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Revista Brasileira de Farmacognosia xxx (2017) xxxx

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Original Article

1 HPLC fingerprint and simultaneous quantitative analysis of

2 phyllanthin and hypophyllanthin for identification and authentication
3 of Phyllanthus niruri from related species
4 Q1 Roni Nasrulloh a,b , Mohamad Rafi a,c,∗ , Wulan Tri Wahyuni a,c , Shuichi Shimma d , Rudi Heryanto a,c
a
5 Department of Chemistry, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Jalan Tanjung Kampus IPB Dramaga, Bogor, Indonesia
b
6 Center for Plant Product Quality Testing, Ministry of Agriculture, Jakarta Selatan, Indonesia
c
7 Tropical Biopharmaca Research Center-Institute of Research and Community Services, Bogor Agricultural University, Jalan Taman Kencana No. 3 Kampus IPB Taman Kencana, Bogor,
8 Indonesia
d
9 Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan
10

11 a r t i c l e i n f o a b s t r a c t
12
13 Article history: A precise and accurate method for the identification and authentication of Phyllanthus niruri L. from P.
14 Received 28 November 2017 debilis Klein ex Willd. and P. urinaria L., Phyllanthaceae, was developed using high-performance liquid
15 Accepted 20 April 2018 chromatography. Chromatographic fingerprint analysis was combined with simultaneous quantifica-
16 Available online xxx
tion of phyllanthin and hypophyllanthin for the developed method. Phyllanthin and hypophyllanthin
17 were successfully separated and quantified under this proposed method. The highest amount of phyl-
18 Keywords: lanthin and hypophyllanthin was found in P. niruri compared to P. debilis and P. urinaria. Fingerprint
19 Authentication
chromatogram of the three Phyllanthus species showed distinct profiles that these may be used to iden-
20 Fingerprint analysis
21 Hypophyllanthin
tify and authenticate each Phyllanthus species, which improved by marker compounds present in each
22 phyllanthin species. The combination of chromatographic fingerprint analysis and discriminant analysis was suc-
23 Phyllanthus cessfully discriminated all three species, including P. niruri adulterated with P. debilis or P. urinaria. The
method can be used for the identification and authentication of P. niruri from related species, such as P.
debilis and P. urinaria.
© 2018 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. This is an open
access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

24 Introduction 2007). It is also used to improve the immune system (immunos- 41

timulant) (Taiwo et al., 2009). P. niruri has numerous secondary 42

25 The genus of Phyllanthus belongs to the family of Phyllanthaceae metabolites such as lignans, alkaloids, flavonoids, hydrolyzable tan- 43

26 comprising of more than 600 species and one of them is Phyllanthus nins (ellagitannins), triterpenoids and polyphenolic compounds 44

27 niruri L., which mostly found in the tropical and subtropical regions. (Patel et al., 2011). The primary bioactive compounds present 45

28 P. niruri has a long history in some traditional medicinal system, in P. niruri are lignans, such as phyllanthin (1), hypophyllanthin 46

29 such as Indonesian Jamu, Indian Ayurveda and Traditional Chinese (2), phyltetralin, nirtetralin and niranthin (Murugaiyah and Chan, 47

30 Medicine, to prevent or treat various diseases (Bagalkotkar et al., 2007; Shanker et al., 2011). Compounds 1 and 2 are two lignans 48

31 2006). In Indonesia, P. niruri is known as meniran and usually grown commonly used as marker compounds in the quality control of P. 49

32 like a weed on agricultural and wastelands. Considering its efficacy niruri as described in Indian Pharmacopoeia (2010). 50

33 to treat many diseases and maintaining health, P. niruri is consid-

34 ered as one of the most important medicinal plants in Jamu. P. niruri
35 has been used to treat health problems such as diarrhea, jaundice,
36 wounds, diabetes, diuretic, stomachache and hepatitis (Joseph and
37 Raj, 2011; Patel et al., 2011). P. niruri denotes many pharmacological
38 activities such as antioxidant (Lim and Murtijaya, 2007), antiviral
39 (Tan et al., 2013), antidiabetic (Adeneye, 2012), anti-inflammatory
40 (Kassuya et al., 2005) and hepatoprotective (Pramyothin et al.,
51

Since the efficacy of a medicinal plant depends on more than 52

∗ Corresponding author. one single chemical compound that work synergistically, there 53

