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Determination of triterpenoids in Psidium

guajava

By

Chen Ying

Master of Science

2012

Institute of Chinese Medical Sciences

University of Macau

Determination oftriterpenoids in Psidium guajava

By

Chen Ying

A thesis submitted in partial fulfillment of the

requirements for the degree of

Master of Science

Institute of Chinese Medical Sciences

University of Macau

2012

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supervisor

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In presenting this thesis in partial fulfillment of the requirement for a Master’s degree at the

University of Macau, I agree that the Library and the Institute of Chinese Medical Sciences shall

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Determination of triterpenoids in Psidium guajava

摘要

番石榴Psidium guajava L. 又名番桃树、鸡屎果、番桃,为桃金娘科

(Myrtaceae)番石榴属植物,为我国番石榴属2个引进种之一,在广东省、广西省及福

建省均有种植。文献报道番石榴果实及叶子内含有的三萜类化合物具有降糖和抗肿

瘤等生物活性。本课题对番石榴叶中的三萜类化合物进行了定量分析研究。

一、同步检测番石榴中9个三萜类含量

建立了HPLC-DAD-ELSD联用的检测方法同步检测番石榴中9个三萜类的成分定量

分析，色谱条件为甲醇（B）-0.1%甲醇水（A）梯度洗脱：0-18分钟, 70% B; 18-

20分钟, 70-83% B; 20-60分钟, 83% B。色谱柱为Cosmosil 5C18 柱 (4.6 × 250

mm, 5 μm) 柱温25℃。蒸发光散射检测器的检测条件为漂移管温度40℃，氮气流

速1.6L/min, 8倍增益比。经过一系列的方法学验证表明，此定量分析方法能达到

良好的线性，并具有较好的精密度，稳定性，灵敏度和准确性，可以应用于番石榴

中三萜类成分的定量分析。

采取单因素考察对加压溶剂提取番石榴的最佳提取条件进行了优化，以9个三

萜类的总峰面积做为评价指标，对番石榴的加压溶剂提取条件做了探索，最后优化

得出的最佳提取条件为：溶剂：甲醇；药材粒径：120-140目；提取温度：100°

C；提取时间：10min；提取一次。并且用已建立的HPLC-DAD-ELSD分析方法对15个

番石榴样品中9个三萜酸类进行了定量评价。

二、番石榴的水解实验

建立了HPLC-PAD快速定量测定科罗索酸的方法。色谱条件为乙腈-0.2%甲酸水

(75-25)等度洗脱,色谱柱为Agilent SB-C18柱 (4.6 × 250 mm, 5 μm)，检测波

长210 nm。12分钟内将科罗索酸与山楂酸获得基线分离度。经过一系列的方法学验

证表明，此定量分析方法能达到良好的线性，并具有较好的精密度，稳定性，灵敏

度和准确性，可以应用于番石榴中三萜类成分的定量分析。

发现番石榴中有科罗索酸的酯类存在，设想酸水解的方法可以用来提高番石榴

中科罗索酸的含量。采用平行蒸发浓缩提取反应器作为提取及水解手段，利用正交

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Master of Science, University of Macau

实验设计找到了最佳水解条件为在 100℃，盐酸浓度为 0.5mol/L 条件下，水解五

个小时，发现番石榴叶提取物在水解后科罗索酸含量均有不同程度的提高，而番石

榴果实提取物中水解前后均未检测到科罗索酸。

三、结论

本实验建立了对番石榴中 9个三萜类成分同时定量的分析方法，对 15 个批次的

番石榴中的三萜类进行了定量分析，实验结果表明，三萜类成分主要存在于番石榴

叶提取物中，而番石榴果实提取物中未检测出三萜类成分；建立了快速检测科罗索

酸的分析方法并对番石榴进行了酸水解实验，实验结果表明，酸水解可以有效提高

番石榴中科罗索酸的含量，为寻找科罗索酸的资源提供了一个可靠，简便的方法。

关键词：番石榴，科罗索酸，三萜类，酸水解，定量分析

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Abstract

Psidium guajava L. belonging to the family of Myrtaceae, has been planted in southern

China, such as Guangxi, Guangdong and Fujian province. Triterpenoids are the main

components existed in P. guajava and they possess extensive pharmacological effects.

