P=0.000) but control
group did not show any change (P=0.149).
Sayyah and Kamalinejad (2006) reported the effect of E. amoenum on depression. They
randomly allocated 35 patients to receive daily either placebo or 375 mg of E. amoenum
aqueous extract in a 6-week double blind, parallel-group trial. They assessed patients for
170
5 times during the study (weeks 0, 1, 2, 4 and 6) by the Hamilton Rating Scale for
depression, the Hamilton Rating Scale for Anxiety and a score sheet on adverse effects. It
was reported that in week 4, the extract showed a significant reduction in depressive
symptoms than placebo. The effect on anxiety was not significant. They also reported
side effects of headache, somnolence, vomiting, dry mouth, constipation and blurred
vision at the dose of 375 mg of E. amoenum aqueous extract in some studies. In the
present study (at the dose of 500 mg dry petals) such side effects were not reported.
Difference may be due to the form in which the herb is used as we have used dry petals,
whereas in their study 375 mg of aqueous extract was used.
5.1.3. Regression
Score of regression initially was 6.43 which reduced to 6.22 after treatment. In control
group it was found to reduce from 5.68 to 5.52.
5.1.4. Fatigue
Score of fatigue reduced from 5.37 to 5.10 in experimental group, whereas in control
group the score was almost similar (5.45 to 5.43). Statistical analysis showed marginally
significant changes in experimental group (P=0.011) but difference were not significant
in control group (P=0.500). As per manual, higher score of fatigue, indicates exhausted,
no energy, sluggish, tired, needing rest, weary, below par in performance state of mood.
Sten score of guilt reduced from 6.88 to 6.20 in experimental group, whereas in control
group, score remained same (5.51). T-test showed highly significant changes in
experimental group (P=0.000).
5.1.5. Guilt
Results showed that score of guilt was significantly reduced in experimental group from
6.88 to 6.2 (P=0.000). In control group, the score were in the average range and did not
change significantly after 30 days supplementation.
5.1.6. Extraversion
As explained in manual of 8SQ, higher score of extraversion is characterized by sociable
outgoing, adventurous, talkative and enthusiastic. As illustrated in figure 21 B, score of
171
extraversion increased from 3.91 to 4.68 in experimental group and from 5.25 to 5.45 in
control group. P value of 0.000 shows highly significant changes in experimental group
whereas control group showed marginally significant difference in score of extraversion
before and after the study.
Figure 65: Effect of borage supplementation on mood states of human subjects
A: Anxiety, stress, depression, regression

8
7.55
6.71 6.45
6.24
5.47 5.24
6
Sten Score
7.22
6.75
5.16
6.43
6.22
5.10 5.00
4.75
5.68 5.52
Before
After
3
2
1
0
B: Fatigue, Guilt, Extraversion, Arousal
6.88
7
Sten Score
6
5
6.20
5.37
5.10
5.45 5.43
5.51 5.51
5.25 5.45
4.68
3.91
5.31 5.25
4.33
4.73
3
2
1
Before
After
E: Experimental group, C: Control group
172
5.1.7. Arousal
Score of arousal improved from 4.33 to 4.73 in experimental group and in control group
sten score was found to be very close to each other (5.31 to 5.25). In experimental group
score of arousal were found to differ significantly after 30 days supplementation
(P=0.001) but significant difference was not observed in control group (P=0.284). Higher
score of arousal were reported in manual as, alert, keyed up, excited, stimulated, keen and
with sharp senses.
5.1.8. Frequency distribution