E-mail: mra@ipb.ac.id (M. Rafi). are two approaches in the quality control of herbal medicine, i.e., 54

https://doi.org/10.1016/j.bjp.2018.04.014
0102-695X/© 2018 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Nasrulloh, R., et al. HPLC fingerprint and simultaneous quantitative analysis of phyllanthin and
hypophyllanthin for identification and authentication of Phyllanthus niruri from related species. Revista Brasileira de Farmacognosia
(2017), https://doi.org/10.1016/j.bjp.2018.04.014
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BJP 496 1–6 ARTICLE IN PRESS
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55 multicomponent-based and pattern-based approaches from the Table 1

Resolution of all common peaks present in P. niruri, P. debilis and P. urinaria samples.
56 chromatograms, spectrum or other graphs from chemical instru-
57 mentation. The pattern-based approach, which is also known as Peak number Resolution
58 the fingerprint analysis, can reveal all detectable compounds in a
P. niruri P. debilis P. urinaria
59 medicinal plant, and therefore, it could be applied in the detection
1 — — —
60 of adulteration of raw materials of herbal medicinal products (Mok
2 15.141 14.914 14.213
61 and Chau, 2005; Ma et al., 2011). On the other hand, quantitative 3 — 13.473 —
62 analysis of multicomponents can reveal the concentration of bioac- 4 — — 16.024
63 tive compounds in a medicinal plant. Therefore, the combination 5 17.919 9.514 3.753
64 of chromatographic fingerprint and quantitative analysis of multi- 6 6.035 5.216 5.836
7 2.177 2.207 1.946
65 components often used for the complementary properties between 8 1.721 1.807 1.748
66 these approaches makes it more comprehensive for identification, 9 3.636 3.786 3.786
67 discrimination and authentication of medicinal herbs. This com- 10 1.954 1.997 2.009
68 bination has been used, for example, in the quality evaluation of 11 18.659 19.495 19.699
69 xiaoyanlidan tablets (Tang et al., 2014), authentication of Curcuma
70 xanthorrhiza from C. longa (Rafi et al., 2015) and chemical char- ®
71 acterization of two morphologically related Espeletia, Asteraceae, purification system of Direct-Q 5 UV-R, and formic acid (purity 115

72 species (Padilla-Gonzalez et al., 2016). 98–100%). All standards and samples were filtered through 0.2 ␮m 116

73 There are two closely related species that have similar mor- nylon membrane filter obtained from Whatman (Kent, UK). The 117

74 phology and grow sympatrically with P. niruri, i.e., P. debilis and P. HPLC mobile phase was prepared fresh daily, filtered through 118

75 urinaria. These may lead to misidentification and quite vulnerable 0.2 ␮m polyvinylidene difluoride (PVDF) membrane filter obtained 119

76 to adulteration of P. niruri from P. debilis and P. urinaria. Therefore, a from Waters Corp. (Milford, Massachusetts, USA) and degassed 120

77 simple and reliable chemical analysis method should be developed before analysis. 121

78 for the identification and authentication of P. niruri using the sec-

79 ondary metabolite profiles. Several analytical methods using HPLC Apparatus and chromatographic conditions 122

80 have been developed for quantitative determination of the two

81 lignans of these species, phyllanthin (1) and hypophyllanthin (2) The HPLC system used was LC-20A series (Shimadzu, Kyoto, 123

82 (Murugaiyah and Chan, 2007; Shanker et al., 2011), as well as for Japan) equipped with a UV detector. The chromatographic separa- 124

83 chromatographic fingerprint analysis (Martins et al., 2011) in some tion was performed on a Luna C18 (2) column (150 mm × 4.6 mm, 125

84 Phyllanthus species. HPLC with ultraviolet (UV) or photodiode array 5 ␮m) (Phenomenex, Torrance, CA, USA). The mobile phase con- 126

85 (PDA) detection is widely applied for those two analytical methods sisted of methanol (solvent A) and aqueous solution of formic acid 127

86 attributed to its convenience and efficiency. 0.5% (v/v, pH 2.3) (solvent B) at a flow rate of 1 ml/min. The gra- 128

87 Previously reported analytical methods for P. niruri were only dient elution program was set as follow: 0–3 min, 23% A; 3–9 min, 129

88 developed for quantitative analysis for its marker compound or 23–35% A; 9–16 min, 35–42% A; 16–17 min, 42–70% A; 17–29 min, 130

89 chromatographic fingerprint analysis alone. There is no reported 70% A; 29–30 min, 70–100% A; 30–40 min, 100% A. This was fol- 131

90 work combining the chromatographic fingerprint and simultane- lowed by a 15 min equilibrium period prior to the next injection. 132