We aim to analyze the triterpenoids in leaves and fruits of P. guajava in present study.

Chapter1. Review on chemical constituents and pharmacological effects of P. guajava.

Chapter2. Simultaneous determination of nine triterpenoids in P. guajava.

An HPLC-DAD-ELSD method was developed for simultaneous determination of nine

triterpenoids, a cosmosil 5C18 column (4.6 × 250 mm, 5 μm) was used, and the

separation was performed with a gradient mobile phase of methanol (B) and 0.1% formic

acid in water (A) at the rate of 1 min/ml. The gradient condition is: 0-18min, 70% B; 18-

20min, 70-83% B; 20-60min, 83% B. The column temperature was maintained at 25oC

and the injection volume is 10µl. The drift tube temperature for ELSD was 40oC with a

nitrogen flow rate of 1.6 L/min and the gain ratio at 8. The method was validated in terms

of calibration curve, sensitivity, precision, accuracy and stability. The results indicated

that the sensitivity, accuracy and precision were reliable for determination of CA and

nine triterpenoids in P. guajava.

We optimized the extraction condition using a univariate approach and found that using

absolute methanol with the particle size of 120-140 mesh under the temperature of 100oC

and extracting for 10min can get a relatively exhausted extraction. And found that the

determined triterpenoids mainly exists in the leaves not fruits of P. guajava

Chapter 3. Hydrolysis of P. guajava.

A simple, precise and accurate high performance liquid chromatography method was

developed to quickly determine CA in P. guajava. An Agilent SB-C18 (4.6 × 250 mm,

I.D., partical size 5 μm) column was used for separation and analysis. The separation was

performed with a constant mobile phase of acetonitrile and 0.2% formic acid in water

(75:25) at a flow rate of 1 min/ml. The analytes were detected at the wavelength of 210

nm. CA and maslinic acid can be baseline separated within 12 minutes. The method

validation results indicated that the sensitivity, accuracy and precision were reliable for

determination of CA and nine triterpenoids in P. guajava.

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iv

A Syncore Polyvap, Analyst and Reactor was used for extraction and hydrolysis,

orthogonal design experiment was conducted and found that samples treated with 0.5

mol/l hydrochloric acid for 5 hours under the temperature of 100℃ was the best

hydrolysis conditions. The results showed that the content of CA in leaf samples

significantly increased up to154%, and the increasing rates were more than 48.6%,

suggesting that P. guajava is a potential resource rich in CA, and hydrochloric acid

hydrolysis might be a cost-effective approach to produce CA from the leaf of P. guajava

Chapter 4. Conclusion


triterpenoids and successfully applied to determine triterpenoids in 15 samples of P.

guajava. The results showed that The determined triterpenoids mainly exists in the leaves

not fruits of P. guajava. An HPLC-PAD method was established to quickly determine

CA in P. guajava and found that the leaf of P. guajava is a potential resource rich in CA,

and hydrochloric acid hydrolysis might be a cost-effective approach to produce CA from

the leaf of P. guajava.

Keywords: Psidium guajava, corosolic acid, triterpenoids, hydrolysis, quantitative

analysis.


Content

Chapter 1: Review on chemical constituents and pharmacological effects of P. guajava 1

Section 1: Chemical constituents in P. guajava ............................................................. 1

1.1 Triterpenoids ......................................................................................................... 1

1.2 Other constituents ................................................................................................. 3

Section2. Pharmacological effects of P. guajava and its triterpenoids ........................... 4

2.1 Pharmacological effects of P. guajava ................................................................ 4

2.1.1 Anti-diabetes .................................................................................................. 4

2.1.2 Anti-cancer ..................................................................................................... 5

2.1.3 Antioxidant .................................................................................................... 5

2.1.4 Anti-diarrhoeal and anti-bacterial .................................................................. 5

2.1.5 Anti-inflammatory and anti-allergic .............................................................. 6

2.2 Pharmacological effects of triterpenoids in P. guajava ....................................... 6

2.2.1 Asiatic acid ..................................................................................................... 6