In experimental group level of anxiety were improved. Before supplementation, none of
the subjects had average score and all subjects had score of high (7 and 8) whereas after
borage supplementation, 20 subjects gained score of average (6) and number of subjects
with score of 8, were drastically reduced from 27 subjects to 7 subjects. Level of stress
also was reduced after interventions. After study, No. of subjects with score of 8 were
reduced from 17 to 0. Score of stress also were to reduce among subjects. As shown in
Table 37 and 38, before study, none of subjects were in the score of 5, whereas after
study, 3 subjects got score of 5 and subject with score of 8 were reduced from 3 to 0 at
the end of the study. At day zero 17 and 30 subjects had score of 6 and 7 respectively
whereas at day 30, it was observed to be 7, 26 and 17 subjects in the score of 5,6 and 7
respectively.
After intervention, 8, 29 and 13 subjects gained score of 4, 5 and 6 in fatigue, whereas
before intervention it was found to be 6, 18 and 26 subjects respectively.
Score of guilt also improved after supplementation. It was observed that 12, 32 and 6
subjects got the score of 6, 7 and 8, whereas after the study, no subject was in the score of
8 and found to be 2, 36 and 12 subjects in the score of 5, 6 and 7 respectively. There were
not much changes in extraversion and arousal, before and after the study.
173
Table 37: Frequency distribution of subjects in experimental group in different

mood state before and after borage supplementation
Before
After
Sten Score
Low 3
Low 4
Average 5
Average 6
High 7
High 8
Low 3
Low 4
Average 5
Average 6
High 7
High 8
Ax
23
27
20
23
7
St
5
28
17
10
18
22
-
De
17
30
3
3
21
26
-
Rg
28
22
7
26
17
-
Fa
6
18
26
8
29
13
-
Gi
12
32
6
2
36
12
-
Ex
13
28
9
16
34
-
Ar
15
12
15
8
19
26
5
-
Ax: anxiety, St: stress, De: Depression, Rg: regression, Fa: fatigue, Gi: guilty, Ex:
extraversion, Ar: arousal
Table 38: Frequency distribution of subjects in control group in different mood
state before and after borage supplementation
Before
After
Sten Score
Low 3
Low 4
Average 5
Average 6
High 7
High 8
Low 3
Low 4
Average 5
Average 6
High 7
High 8
Ax
1
25
24
3
32
15
-
St
9
24
17
15
33
2
-
De
8
29
13
4
42
4
-
Rg
1
19
25
5
28
18
4
-
Fa
28
22
29
21
-
Gi
25
25
24
26
-
Ex
4
29
17
27
23
-
Ar
34
16
2
33
15
-
Ax: anxiety, St: stress, De: Depression, Rg: regression, Fa: fatigue, Gi: guilty, Ex:
extraversion, Ar: arousal
It was observed that, borage supplementation improved eight mood states significantly.
Improvement was more significant in subjects who had higher score of stress than
subjects with normal level. The present study proves the traditional belief regarding the
role of borage in improving mood states.
174
5.2. EFFECT OF VALERIAN AND GINGER SUPPLEMENTATION ON

ANIMAL WEIGHT GAIN AND FOOD INTAKE
Traditionally in India ginger is used to facilitate food digestion. In China ginger is used to
reduce weight. On the other hand, valerian is used as sleep aid medicine which is known
to increase release of GABA (gamma aminobutyric acid) and inhibit enzyme induced
breakdown of GABA (Cavadas et al., 1995; Ortiz et al., 1999; Imbalance, 2009) and
GABA has direct relationship with serotonin which by its secretion inhibits appetite and
food intake (Blundell, 1992). To study the effect of valerian and ginger on weight gain,
food intake and blood parameters, 40 adult wistar rat were selected and divided to 5
group of 8. Corn starch, 3 mg ginger, 6 mg ginger, 3.0 mg valerian and 6.0 mg valerian
was administered to control and experimental group respectively. Food intake and weight
gain was measured and at the end of the study, animals were sacrificed and blood and 4
vital organs were collected for further analysis.
5.2.1. Body weight