91 ous quantitative analysis of 1 and 2 for the identification and The chromatogram was monitored at a wavelength of 210 nm dur- 133

92 authentication of P. niruri from P. debilis and P. urinaria. In this study, ing experiment. The column temperature was maintained at 34 ◦ C 134

93 we developed a simple and reliable method for the identification and the injection volume was 20 ␮l. 135

94 and authentication of P. niruri by combining the two approaches

95 (simultaneous quantification and HPLC fingerprint analysis) with Preparation of samples and standard solutions 136

96 one multivariate analysis, i.e., discriminant analysis. The developed

97 method can be used for the identification and authentication of P. Powdered samples of 200 mg each were accurately weighed and 137

98 niruri from related species, such as P. debilis and P. urinaria. sonicated with 10 ml methanol for 60 min at room temperature. 138

The samples were then filtered through a 0.2 ␮m nylon membrane 139

filter, and methanol was added to a final volume of 10 ml before 140

99 Materials and methods injected into the HPLC system. A mixed of standard stock solution 141

of PTN and HPN was prepared in methanol at a concentration of 142

100 Plant material 1000 ␮g/ml. An appropriate amount of standard stock solutions 143

was diluted with methanol to obtain six concentrations in the range 144
101 Twenty samples consisting of six samples of P. niruri L., eight of 1.05–83.87 ␮g/ml for 1 and 2.10–83.87 ␮g/ml for 2. The cali- 145
102 samples of P. debilis Klein ex Willd. and six samples of P. urinaria L., bration curve was obtained by plotting the peak area (y) against 146
103 Phyllanthaceae, were collected in the year 2017 from eight different concentration (x) of each compound and were fitted to a linear 147
104 locations in West Java province, Indonesia (Box 1). All samples were regression equation of y = ax + b. 148
105 identified in Herbarium Bogoriense, Research Center for Biology,
106 Indonesian Institute of Science. All samples were dried at 40 ◦ C,
Method validation 149
107 pulverized, and sieved (60-mesh) before analysis. Three simulated
108 samples of P. niruri adulterated with 25, 50 and 75% of P. debilis and
Quantitative analysis of phyllanthin (1) and hypophyllanthin (2) 150
109 P. urinaria were also used.
was validated following the International Conference on Harmo- 151

nization (ICH) guidelines (ICH, 2005) in terms of linearity of the 152

110 Chemicals and reagents calibration curves, accuracy, precision, stability, limit of detection 153

(LOD) and limit of quantification (LOQ). The linearity of the calibra- 154

111 Phyllanthin (1) and hypophyllanthin (2) were purchased from tion curve of each analyte was evaluated by plotting the peak areas 155

112 ChromaDex Inc. (Santa Ana, CA, USA) with about 98.9% purity. All (y) versus the concentrations (x, ␮g/ml). Recovery test was used 156

113 solvents used for the analysis were obtained from Merck (Darm- to determine the accuracy of the analytical method. The accuracy 157

114 stadt, Germany), i.e., methanol (HPLC grade), ultrapure water from (recovery test) was performed using standard addition method. 158

Please cite this article in press as: Nasrulloh, R., et al. HPLC fingerprint and simultaneous quantitative analysis of phyllanthin and
hypophyllanthin for identification and authentication of Phyllanthus niruri from related species. Revista Brasileira de Farmacognosia
(2017), https://doi.org/10.1016/j.bjp.2018.04.014
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uV (x1.000.000)

1.75

1.50 9 10
7
8
1.25
6
1.00

0.75 a
10
0.50 b 78 9

0.25
c
1 2 6
5
0.00
d

5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 min

Figure 1. HPLC chromatograms of PN-1 sample using various solvent compositions: (a) methanol–water, (b) methanol–0.5% acetic acid, (c) methanol–0.5% phosphoric acid
and (d) methanol–0.5% formic acid.

159 The known amount of the mixed standard solutions with the high, a uV (x1,000,000)
160 intermediate and low level were spiked into the sample (PN-1) in 1.0

161 triplicates of each level. The precision of the method was evalu- 0.9

162 ated by intra- and inter-day repeatability of six individual samples 0.8

0.7
163 (PN-1) each day within three consecutive days. Stability of analytes 0.6
164 was assessed by analyzing the same sample solution (PN-1) at 0, 4, 0.5
7
165 8, 12, 24 and 48 h after preparation. Validation of the HPLC finger- 0.4

166 print analysis was performed by evaluating intra- and inter-day 0.3
8
167 precision and stability which expressed as relative standard devi- 0.2

