Precision, Stability and Recovery

The intra- and inter-day variations chosen to determine the precision of the developed method were investigated by determining thirteen analytes in six replicates in a single day and by duplicating the experiments on three consecutive days. Variations of the peak areas were taken as the measures of precision and expressed as percentage relative standard deviations (%RSD=standard deviation/mean*100). The overall intra- and inter-day precision was not more than 1.66. Stability of sample solutions stored at room temperature was investigated by replicating injections of the sample solution at 0,2,4,8, 12 and 24 h. The %RSD value of stability of the 13 analytes is < 3.14. A recovery test was carried out to evaluate the accuracy of this method. Three different concentration levels (high, middle and low) of the analytical standards were added into the samples in triplicate, and average recoveries were determined by comparing the response of the extract with the response of the reference material dissolved in a pure solvent. The analytical method developed had good accuracy with overall recovery in the range from 96.95 to 102.39 (%RSD < 2.78) for all the analytes (Table 3.2).

Quantitative Analysis of Samples

The newly developed UPLC-ESI-MS/MS method was applied to quantify 13 bioactive ingredients in the fruit and leaf extracts of ten Piper species, namely, P. nigrum, P. longurn, P. chaba, P. umbellatum, P. mullesa, P. colubrinum, P. hymenophyllum, P. argyrophyllum, P. attenuatum and P. galeatum. The Piper species such as P. nigrum, P. longurn and P. chaba are commercially well known and much explored as they are used in various herbal preparations, whereas the rest of the Piper species are less explored in terms of the bioactive phytoconstituents such as piperamides, phenolics, flavonoids and terpenoids and their diversities and variations. The contents of the 13 compounds in Piper species are listed in Table 3.3. The results indicated remarkable differences in the contents of piperamides, phenolics, flavonoids and terpenoid among the ten different species of Piper. Graphical representation of these observations shown in Figure 3.4 clearly explains the variations among the Piper species and between fruits and leaves.

Among the three piperamides, piperine was the major compound in most of the Piper species. P. nigrum fruits had the highest piperine content (7,220 pg/g), followed by P. chaba (5,530 pg/g), P. longurn (2,150 pg/g) and P. hymenophyllum (309 pg/g). Only low piperine content was detected in P. umbellatum (2.10 pg/g) and P. attenuatum (1.65 pg/g). These results were consistent with the previous reports of determination of piperine (ref). Among the leaves, P. chaba (203 pg/g) and P. mullesa (271 pg/g) leaves yielded high content of piperine, whereas the lowest content was obtained in P. umbellatum (1.09 pg/g). Piperine was below the detection limit in both fruits and leaves of P. argyrophyllum. The contents of other piperamides, namely, piperlongumi- nine and piperlongumine, were the highest in fruits of P. longurn (15,200 pg/g and 162.00 pg/g, respectively). Thus, piperamides could serve as the signature constituents to distinguish among the Piper species. As shown in Table 3.3, the total content of phenolics was in close proportion and highest when compared to others in fruits of P. mullesa (1,129 pg/g) and leaves of P. longurn (1,094 pg/g), whereas it was found lowest in both fruits and leaves of P. chaba (82 pg/g). The total content of flavonoids was found higher in fruits and leaves of P. umbellatum (1,124 pg/g and 625 pg/g, respectively), while components such as kaempferol and luteolin could not be quantified or detected in most

TABLE 3.2 Regression Equation, Correlation Coefficients, Linearity Ranges and Lower LOD and LOQ for 13 Reference Analytes

ANALYTES

REGRESSION

EQUATION

R2

LINEAR

RANGE

NG/ML

LOD

NG/

ML

LOQ

NG/

ML

PRECISION RSD (%)

STABILITY

RECOVERY RSD (%)

Intra-day (n = 6)

Inter-day (n = 6)

RSD,

(n = 5)