The mean body weights of the animals were similar in all 5 experimental groups at the
beginning of the study (192.1-193.1 g), since animals were arranged in groups with
random design (Figure 66). After 15 days, there were differences in weight of animals.
Control group gained weight (213.2 g) whereas ginger group showed weight reduction in
both level of ginger (184.2 and172.37 g respectively) and valerian group gained more
weight than control. After 30 days of treatment, the weights of animals were taken and
reported as 219.4 g for control group, 176.5 and 157.1 g in 3.0 mg and 6.0 mg ginger
group respectively and 239.1 and 246.2 g in 3.0 mg and 6.0 mg valerian group
respectively. Statistically, there was significant weight reduction in both level of ginger
group compared to control group whereas in group 3 and 4 there was a significant weight
gain after 30 days of treatment with valerian but difference between 3.0 mg and 6.0 mg
valerian was not significant. The results of other studies support this study data. In a
study on plant foodstuffs with anti-obese activity on adipocytes 3T3-L1 cells, ginger
showed inhibition in adipogeneisis of 3T3-L1 cells (Niwano et al., 2009). In other study,
175
ethanolic extract of ginger (100,200 and 400 mg/kg body weight) was given to rats along
with high fat diet. It was reported that feeding ethanolic extract of ginger can
significantly suppress the rat body weight gain (Nammi et al., 2009). Han et al, (2005) in
an in vitro study reported that, an aqueous extract of Z. officinale Roscoe could inhibit the
hydrolysis of triolein emulsified with phosphatidylcholine by pancreatic lipase and it
reduced the elevation of rat plasma triglyceride one and two hours after oral
administration of a lipid emulsion containing corn oil. Also they observed that feeding Z.
officinale significantly reduces mice weight and final parametrical adipose tissue weights.
They suggested aqueous extract of Z. officinale Roscoe might inhibit the intestinal
absorption of dietary fat by inhibiting its hydrolysis by the active compounds of Z.
officinale Roscoe. David et al (2007) studied the effect of a Chinese herbal extract- 7% Z.
officinale- on weight gain of the rat and found a significant weight reduction when
animals received 1.5 and 0.75 g dose/day. They found a significant reduction in leptin
hormone in both treatment groups (27.5% to 46.2) vs. control group and reduction of
parametrical fat was observed at both dose of treatment (14.1% and 55.5%).
Figure 66: Effect of ginger & valerian supplementation on body weight gain of adult
Wistar rat before and after the study (g)
300
250
200
Weight (g)
A
A-B
B
C
Day 1
150
Day 15
100
Day 30
50
0
control
G3
G6
V3
V6
Weight gain
G3, 3.0 mg ginger; G6, 6.0 mg ginger; V3, 3.0 mg valerian; V6, 6.0 mg valerian.
Significant differences between samples on application of post test (Tukeys) indicated by
different alphabets.
176
To the group 4 and 5, 3 and 6 mg of dry valerian was given for 30 days and significant
weight gain was seen in both groups. Also animals were found to be more sleepy than
control group. This may be one more reason for animal weight gain.
5.2.2. Food intake

The animals were fed on regular commercial standardized food during the study ad
libitum. Food intake of animals was measured for the period of 30 days and as shown in
Figure 14, control group consumed 598 g and group 2 and 3, 597 and 595 g of food
respectively. Statistically there were no significant difference in food intake in control
and ginger group. It can be concluded that ginger may not affect appetite of animal but as
Han et al (2005) suggested it might inhibit the intestinal absorption of dietary fat. On the
other hand in group 4 and 5, food intake significantly increased so weight gain may be
due to effect of valerian on rats appetite.
Figure 67: Effect of ginger and valerian supplementation on Food intake of adult
Wistar rat in duration of one month
620
Intake (g)
610
600
590
580
control
G3
G6
V3
V6
G3: 3.0 mg ginger, G6: 6.0mg ginger, V3: 3.0 mg valerian, V6:6.0 mg valerian
177
5.2.3. Feed Efficiency