0.1
168 ation (RSD) of the relative retention time (RRT) and relative peak
0.0
169 area (RPA) of the characteristic peaks to the reference peak (peak -0.0
170 number 9). 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 min

b uV (x1,000,000)
1.0

171 Statistical analysis 0.9

0.8

172 Discriminant analysis was performed in XLSTAT ver 2012 0.7

78 9 11
0.6
10
173 software (Addinsoft, New York, USA) for the identification and
0.5 2
174 authentication of P. niruri from P. debilis and P. urinaria. 1 6
0.4 5
0.3

4
175 Results and discussion 0.2

0.1
3
0.0
176 Optimization of HPLC condition -0.0
2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 min

177 To obtain the most chemical information and good separa- c1.0
uV (x1,000,000)
78 9
178 tion for chromatographic fingerprint and quantitative analyses, we 0.9 10 11
179 optimized the mobile phase composition, gradient elution proce- 0.8
2
180 dure and detection wavelength. We used mixed standard solution 0.7
1
6
0.6 5
181 and PN-1 sample in this step to obtain the optimum condition. 1
0.5
182 The mobile phase containing methanol–water gave better sepa- 0.4
2
4
183 ration for phyllanthin (1) and hypophyllanthin (2) compared with 0.3
3
4
184 acetonitrile–water. In order to give a better resolution, formic acid, 0.2
5
185 phosphoric acid and acetic acid were added to the aqueous phase 0.1
3
6
186 of a binary mixture of methanol–water. As a result, mobile phase 0.0

-0.0
187 consisting of methanol–0.5% formic acid in water (v/v, pH 2.3) was 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 min

188 found to be optimum for chromatographic fingerprint and quanti-

189 tative analyses of 1 and 2 (Fig. 1). Figure 2. HPLC chromatograms of standard (a) phyllanthin (1) 1.05 ␮g/ml and (b)
hypophyllanthin (2) 2.10 ␮g/ml for evaluation of LOQ.
190 The resolution of phyllanthin (1) (peak 7) and hypophyllanthin
191 (2) (peak 8) in the mixed standard solution was >1.5 (acceptance
192 criteria for resolution >1.3) with a total analysis time of 40 min Validation of the developed method 201
193 (Fig. 2a). The resolution of all common peaks in P. niruri, P. debilis
194 and P. urinaria samples was also >1.5 (Table 1). Chromatograms The linear regression equation was expressed as follow: 202
195 of 1 and 2 in P. niruri sample and the mixed standard solution y = 123 450.5608x − 1277.5080, r2 = 0.9988 (phyllanthin (1) linear 203
196 at detection wavelength between 210 and 370 nm were observed. range was 1.05–83.87 ␮g/ml) and y = 71 488.0543x + 4591.6544, 204
197 Detection wavelength of 210 nm was selected for the chromato- r2 = 0.9974 (for hypophyllanthin (2), linear range was 205
198 graphic fingerprint and quantitative analyses because it provided 2.10–83.87 ␮g/ml) (Fig. 3). All calibration curves showed a 206
199 higher signal intensities of target compounds and all detectable good linear regression within the test ranges (acceptance criteria: 207
200 separated compounds. r2 ≥ 0.995). Data of calibration curves of 1 and 2 are shown in 208

Please cite this article in press as: Nasrulloh, R., et al. HPLC fingerprint and simultaneous quantitative analysis of phyllanthin and
hypophyllanthin for identification and authentication of Phyllanthus niruri from related species. Revista Brasileira de Farmacognosia
(2017), https://doi.org/10.1016/j.bjp.2018.04.014
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Table 2
Analytical data of calibration curves of phyllanthin (1) and hypophyllanthin (2).

Phyllanthin (1) Hypophyllanthin (2)

Concentration (␮g/ml) Peak area Concentration (␮g/ml) Peak area

1.05 8971 2.10 11 022

10.48 1 339 581 10.48 809 575
20.97 2 538 863 20.97 1 511 167
41.93 5 326 690 41.93 3 127 475
62.90 7 912 859 62.90 4 593 076
83.87 10 172 635 83.87 5 863 455

a a uV (x10,000)
12 1.2
Millions

1.1
1.0
7
10
0.9
0.8
8 0.7
0.8 8
Peak area

6 0.5
0.4

4 0.3
0.2
y = 123,450.5608x - 1,277.5080 0.1
2 R2 = 0.9988 0.0
-01
0 -02
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5min
0 20 40 60 80 100 b uV (x10,000)
2.00
b Concentraion, µg/ml 7
1.75
7
1.50
Millions