Protocatechu ic acid

y = 8.3201x-0.0364

0.9993

1-50

0.12

0.36

1.66

1.05

2.78

1.22

Caffeic acid

у = 18.2x +0.00859

0.9989

1-50

0.16

0.48

0.86

2.63

1.66

1.56

Ferulic acid

y = 2.685x- 0.0209

0.9991

1-50

0.19

0.56

2.22

2.94

1.68

1.55

Rosmarinic acid

y= 12.68x + 0.0914

0.9995

1-50

0.08

0.28

1.72

1.82

2.11

1.27

Vanillic acid

y= 37.795x+ 0.1116

0.9998

1-50

0.12

0.40

1.24

1.36

2.15

2.78

Luteolin

y=3.6406x-0.0064

0.9995

1-50

0.18

0.52

1.13

1.14

3.14

1.66

Quercetin

y=9.7543x-0.0102

0.9991

5-100

0.10

0.33

2.51

1.23

2.86

0.86

Kaempferol

y=0.7303x- 0.0006

0.9993

10-250

1.34

3.88

0.63

2.36

1.89

0.78

Apigenin

y= 13.426x-0.0527

0.9992

1-50

0.18

0.52

1.02

1.17

1.66

1.45

Pi perlongumine

y=0.0186x-0.0076

0.9998

0.5-50

0.06

0.20

0.77

2.25

1.59

2.38

Piperlonguminine

у = 1.133x+ 0.221

0.9998

0.5-100

0.04

0.12

3.37

0.93

2.68

1.89

Pi perine

y=0.0063x-0.0033

0.9999

0.5-100

0.02

0.06

0.88

0.91

2.45

1.87

Ursolic acid

y=0.6281x-0.0862

0.9986

5-100

0.62

1.92

1.59

4.09

2.16

1.59

Source: Reproduced from Ref. Chandra et al. 2015 with permission from Elsevier.