The, feed efficiency is expressed as weight gain (g) divided by weight of food consumed
(Figure 68). The feed efficiency of the valerian group (4 & 5), was higher than control
and both level of ginger groups. Statistical analysis did not show significant difference
between control and both level valerian groups. Feed efficiency of the group 4 and 5 were
5.7% and 7.3% higher than control group and in group 2 and 3 were 18.2% and 28%
lower than that of the control group respectively. R2 values for each group were: Control,
0.346; 3 mg valerian, 0.780; 6 mg valerian, 0.348; 3 mg ginger, 0.052 and 6 mg ginger, 0.441. Correlation is shown in Figure 69.
Figure 68: Effect of ginger and valerian supplementation on Feed efficiency in adult
Wistar rat duration of one month
0.5
Feed efficiency
0.4
0.3
0.2
0.1
0
control
G3
G6
V3
V6
Treatment group
G3: 3.0 mg ginger, G6: 6.0mg ginger, V3: 3.0 mg valerian, V6:6.0 mg valerian
178
Figure 69: Correlation between weight gain and food intake of Wistar rat treated
with ginger and valerian
Total food intake (g)
Control
800
600
400
200
0
195
200
205
210
215
220
225
230
235
Changes in body weight (g)
3 mg valerian
6 mg valerian
800
800
600
600
400
400
200
200
0
220
230
240
250
260
230
3mg Ginger
800
600
600
400
400
200
200
0
170
180
250
260
270
6 mg Ginger
800
160
240
190
200
50
100
150
200
179
5.2.4. Organ weight

After the study, weight of four vital organs (spleen, pancreas, liver and kidney) was
measured and values are presented in Figure 70. Liver weight was found to be 7.5, 7.3,
8.3, 7.3 and 7.7 g in control, group 2, 3, 4 and 5 respectively.
Weight of spleen was found to be in the range of 0.42 to 0.48 g, pancreas 1.002-1.22 g
and kidneys, 1.65- 1.76 g. When statistically analyzed, there was no significant difference
between control group and other groups, in any organ weight.
Figure 70: Effect of one month ginger and valerian supplementation on organ
weight of adult Wistar rat
10
8
Control
G3
G6
V3
V6
0
Liver
Spleen
Pancreas
Kidney
Control: A, 3.0 mg ginger: B, 6.0 mg ginger: C, 3.0 mg valerian: D, 6.0 mg valerian: E.
5.2.5. Blood chemistry

a. Blood electrolytes
Blood electrolytes were analyzed at the end of the study. Sodium level of females in
control group was 141.11 (mEq/l) and in all other groups was in the range of 140.58 to
142.33 (mEq/l) and control group males had 140.71(mEq/l) sodium in blood and in other
groups it varied between 140.9- 145.1 (mEq/l). Blood potassium of female and male of
control group was 6.97 and 5.10 (mEq/l), respectively and in other groups, it was in the
180
range of 6.0 7.2 and 5.3- 6.1(mEq/l) respectively. Blood chloride in female and male in
control group was 104.4 and 101.9 (mEq/l) respectively and in other groups, it was in the
range of 104.4-105.1 and 101.7- 103.0 (mEq/l) respectively. Statistically there were no
significant differences between the study group and control group in 3 blood electrolytes
analyzed.
Table 39: Effect of ginger and valerian supplementation for one month on blood
electrolytes of adult Wistar rat (mEq/l)
Electrolytes
Control
Ginger
(3.0 mg)
Ginger
(6.0 mg)
Valerian
(3.0 mg)
Valerian
(6.0 mg)
Sodium
Female
Male
141.11
140.71
2.41
4.10
142.33
140.90
2.56
1.50
142.33
145.10
2.80
0.99
140.58
143.60
3.50
3.30
140.76
142.23
1.56
1.89
Potassium
Female
Male
6.97
5.10
2.59
0.28
6.80
5.96
3.14
0.60
6.97
5.30
1.33
0.30
6.00
5.90
1.29
0.26
7.20
6.10
2.37
0.35
Chloride
Female
Male
104.40
101.95
1.80
3.60
105.12
101.76
2.70
2.26
104.67
102.50
0.87
0.92
104.80
103.03
0.86
0.95
104.45
102.50
0.66
2.18
b. Complete Blood Count

Complete blood count was determined at the end of study with automated analyzer set for
animal blood count. WBC of female and male in control group was found to be 5.6 and
7.4 respectively and in other groups was in the range of 5.5-9.0 and 6.0-11.9 respectively.
RBC value in control group was 6.98 and 8.7 in female and male respectively and in
study groups it was in the range of 7.3-8.0 and 7.5 -8.9106 l respectively. Hemoglobin
was found to be 13.06 and 13.65 in control group female and male respectively and in
other study groups it was in the range of 13.7-14.8 and 15.7-16.4 g/dl respectively.
Haematocrit values (%) were 39.8 and 50.8 in control group female and male
respectively and in study groups were in the range of 42.5-46.7 and 50.1-52.1%
respectively. All analyzed blood cells were in the normal range.
181
Table 40: Complete blood count of animals after 30 days treatment