6 1.25 8
1.00
5
0.75
4
Peak area

0.50

3 0.25

0.00
2
y = 71,488.0543x - 4,591.6544 -0.25
1 R2 = 0.9974
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5min

0
Figure 4. HPLC chromatograms of (a) mixed standard solutions of phyllanthin
0 20 40 60 80 100
(1) and hypophyllanthin (2), (b) individual Phyllanthus sample and (c) adulterated
Concentraion, µg/ml
samples. Composition: (b1) PN-5, 100%; (b2) PU-5, 100%; (b3) PD-5, 100%; (c1) PN-
5:PU-5, 75:25%; (c2) PN-5:PU-5, 50:50%; (c3) PN-5:PU-5, 25:75%; (c4) PN-5:PD-5,
Figure 3. Plot of calibration curves of standard (a) phyllanthin (1) and (b) hypophyl-
75:25%; (c5) PN-5:PD-5, 50:50%; (c6) PN-5:PD-5, 25:75%.
lanthin (2).

4
209 Table 2. The LOD and LOQ of the two analytes were determined
3
210 at a signal
Q2 to noise ratio (S/N) of 3 and 10, respectively (Fig. 4). PN
2
211 The LOQ of 1 and 2 were 1.05 and 2.10 ␮g/ml, respectively. The
F2 (3.23%)

PD
212 chromatograms used for LOQ evaluation are shown in Fig. 5. The 1 PU

213 LOD of 1 and 2 were 0.31 and 0.63 ␮g/ml, respectively. 0 PN:PD 25:75

214 The recovery of 1 and 2 were found to be 98.78–100.20 and -20 -10 0 10 20 30
1 PN:PU 25:75

215 97.72–99.97%, respectively, and in accordance with acceptance cri- PN:PD 50:50
-2
216 teria of recovery about 80–120%. These results indicated that the PN:PU 50:50
-3
217 assessed method was accurate and reliable. The RSD values of intra- PN:PD 75:25
218 and inter-day repeatability were less than 1.83 and 1.59%, respec- -4
PN:PU 75:25
219 tively, and below the acceptance criteria value for repeatability -5
F1 (95.32%)
220 (RSD ≤ 2%). These results showed excellent repeatability of the ana-
221 lytical method. The RSD values of stability were <1.02% with the Figure 5. DA plots of individual samples (P. niruri, P. debilis and P. urinaria) and
222 acceptance criteria of RSD for stability ≤2%) which indicated that adulterated samples (P. niruri with P. debilis and P. urinaria).

Table 3
Analytical data of recovery, precision and stability for quantitative determination of lignans in PN-1samples.

Analyte Recovery (n = 3) Precision (RSD, %) Stability (n = 6)

Average (%) RSD (%) Intra-day (n = 6) Inter-day (n = 3) RSD (%)

(1) 99.81 0.24 Day 1 = 0.52 1.42 0.82

100.20 0.49 Day 2 = 1.41
98.78 0.55 Day 3 = 0.49
(2) 99.72 0.17 Day 1 = 0.71 1.58 1.01
99.97 0.40 Day 2 = 1.82
97.72 0.59 Day 3 = 0.34

Phyllanthin (1) and hypophyllanthin (2).

Please cite this article in press as: Nasrulloh, R., et al. HPLC fingerprint and simultaneous quantitative analysis of phyllanthin and
hypophyllanthin for identification and authentication of Phyllanthus niruri from related species. Revista Brasileira de Farmacognosia
(2017), https://doi.org/10.1016/j.bjp.2018.04.014
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Table 4
Analytical data of precision and stability of nine characteristic peaks in PN-1 sample.

Peak number RSD of RRT (%) RSD of RPA (%)

Intra-day precision (n = 6) Inter-day precision (n = 3) Stability (n = 6) Intra-day precision (n = 6) Inter-day precision (n = 3) Stability (n = 6)

1 0.01 0.03 0.00 0.79 1.52 1.60

2 0.02 0.06 0.08 0.51 0.62 0.93
5 0.13 0.37 0.52 1.01 0.71 1.53
6 0.01 0.02 0.00 0.06 0.40 0.12
7 0.01 0.01 0.00 0.12 0.44 0.30
8 0.01 0.01 0.00 0.23 0.65 0.56
9a 0.00 0.00 0.00 0.00 0.00 0.00
10 0.00 0.00 0.00 0.18 0.24 0.08
11 0.03 0.05 0.08 0.60 1.12 1.21
a
Reference peak.