2.2.2 Maslinic acid .................................................................................................. 6

2.2.3 Corosolic acid ................................................................................................ 7

2.2.4 Oleanolic acid and ursolic acid ...................................................................... 7

Section3. Analytical methods for determination of chemical constituents in P. guajava

......................................................................................................................................... 8

3.1 Thin Layer Chromatography ................................................................................. 8

3.2 High Performance Liquid Chromatography. ........................................................ 8

Chapter2. Simultaneous determination of nine triterpenoids in P. guajava ..................... 21

Section 1 Materials and instruments ............................................................................. 21

1.1 Materials and reagents. ....................................................................................... 21

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1.2 Instruments .......................................................................................................... 22

Section2: Methods ........................................................................................................ 22

2.1 Sample preparation ............................................................................................. 22

2.2 HPLC analysis .................................................................................................... 23

2.3 Method validation ............................................................................................... 23

Section3: Results and discussion .................................................................................. 24

3.1 HPLC conditions ................................................................................................. 24

3.2 Optimization of PLE procedure .......................................................................... 27


3.2.1 Calibration curves ........................................................................................ 28

3.2.2 Sensitivity .................................................................................................... 28

3.2.3 Precision ....................................................................................................... 29

3.2.4 Accuracy ...................................................................................................... 30

3.2.5 Stability ........................................................................................................ 31

3.4 Sample determination ......................................................................................... 31

Section 4: Summary ...................................................................................................... 33

Chapter3. Hydrolysis of P. guajava .................................................................................. 35

Section1. Materials and instruments ............................................................................. 35

1.1 Materials and Reagents ....................................................................................... 35

1.2 Instruments and apparatus................................................................................... 35

Section 2: Methods ....................................................................................................... 36

2.1 Sample preparation ............................................................................................. 36

2.2 HPLC analysis .................................................................................................... 36


Section 3: Results and discussion ................................................................................. 37

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3.1 Optimization of hydrolysis conditions ................................................................ 37

3.2 Optimization of chromatographic conditions ..................................................... 38


3.4 Sample determination ......................................................................................... 39

Section 4. Summary ...................................................................................................... 42

Chapter4. Conclusion ........................................................................................................ 43

References ......................................................................................................................... 44

Appendix A: HPLC chromatograms of 9 triterpenoids P. guajava samples .................... 51

Appendix B: HPLC chromatogram of hydrolysis samples of P. guajava ........................ 59

Publications ....................................................................................................................... 66

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List of Tables

Table 2.1.1Details of 15 different Psidium guajava ......................................... 21

Table 2.3.1linear regression data, LOD and LOQ of the 9 triterpenoids..........28

Table 2.3.2Intra- and inter- day precision of the 9 triterpenoids ...................... 29

Table 2.3.3Recoveries of the 9 triterpenoids in P. guajava .............................. 30

Table 2.3.4Contents of 9 triterpenoids in P. guajava ....................................... 32

Table 3.3.1Results of the orthogonal design experiments for sample hydrolysis

..............................................................................................................................37

Table 3.3.2Content of CA in samples of P. guajava with and without

hydrolysis .......................................................................................................... 40

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List of Figures

Fig.1.1.1 Chemical structures of 9 determined triterpenoids ............................. 3

Fig.2.3.1 Typical chromatograms of simultaneous determination of 9

triterpenoids..............................................................................................27

Fig.2.3.2 Influence of temperature, extraction duration, particle size and

extraction cycles on PLE (n=3)................................................................. 28

Fig.3.3.1Typical chromatograms of quick determination of CA.......................39

Fig.3.3.2Typical chromatograms of quick determination of CA in P. guajava 41

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List of Abbreviations

HPLC High Performance Liquid Chromatography

DAD Diode Array Detector

ELSD Evaporative Light Scattering Detector

PLE Pressurized Liquid Extraction

ASE Accelerated solvent extraction

LOD Limit of Detection

LOQ Limit of Quantification

RSD Relative Standard Deviation

TLC Thin Layer Chromatography

UV Ultraviolet Visible

PG Psidium guajava

CA Corosolic Acid

S/N Signal-to-Noise

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viii

Acknowledgements

Here I shall take this special opportunity to express my sincerest grateful attitude to my

supervisor Dr. Zhang Qingwen, who has enlightened and guided me using his ultimate