CBC
WBC 10
Neutrophils %
Lymphocytes
%
Monocytes %
Eosinophils %
Basophils %
RBC 106/l
Hb g/dl
Hematocrit %
MCV fL
MCH pq
MCHC %
RCDW fL
Platelet 10/
L
PDW
MPV
Sex
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
Control
5.60.98
7.451.20
14.200.72
16.10.42
81.51.3
79.251.4
2.861.80
2.751.06
1.000.50
1.051.00
0.330.20
1.300.35
6.980.88
8.700.16
13.062.41
13.652.89
39.837.29
50.850.21
57.063.15
58.01.27
18.71.40
15.53.60
32.82.36
26.85.60
14.762.23
16.000.28
407128
48495
16.430.75
16.901.13
8.702.55
6.950.35
Ginger
3.0 mg
6.550.42
9.91.01
15.323.70
13.80.91
79.83.51
82.661.2
3.720.92
2.500.49
0.570.68
0.530.37
0.550.57
0.460.30
8.001.20
8.620.60
14.821.60
15.860.75
46.776.28
52.133.81
58.621.34
60.52.71
18.60.97
18.40.43
31.71.03
30.41.30
14.101.65
17.401.58
576122
478211.3
12.457.65
17.200.96
7.124.60
7.300.34
6.0 mg
7.723.03
10.52.20
14.671.55
16.61.7
78.82.49
77.22.3
4.172.64
3.601.81
1.071.20
1.000.47
1.201.50
1.600.79
7.830.70
8.900.11
14.721.22
16.404.12
45.354.61
51.403.71
57.851.73
57.82.4
18.80.65
18.41.82
32.50.64
31.81.99
14.071.83
14.702.52
455166
483120
17.970.61
16.603.21
7.451.23
6.701.10
Valerian
3.0 mg
6.0 mg
9.0751.75
5.5752.46
11.90.90
62.06
16.003.59
11.550.98
12.91.06
15.83.2
80.03.24
81.83.39
77.554.5
76.83.7
2.9750.73
3.971.93
4.300.56
4.462.40
0.300.14
1.321.70
0.250.07
1.761.07
0.420.12
1.321.49
1.451.12
1.10.95
7.470.78
7.321.00
7.501.13
8.191.37
13.771.30
13.921.49
15.750.91
16.261.46
42.674.44
42.525.50
50.153.32
52.004.68
57.151.88
58.121.98
68.014.63
60.03.89
18.40.72
19.00.85
21.34.38
18.40.62
32.30.42
32.70.75
31.40.28
31.30.00
14.020.84
13.570.67
15.500.84
16.361.52
524182
513146
48095
421165
16.600.98
17.650.73
17.350.77
180.80
7.050.79
7.400.66
8.051.06
7.261.15
RBC: Red blood cell

MCV: Mean corpuscular volume
MCH: Mean corpuscular hemoglobin
MCHC: Mean corpuscular hemoglobin concentration
PDW: Platelet distribution width
MPV: Mean platelet volume
Hb: Hemoglobin
CBC: Cell blood count
WBC: White blood cell
182
c. Liver function test