Box 1 Authentication and determination of P. niruri from P. debilis and 256

Origin and code of the sample.
P. urinaria 257

No. Origin (sub-district, regency) Sample code

P. niruri P. debilis P. urinaria

The proposed HPLC fingerprint method was successfully applied 258

to the three Phyllanthus species obtained from Rajapolah, Tasik- 259

1 Dramaga-1, Bogor PN-1 PD-1 PU-1
malaya (PN-5, PD-5 and PU-5). There were 11 common peaks found 260
2 Dramaga-2, Bogor PN-2 PD-2 PU-2
3 Central Bogor, Bogor PN-3 PD-3 PU-3 in all chromatograms. There are nine characteristic peaks (peaks 1, 261

4 Southern Bogor, Bogor PN-4 PD-4 PU-4 2, 5, 6, 7, 8, 9, 10 and 11) for P. niruri (Fig. 2b1), five characteristic 262
5 Rajapolah, Tasikmalaya PN-5 PD-5 PU-5 peaks (peaks 1, 2, 4, 5 and 11) for P. urinaria (Fig. 2b2) and four 263
6 Purwadadi, Subang PN-6 PD-6 PU-6 characteristic peaks (peaks 1, 2, 3 and 11) for P. debilis (Fig. 2b3). 264
7 Pondok Gede, Bekasi — PD-7 —
8 Ciomas, Bogor — PD-8 —
In order to discriminate P. niruri, P. debilis and P. urinaria, the fin- 265

gerprint chromatograms of all samples were compared. The result 266

showed that six peaks with relatively high intensities (peaks 5, 6, 267

7, 8, 9 and 10) were typical peaks for P. niruri. These peaks may 268

be used for identification and discrimination of P. niruri from P. 269

223 the analytes were stable within 48 h. The results of accuracy, pre- debilis and P. urinaria (Fig. 2b). Peaks 3 and 4 were typical peaks for 270
224 cision and stability are summarized in Table 3. P. debilis and P. urinaria, respectively. These peaks can be used for 271
225 Peaks existed in samples with narrow peak shapes (tailing fac- authentication of P. niruri if there is a contamination from P. debilis 272
226 tor < 1.2), good resolutions (resolution > 1.3) and high intensities and P. urinaria as can be seen in Fig. 2c when P. niruri adulterated 273
227 compared to the other peaks were assigned as characteristic peaks. with P. debilis and P. urinaria. The intensity of typical peaks for P. 274
228 There were nine characteristic peaks (peaks 1, 2, 5, 6, 7, 8, 9, 10 and niruri was linearly decreased with the increasing adulterant con- 275
229 11) found in the fingerprint chromatogram of P. niruri (Fig. 2b1). centrations (P. debilis and P. urinaria). These results showed that 276
230 Peak 9 was chosen as the reference peak since it had a large peak the chromatographic fingerprint might be used for authentication 277
231 area. The RSD of RRT and RPA of the characteristic peaks for intra- of P. niruri from P. debilis and P. urinaria. 278
232 and inter-day precision and stability test were less than 0.53 and Authentication of P. niruri from P. debilis and P. urinaria could be 279
233 1.61%, respectively. The acceptance criteria for precision is ≤2%. The indicated by visual observation of their fingerprint chromatograms 280
234 analytical results of the precision and stability for chromatographic in addition to discriminant analysis (DA) used in this study. DA is 281
235 fingerprint are shown in Table 4. These results indicated that the one of the supervised pattern recognition analyses and mostly uti- 282
236 developed method was reliable for simultaneous determination of lized for a discrimination or classification purpose of the object. DA 283
237 1 and 2 and HPLC fingerprint analysis of P. niruri. will generate a discriminant function (DF) for each group by search- 284

ing a linear combination of data that will give separation of two or 285

more observation groups. In this study, DA was used for authentica- 286

238 Quantitative determination of phyllanthin (1) and tion and discrimination of P. niruri, P. debilis and P. urinaria samples 287

239 hypophyllanthin (2) based on the peak areas of 11 common peaks. DA was performed 288

on 38 objects (20 individual and 18 adulterated samples) and 11 289

240 The proposed HPLC method was successfully applied to simulta- variable data matrices. 290

241 neously determined 1 and 2 in the three Phyllanthus species used in The individual sample used is six P. niruri samples, eight P. debilis 291

242 this work. About 20 samples comprising of six samples of P. niruri, samples and six P. urinaria samples. The adulterated samples used 292

243 eight samples of P. debilis and six samples of P. urinaria were ana- were from six adulterant samples (PD-3, PD-5, PD-6, PU-3, PU-5 293