SAMPLE

CODE

PIPERAMIDES

TOTAL

PHENOLICS

TOTAL

FLAVONOIDS

TOTAL

TERPE

NOID

PIPERINE

PIPERLONGU-

MINE

PIPERLONGU-

MINE

PROTOCATE- CHUIC ACID

CAFFEIC

ACID

FERULIC

ACID

ROSMARINIC

ACID

VANILLIC

ACID

LUTEOLIN

QUERCETIN

KAEMPFEROL

APIGENIN

URSOLIC

ACID

1 FR

bdl

36.0 ± 0.28

bdl

36.0 ± 0.28

50.50 ± 0.02

nd

26.45 ± 0.30

5.30 ± 0.05

3.30 ± 0.01

85.55 ± 0.38

nD

91.1 ± 0.28

nd

5.30 ± 0.69

96.40 ± 0.97

nd

1 LF

bdl

Bdl

bdl

0.00

11.40 ± 0.41

bdl

21.35 ± 1.02

108.0 ± 1.27

nd

140.75 ± 2.70

ND

93.9 ± 1.01

nd

5.90 ± 0.40

99.80 ± 1.41

nd

2 FR

1.65 ± 0.05

40.0 ± 0.66

bdl

41.65 ± 0.71

135.0 ± 1.06

bdl

22.05 ± 0.82

  • 21.20±
  • 0.02

3.24 ± 1.01

181.49 ± 2.91

0.78 ± 0.02

22.2 ± 1.22

nd

5.35 ± 1.05

28.33 ± 2.29

nd

2 LF

18.54 ± 1.12

36.30 ± 1.31

bdl

54.84 ± 2.43

88.50 ± 1.21

bdl

72.0 ± 0.44

105.0 ± 0.08

3.69 ± 0.54

269.19 ± 2.27

0.56 ± 0.11

nd

nd

5.90 ± 1.34

6.46 ± 1.45

nd

3FR

bdl

14.10 ± 0.80

nd

14.10 ± 0.80

77.0 ± 0.63

bdl

31.35 ± 0.03

111.0 ± 1.66

3.37 ± 0.81

222.72 ± 3.13

38.2 ± 0.27

885.0 ± 0.48

  • 11 ±
  • 0.03

9.05 ± 0.05

943.25 ± 0.83

2920.0 ± 0.49

3 LF

4.67 ± 0.07

36.80 ± 2.01

nd

41.47 ± 2.08

57.0 ± 2.05

18.0 ± 1.05

59.0 ± 1.02

5.45 ± 0.25

3.25 ± 0.73

142.70 ± 5.10

4.24 ± 0.76

bdl

15.0 ± 0.06

8.25 ± 0.93

27.49 ± 1.75

107.0 ± 0.22

4 FR

  • 10.0±
  • 1.03

11.80 ± 1.22

nd

21.8 ± 2.25

5.10 ± 0.09

5.6 ± 0.02

23.0 ± 0.05

4.22 ± 0.04

nd

37.92 ± 0.20

1.33 ± 0.05

10.98 ± 0.64

11.0 ± 0.48

nd

23.31 ± 1.17

1235.0 ± 1.11

4 LF

28.9 ± 0.52

14.50 ± 0.55

nd

43.40 ± 1.07

139.50 ± 0.46

39.35 ± 0.29

42.7 ± 0.57

16.56 ± 0.41

1.71 ± 0.90

239.82 ± 2.63

0.81 ± 0.03

nd

5.65 ± 0.27

6.05 ± 0.06

12.51 ± 0.36

158.0 ± 0.32

5 FR

309.0 ± 3.12

bdl

bdl

309.0 ± 3.12

13.50 ± 1.20

bdl

45.1 ± 1.06

733.0 ± 0.62

1.61 ± 1.02

793.21 ± 3.90

1.03 ± 0.08

527.0 ± 0.80

4.0 ± 0.01

5.95 ± 1.03

537.98 ± 1.92

2420.0 ± 0.50

5 LF

29.30 ± 2.04

  • 10.60±
  • 1.77

nd

39.90 ± 3.81

Nd

bdl

27.7 ± 0.10

15.14 ± 0.20

1.65 ± 0.06

44.49 ± 0.36

1.4 ± 0.18

11.44 ± 0.02

16.0 ± 0.04

6.40 ± 0.11

35.24 ± 0.35

810.0 ± 0.44

6 FR

5530.0 ± 3.12

407.0 ± 2.03

bdl

5937.0 ± 5.15

18.50 ± 0.07

15.6 ± 0.03

nd

8.60 ± 0.11

nd

42.70 ± 0.21

3.15 ± 1.01

18.38 ± 0.07

bdl

7.45 ± 0.73

28.98 ± 1.81

nd

('Continued)