Liver function test was done at the end of study and results are shown in Table 41. In
female, rat total bilirubin was found to be in the range of 0.247- 0.333 mg/dl in study
group and control group was 0.35 mg/dl. And male animal was 0.242-0.340 mg/dl and in
control group it was 0.331 mg/dl. SGOT was 624 and 306.45 u/l in control group and in
female and male respectively. In study groups it was ranged between 442-571 and 372537 u/l respectively. In control group SGPT was 137.7 and 64.8 u/l in female and male
respectively, whereas in study groups, it ranged between 97.6 to 109.4 and 81.6 to 84.1
u/l respectively. All values were close to the reported normal values.
Zahedi et al (2004) administered the percolated extract of this plant with doses of 100 and
200 mg/kg to 5 groups of rats via orogastric tube for 7 days. At the 8th day, the blood
samples were taken for biochemical studies. The results showed significant variation of
the levels of AST, ALT and AlkP in comparisons with control group. AlkP was increased
significantly after oral administration of extract with dose of 100 mg/kg and 200 mg/kg.
ALT was decreased with dose of 100 and 200 mg/kg of valerian significantly but AST
increased significantly only with dose of 100 mg/kg. AST did not change with dose of
100 mg/kg, but decreased significantly with dose of 200 mg/kg (P < 0.01). They reported
that renal function tests including blood urea nitrogen and creatinine also did not change
significantly after oral administration of extract.
183
Table 41: Effect of ginger and valerian supplementation on Liver function test of
adult Wistar rat
Constituents
Sex
F
Bilirubin T mg/dl
M
Bilirubin direct
mg/dl
Bilirubin indirect
mg/dl
F
M
F
M
F
SGOT u/l
M
F
SGPT u/l
M
Alkaline
phosphatase u/l
F
M
GGT u/l
F
M
Total protein g/dl
F
M
Albumin g/dl
F
M
Serum
albumin/globulin
ratio
F
M
Control
0.350
0.073
0.331
0.028
0.077
0.033
0.075
0.035
0.272
0.063
0.25
0
624.0
270.20
306.4
67.6
137.7
44.0
64.8
8.34
352.9
146.21
349.4
36.41
5.77
5.53
1.6
0.14
7.13
0.65
6.57
0.20
2.86
0.392
2.97
0.37
0.67
0.104
0.85
0.240
Ginger
3.0 mg
0.325
0.017
0.242
0.121
0.087
0.022
0.166
0.167
0.237
0.020
0.246
0.015
501.7
247.44
497.1
197.2
106.3
34.6
82.7
14.81
359.6
48.14
350.8
46.58
4.85
4.46
2.6
1.47
7.11
0.53
6.81
0.25
2.97
0.232
3.02
0.20
0.72
0.074
0.80
0.111
6.0 mg
0.333
0.070
0.340
0.100
0.085
0.026
0.111
0.080
0.245
0.058
0.29
0.020
539.5
283.48
372.0
11.5
109.4
44.5
83.5
7.21
296.4
31.46
351.5
8.60
4.60
4.03
3.2
1.01
7.30
0.40
6.41
0.80
3.17
0.211
3.09
0.09
0.76
0.049
0.93
0.040
Valerian
3.0 mg
6.0 mg
0.247
0.301
0.096
0.040
0.303
0.312
0.011
0.099
0.142
0.071
0.139
0.021
0.086
0.062
0.023
0.025
0.225
0.231
0.034
0.043
0.216
0.250
0.011
0.077
442.8
571.3
219.64
167.54
537.4
416.3
61.7
212.0
97.6
104.5
35.3
38.8
84.1
81.6
12.4
24.57
271.5
203.1
75.47
17.04
303.7
291.3
54.05
67.44
2.15
3.87
0.85
1.83
4.5
2.3
1.70
1.90
6.78
7.35
0.56
0.24
6.80
6.73
0.57
0.48
2.87
3.27
0.087
0.186
3.34
3.13
0.47
0.26
0.74
0.80
0.121
0.044
0.96
0.87
0.112
0.114
184
d) Lipid profile
Lipid profile of all animals was measured at the end of the study. Total cholesterol of
animals in control group was 46.45 and 44.5 mg% in female and male respectively. All
study group values were close to control group. And significant difference was not seen.
A mild increase was seen in triglyceride level of male animals in group 3, 4 and 5 but it
was not statistically significant. HDL level was in the range of 30.35 to 37.85 mg % in
female animals and in the range of 24.7 to 29.5 mg%.
Many studies reported the hypolipidemic effect of ginger (Bhandari et al., 1998;
Bhandari, 2005). It is said that ginger can increase the pancreatic lipase (Platel and
Srinivasan, 2000) and when ginger was incorporated in rats diet, it significantly
increased the activity of hepatic cholesterol 7a-hydroxylase (Srinivasan, 1991). Bhandari
(2005) used 200 mg/kg ethanolic extract and observed the lipid lowering effect of ginger
on rat and the same effect on rabbit (Bhandari et al., 1998). At the dose which ginger was
given to animals in present study, we did not find any effect on cholesterol level of
animals
5.2.6. Safety and toxicity