244 lyzed. The analysis of each sample was performed in triplicates. The and PU-6) with three-level adulteration concentrations (25, 50 and 294

245 contents of 1 and 2 in the six batches of P. niruri were in the range 75%). The result of DA showed that the total variance of two discrim- 295

246 0.73–3.05 and 2.58–6.27 ␮g/mg, respectively. The large variation inant functions (DF) was 98.55% (DF1 = 95.32% and DF2 = 3.23%). 296

247 of 1 and 2 contents in samples from different areas may be due This result showed that the individual samples (P. niruri, P. debilis 297

248 to many factors, such as geographical conditions, environmental and P. urinaria) and adulterated samples (P. niruri with P. debilis 298

249 growth conditions and the age of the plants. Meanwhile, the con- and P. niruri with P. urinaria) could be classified into their own 299

250 tents of 1 and 2 in P. debilis and P. urinaria are mostly not detected groups as shown in Fig. 5. Leave one out cross-validation was used 300

251 except in the samples of PD-2 and PU-5. Overall, the contents of to validate the DA model and gave about 74% of samples were cor- 301

252 1 and 2 in P. niruri are significantly higher than in P. debilis and P. rectly classified into their groups. In this case, peak areas of the 11 302

253 urinaria (Fig. 5). These results showed that the proposed method common peaks are adequate predictors for authentication and dis- 303

254 could be used to identify and discriminate P. niruri from P. debilis crimination of P. niruri, P. debilis, P. urinaria and P. niruri adulterated 304

255 and P. urinaria based on the contents of 1 and 2 in the samples. with P. debilis and P. urinaria. So, by using a combination between 305

Please cite this article in press as: Nasrulloh, R., et al. HPLC fingerprint and simultaneous quantitative analysis of phyllanthin and
hypophyllanthin for identification and authentication of Phyllanthus niruri from related species. Revista Brasileira de Farmacognosia
(2017), https://doi.org/10.1016/j.bjp.2018.04.014
 G Model
BJP 496 1–6 ARTICLE IN PRESS
6 R. Nasrulloh et al. / Revista Brasileira de Farmacognosia xxx (2017) xxxx

306 marker peaks in the three samples and DA plot, we could detect an Bagalkotkar, G., Sagineedu, S.R., Saad, M.S., Stanslas, J., 2006. Phytochemical from 342

307 adulteration of P. niruri with P. debilis and P. urinaria. Phyllanthus niruri Linn. and their pharmacological properties: a review. J. Pharm. 343
Pharmacol. 58, 1559–1570. 344
ICH, 2005. Validation of analytical procedures: text and methodology Q2 (R1). Inter- 345
308 Conclusions national Conference on Harmonization, Geneva. http://www.ich.org/fileadmin/ 346
Public Web Site/ICH Products/Guidelines/Quality/Q2 R1/Step4/Q2 R1 347
Guideline.pdf/ (accessed 10 August 2016). 348
309 In this study, a simple, accurate, and reliable method was devel- Indian Pharmacopoeia, 2010. The Indian Pharmacopoeia Commission, Ghaziabad, 349
310 oped for the identification and authentication of P. niruri from 2488 pp. 350
311 P. debilis and P. urinaria using the established HPLC fingerprint Joseph, B., Raj, S.J., 2011. An overview: pharmacognostic properties of Phyllanthus 351
amarus Linn. Int. J. Pharmacol. 7, 40–45. 352
312 combined with simultaneous quantification of phyllanthin (1) and Kassuya, C.A., Leite, D.F., de Melo, L.V., Rehder, V.L.G., Calixto, J.B., 2005. Anti- 353
313 hypophyllanthin (2). The results showed that the method was inflammatory properties of extracts, fractions and lignans isolated from 354
314 reliable with high accuracy and precision. For the quantitative Phyllanthus amarus. Planta Med. 71, 721–726. 355
Lim, Y., Murtijaya, J., 2007. Antioxidant properties of Phyllanthus amarus extract as 356
315 determination, it was found that 1 and 2 contents in P. niruri were
affected by different drying methods. Food Sci. Technol. 40, 1664–1669. 357
316 much higher than P. debilis and P. urinaria. HPLC fingerprint anal- Ma, T., Huang, C., Meng, X., Zhang, Q., Zhang, L., Lv, X., Jin, Y., Xie, J., Li, J., 2011. 358
317 ysis combined with DA could be used for the discrimination and Fingerprint analysis of Hawk-tea by high-performance liquid chromatography. 359
Food Chem. 129, 551–556. 360
318 authentication of P. niruri from P. debilis and P. urinaria. The devel-
Martins, L.R.R., Pereira-Filho, E.R., Cass, Q.B., 2011. Chromatographic 361
319 oped method, therefore, has a great potential to be widely used profiles of Phyllanthus aqueous extracts samples: a proposition of 362
320 for the identification and authentication of P. niruri in the quality classification using chemometric models. Anal. Bioanal. Chem. 400, 363