SAMPLE

CODE

PIPERAMIDES

TOTAL

PHENOUCS

TOTAL

FLAVONOIDS

TOTAL

TERPE

NOID

PIPERINE

PIPERLONGU-

MINE

PIPERLONGU-

MINE

PROTOCATE- CHUIC ACID

CAFFEIC

ACID

FERULIC

ACID

ROSMARINIC

ACID

VANILLIC

ACID

LUTEOLIN

QUERCETIN

KAEMPFEROL

APIGENIN

URSOLIC

ACID

6 LF

203.20 ± 0.98

bdl

65.10 ± 0.21

268.30 ± 1.19

Nd

bdl

30.8 ± 2.03

5.25 ± 1.06

3.28 ± 0.36

39.33 + 3.45

nd

nd

bdl

5.30 ± 0.48

5.3 ± 0.48

nd

7 FR

2150.0 ± 1.23

15200.0 + 2.81

162.0 ± 0.09

20512.0 + 4.13

15.70 ± 0.14

bdl

51.0 ± 0.10

21.20 ± 1.50

nd

87.90 + 1.74

nd

nd

nd

5.75 ± 0.82

5.75 ± 0.82

1775.0 ± 0.38

7 LF

bdl

43.90 ± 0.11

39.0 ± 0.02

82.90 ± 0.13

15.10 ± 0.64

bdl

15.75 ± 0.23

1060.0

±2.01

3.25 ± 1.26

1094.1 ± 4.14

nd

88.8 ± 0.33

nd

5.75 ± 0.29

94.55 + 0.62

nd

8 FR

7220.0 ± 0.62

62.90 ± 1.09

34.50 ± 0.88

7317.40 ± 2.59

  • 20.10±
  • 0.68

bdl

32.30 ± 0.06

5.35 ± 1.92

1.56 ± 0.03

59.31 ± 2.69

nd

nd

8.0 ± 0.22

5.40 ± 0.10

13.40 ± 0.32

193.0 ± 0.05

8 LF

27.60 ± 1.45

48.80 ± 2.33

17.10 ± 1.6

93.50 ± 5.38

18.05 ± 0.02

bdl

55.0 ± 0.12

23.0 ± 0.06

1.59 ± 0.46

97.64 ± 0.66

nd

18.86 ± 1.36

12.0 ± 1.01

6.0 ± 0.51

36.86 ± 2.88

580.0 ± 1.04

9 FR

28.80 ± 0.83

40.0 ± 0.49

nd

68.80 ± 1.32

96.5 ± 0.04

nd

132.5 ± 1.07

897.0 ± 0.71

3.28 ± 0.73

1129.28 ± 2.55

nd

bdl

bdl

5.20 ± 0.03

5.20 ± 0.03

nd

9 LF

271.0 ± 3.02

36.0 ± 0.53

nd

307.0 ± 3.55

37.55 ± 0.61

20.3 ± 0.49

165.0 ± 0.29

762.0 ± 0.38

3.27 ± 1.40

988.12 ± 3.17

nd

bdl

bdl

6.40 ± 0.17

6.40 ± 0.17

389.0 ± 1.36

10 FR

2.10 ± 0.72

17.10 ± 1.04

  • 64.30±
  • 0.04

83.50 ± 1.80

42.6 ± 0.03

7.65 ± 1.13

111.5 ± 0.43

21.40 ± 0.02

nd

183.15 ± 1.61

1.43 ± 0.17

1410.0 ± 1.27

6.0 ± 0.31

7.0 ± 1.82

1424.43 ± 3.57

8600.0 ± 1.20

10 LF

1.09 ± 0.05

bdl

bdl

1.09 ± 0.05

55.0 ± 0.13

28.25 ± 1.50

131.5 ± 0.06

21 20 ± 1.04

3.21 ± 0.06

239.16 ± 2.79

2.35 ± 0.21

605.0 ± 0.90

1.0 ± 0.55

16.70 ± 0.04

625.05 ± 1.70

3965.0 ± 0.72

nd = not detected; bdl = below detection limit; FR = fruit; LF = leaf; P. argyrophyllum (1); P. attenuatum (2); P. colubrinum (3); P. galeatum (4); P. hymenophyllum (5); P. chaba (6); P. longum (7); P. nigrum (8); P. mullesa (9); P. umbellatum (10).

Source: Reproduced from Ref. Chandra et al. 2015 with permission from Elsevier.

50 Phytochemistry of Plants of Genus Piper

(a) Graphical representation of 13 compounds plant part of different Piper species,

FIGURE 3.4 (a) Graphical representation of 13 compounds plant part of different Piper species, (b) Graphical representation of compounds contents in plant part of ten Piper species. (Reproduced from Ref. Chandra et al. 2015 with permission from Elsevier.) of the Piper species. Terpenoid (ursolic acid) content was quantitatively found highest in fruits followed by leaves of P. umbellatum (8,600 pg/g and 3,965 pg/g, respectively) and lowest in the leaf of P. colubrinum (107 pg/g). This characteristic observation helps in differentiating these species among others.

The genus Piper is in great demand in herbal industry, and because of the high content of piperamides, Piper fruit extracts are incorporated in various formulations. Our findings indicated that piperamides are the signature class of compounds Piper species, and their contents are altogether the highest in the fruits of P. longum (21,500 pg/g), P. nigrum (7,317.40 pg/g) and P. chaba (5,937 pg/g) as compared to the other seven species. There were significant differences among the collected plant samples of Piper species in terms of individual contents of the investigated bioactive compounds.

3 • Chemical Constituents of Piper Species 51

 
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