During the 30 days of the experiment, all rats were observed daily. Notations were made
regarding their physical activity/lethargy, fur condition, skin condition, eye condition,
stools and others. During the study alopecia was not seen in any group. Fur condition,
skin condition, eye condition, stools were found to be normal. In group 4 and 5, rats
found to be more sleepy than control group. And group 1, 2 and 3 were found to be
normally active.
Histology of liver, kidney, pancreas and spleen carried after the study. It was observed
that there were no changes in tissue of theses organs in experimental group compare to
the control group.
185
Table 42: Effect of ginger and valerian supplementation on Lipid profile of adult
wistar rat
Ginger
Valerian
3.0 mg
6.0 mg
3.0 mg
6.0 mg
39.45
41.92
45.47
46.50
49.60
F
8.54
2.42
10.44
12.12
17.84
Total
cholesterol
44.5
37.6
40.3
47.1
38.3
M
1.83
2.53
2.10
7.59
10.85
30.35
30.45
35.22
33.12
37.85
F
5.43
2.22
1.92
7.54
5.76
HDL
26.95
26.43
29.50
24.70
26.87
M
0.91
4.79
1.80
3.21
3.86
87.8
81.7
100.9
52.8
95.4
F
12.20
27.73
35.84
22.46
24.80
TG
53.9
68.6
101.1
102.9
70.0
M
7.9
25.8
15.8
26.3
16.5
17.56
16.34
20.19
10.56
22.08
F
2.44
5.54
7.16
4.49
8.34
VLDL
10.78
13.72
19.32
17.59
14.96
M
1.58
5.16
2.41
39.00
4.53
1.32
1.39
1.28
1.39
1.28
F
0.334
0.176
0.260
0.111
0.278
TG/HDL
ratio
1.65
1.44
1.37
1.91
1.41
M
0.120
0.222
0.900
0.321
0.285
VLDL: Very low density lipoprotein, HDL: High-density lipoprotein
TG: Triglyceride
Lipid profile
Sex Control
Traditionally, ginger is used in herbal preparations for weight reduction. Present study
demonstrated that ginger supplementation lead to weight reduction without significant
reduction in food intake, whereas valerian showed a significant weight gain with
significant increase in food intake. Blood biochemical analysis demonstrated that either
ginger or valerian did not affect lipid profile, liver function, blood electrolyte and
complete blood count.
186
Figure 71: Histology of Pancreas of animals in 5 groups after treatment
A: Control; B:3.0mg ginger; C:6.0 mg ginger; D:3.0 mg valerian, E:6.0 mg valerian

187
Figure 72: Histology of spleen of animals in 5 groups after treatment
A: Control; B:3.0mg ginger; C:6.0 mg ginger; D:3.0 mg valerian, E:6.0 mg valerian
188
Figure 73: Histology of kidney of animals in 5 groups after treatment
A:control; B:3.0mg ginger; C:6.0 mg ginger; D:3.0 mg valerian, E:6.0 mg valerian
189
Figure 74: Histology of liver of animals in 5 groups after treatment
A:control; B:3.0mg ginger; C:6.0 mg ginger; D:3.0 mg valerian, E:6.0 mg valerian

190

14 Chapter 5

Diunggah oleh

Informasi Dokumen

Hak Cipta

Format Tersedia

Bagikan dokumen Ini

Bagikan atau Tanam Dokumen

Opsi Berbagi

Apakah menurut Anda dokumen ini bermanfaat?

Apakah konten ini tidak pantas?

Hak Cipta:

Format Tersedia

14 Chapter 5

Diunggah oleh

Hak Cipta:

Format Tersedia

1.