321 control process of its raw material. 469–481. 364

Mok, D.K.W., Chau, F.T., 2005. Chemical information of Chinese medicines: a chal- 365
lenge to chemist. Chemom. Intell. Lab. Syst. 82, 210–217. 366
322 Authors’ contributions Murugaiyah, V., Chan, K.L., 2007. Determination of four lignans in Phyllanthus niruri L. 367
by a simple high-performance liquid chromatography method with fluorescence 368
detection. J. Chromatogr. A 1154, 198–204. 369
323 R.N. contributed in collecting and identifying plant samples, Patel, J.R., Tripathi, P., Sharma, V., Chauhan, N.S., Dixit, V.K., 2011. Phyllanthus 370
324 herbarium confection, running the laboratory work, analysis of the amarus: ethnomedicinal uses, phytochemistry and pharmacology: a review. J. 371

325 data, performing chromatographic analysis and drafting the paper. Ethnopharmacol. 138, 286–313. 372
Padilla-Gonzalez, G.F., Aldana, J.A., Da Costa, F.B., 2016. Chemical characterization 373
326 M.R. designed the study, supervised the laboratory work and con- of two morphologically related Espeletia (Asteraceae) species and chemomet- 374
327 tributed in collecting plant samples, statistical analysis and critical ric analysis based on essential oil components. Rev. Bras. Farmacogn. 26, 375
328 reading of the manuscript. W.T.W. supervised the laboratory work 694–700. 376
Pramyothin, P., Ngamtin, C., Poungshompoo, S., Chaichantipyuth, C., 2007. 377
329 and contributed in critical reading of the manuscript. S.S. con- Hepatoprotective activity of Phyllanthus amarus Schum Thonn extract in 378
330 tributed in critical reading and improvement of the manuscript. ethanol treated rats: in vitro and in vivo studies. J. Ethnopharmacol. 114, 379
331 R.H. contributed in multivariate analysis. All authors have read the 169–173. 380
Rafi, M., Wulansari, L., Heryanto, R., Darusman, L.K., Lim, L.W., Takeuchi, T., 2015. 381
332 final manuscript and approved the submission.
Curcuminoid’s content and fingerprint analysis for authentication and discrimi- 382
nation of Curcuma xanthorrhiza from Curcuma longa by high-performance 383
333 Conflicts of interest liquid chromatography-diode array detector. Food Anal. Methods 8, 384
2185–2193. 385
Shanker, K., Singh, M., Srivastava, V., Verma, R.K., Gupta, A.K., Gupta, M.M., 2011. 386
334 The authors declare no conflicts of interest. Simultaneous analysis of six bioactive lignans in Phyllanthus species by reversed 387
phase hyphenated high performance liquid chromatographic technique. Acta 388
Chromatogr. 23, 321–337. 389
335 Acknowledgement
Tan, W., Jaganath, I., Manikam, I., 2013. Evaluation of antiviral activities of four 390
local Malaysian Phyllanthus species against Herpes simplex viruses and possible 391
336 Roni Nasrulloh gratefully acknowledges the Ministry of Agri- antiviral target. Int. J. Med. Sci. 10, 1817–1892. 392
Tang, D.Q., Li, Z., Jiang, X.L., Li, Y.J., Du, Q., Yang, D.Z., 2014. Fingerprint analysis 393
337 culture Republic of Indonesia for the Master scholarship in Bogor
and multi-ingredient quantitative analysis for quality evaluation of Xiaoyanli- 394
338 Agricultural University, Indonesia. dan tablets by ultra high performance liquid chromatography with diode array 395
detection. J. Sep. Sci. 37, 2131–2137. 396
Taiwo, I.A., Oboh, B.O., Francis-Garuba, P.N., 2009. Haematological proper- 397
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Please cite this article in press as: Nasrulloh, R., et al. HPLC fingerprint and simultaneous quantitative analysis of phyllanthin and
hypophyllanthin for identification and authentication of Phyllanthus niruri from related species. Revista Brasileira de Farmacognosia
(2017), https://doi.org/10.1016/j.bjp.2018.04.014