1.1. ANTHOCYANIN CONTENT

2. IN VITRO ANTIOXIDANT POTENTIAL

Table 18: Antioxidant components of borage in different extracts (g/100 g)

Table 19: Antioxidant components of valerian (g/100 g)

2.1.4. Whole Lime

Table 21: Antioxidant components of lime (g/100 g)

2.2. ANTIOXIDANT ACTIVITY

2.2.1. Total antioxidant activity

2.2.2. Antioxidant activity by reducing power assay

Figure 11: Reducing power of borage in different extracting media

Optical Density (nm)

[Concentration, A: 2000 ppm, B: 4000 ppm, C: 6000 ppm, D: 8000 ppm]

Optical Density (nm)

[Concentration, A: 2000 ppm, B: 4000 ppm, C: 6000 ppm, D: 8000 ppm]

Figure 13: Reducing power of ginger in different extracting media

[Concentration, A: 2000 ppm, B: 4000 ppm, C: 6000 ppm, D: 8000 ppm]

Optical Density (nm)

[Concentration, A: 2000 ppm, B: 4000 ppm, C: 6000 ppm, D: 8000 ppm]

Figure 15: Reducing power of shallot in different extracting media

Optical Density (nm)

[Concentration, A: 2000 ppm, B: 4000 ppm, C: 6000 ppm, D: 8000 ppm]

Methanol Ethanol Methanol Ethanol

Concentration- All extracts, A: 2.5, B: 5.0, C: 7.5 and D: 10 mg of sample

Concentration- All extracts, A: 5, B: 10, C: 15 and D: 20 mg of sample

Table 24: Correlation coefficient between antioxidant compound and antioxidant

3.1. ANTIBACTERIAL ACTIVITY OF AQUEOUS EXTRACT

+C: positive control, -C: negative control, A: 5 l, B: 10 l, C:25 l and D: 50l

Sh: Shallot, L: Lime, +C: positive control, -C: negative control

Sh: Shallot, L: Lime, +C: positive control, -C: negative control

+C: positive control, -C: negative control, A: 5 l, B: 10 l, C: 25 l and D: 50l

+C: positive control, -C: negative control, A: 5 l, B: 10 l, C:25 l and D: 50l

+C: positive control, -C: negative control, A: 5 l, B: 10 l, C:25 l and D: 50l

+C: positive control, -C: negative control, A: 5 l, B: 10 l, C:25 l and D: 50l

Table25: Antibacterial activity of aqueous

Concentration: 50l/ well.

Table 26: MIC of aqueous extracts against gram positive bacteria

Table 27: MIC of aqueous extracts against gram negative bacteria

3.2. ANTIBACTERIAL ACTIVITY OF METHANOLIC EXTRACT

Sh: Shallot, L: Lime, +C: positive control, -C: negative control

+C: Positive control, -C: Negative control, A: 5 l, B: 10 l, C: 25 l and D: 50l

Sh: Shallot, L: Lime, +C: positive control, -C: negative control

evaluated in different concentration, a range of 25-18 mm and 26-16 mm were observed

Table 28: Antibacterial activity of methanol extract

Table 29: MIC of methanolic extracts against gram negative bacteria

Concentration: 50l/ well.

Table 30: MIC of methanolic extracts against gram positive bacteria

Concentration: 50l/ well.

4. IN VIVO ANTIOXIDANT POTENTIAL OF BORAGE AND

4.1.1. Total Thiol

Total thiol mol/ml

4.1.2. Serum lipid peroxide level

no significant difference. Lipid peroxidation, glutathione levels, glutathione peroxidase

Lipid peroxidation nmol/ml

4.1.3. Total antioxidant capacity (TAC) by FRAP assay

Figure 51: Effect of borage supplementation on Serum total antioxidant activity of

4.1.4. Food intake

E: experimental group, C: Control group

E: experimental group, C: Control group

Figure 54: Effect of borage supplementation on Serum Blood glucose of diabetic

FBG: Fasting blood glucose in study group

Figure 55: Effect of borage supplementation on Lipid profile of diabetic patients

-S: Study group

Table 33: Effect of borage supplementation on Lipid profile of diabetic patients