sense of responsibility throughout this two-year research project: You have broadened

my vision with your unpredictable depth of heart; You have given me knowledge and

skill right from the zero line with your unexhausted patience; You have given me wise

advise when I found it nowhere to go, even when I didn’t believe in myself, you are

always there offering help and encouragement. This work will never be fulfilled without

your kindly consideration and steady support. And my hearted thank to Dr. Li Songlin,

who have shared many wise advice and endless inspiration with me. Also deliver my

great thanks to Prof. Wang Yitao, who give me the chance to study here and build such

modern and warm atmosphere for us to conduct our experiments peacefully with passion.

And Prof. Li Shaoping, Dr. Zheng Ying, Dr. Zhao Jing, Dr. Simon Lee, Dr Wan Jianbo,

Dr. Yan Ru, Dr. Maggie Hoi for the support during the two-year’s learning.

This research is supported by grants from Macao Science and Technology Development

Fund (013/2008/A1), grant of University of Macau (MYRG191 (Y1-L3) –ICMS11-ZQW)

and the Team Project of the Natural Science Foundation of Guangdong

(8351063201000003). And thanks Mr. Leon Lai from our institute for his technical

assistance.

Sincerely thank Dang Yuanye, Song Yuelin, Yi Yan, Chu Jun, Zhou Yanqing, Li Ping,

Chen Yangan, Hong Hinchu, Zhu Kan and Xu Faxiang, they have given me endless help

during my two-year study. And my roommate Chen Xiaojia, who is more than a sister

because she has offered me help in both living and learning.

Thanks to my family for offering great support during this research project.


Chapter 1: Review on chemical constituents and

pharmacological effects of P. guajava

Psidium guajava, an important food and medicine dual purposes plant cultivated in

tropical and subtropical regions, has been widely used as food crop and folk medicine,

such as anti-diabetes agent, around the world. [1], and it has been planted in southern

China, such as Guangxi, Guangdong and Fujian province [2].

Section 1: Chemical constituents in P. guajava

1.1 Triterpenoids

Triterpenoids are major components of P. guajava and they contribute a lot to the

pharmacological effects of P. guajava. In 2002, Sabira Begum et al. obtained guavanoic

acid and guavacoumaric acid along with known compounds 2 α -hydroxyursolic acid

(PG-3), jacoumaric acid, isoneriucoumaric acid, asiatic acid (PG-1) from P. guajava [3].

In the same year, they also found one new triterpenoids guajavanoic acid and known

compounds goreishic acid I [4] from the leaves of P. guajava. Oleanolic acid (PG-8),

maslinic acid (PG-2) [5] and ursolic acid (PG-9) [6] were isolated from P. guajava as

well. Recently, four new triterpenes, psiguanins A–D, together with 13 known ones,

jacoumaric acid , 3 β -O-trans-p-coumaroyl maslinic acid (PG-6) , 3 β -O-trans-ferulyl-

2α-hydroxy-urs-12-en-28-oic acid (PG-7), eucalyptolic acid , 3 β -O-cis-coumaroyl-2 α -

hydroxy-urs-12-en-28-oic acid (PG-5), 3 β -O-cis-p-coumaroyl maslinic acid (PG-4), 3 β

-O-cis-ferulyl-2 α -hydroxy-urs-12-en-28-oic acid, 6 β –hydroxy maslinic acid, asiatic

acid, urjinolic acid, 3 β -acetylursolic acid, 3 β,13 β -dihydroxyurs-11-en-28-oic acid, and

3 β -hydroxyurs-11-en-28,13 β -olide were isolated from the leaves of P. guajava [7].

Nine triterpenoids, namely ursolic acid, 1β, 3β-dihydroxyurs-12-en-28-oic acid , 2α, 3β-

dihydroxyurs-12-en-28-oic acid, 3β, 19α-dihydroxyurs-12-en-28-oic acid, 19α-

hydroxylurs-12-en-28-oic-acid-3-O-α-L-2-arabinopyranoside, 3β, 23-dihydroxy-urs-12-

1


en-28-oic acid, 3β, 19α, 23β-tri-hydroxylurs-12-en-28-oic acid, 2α, 3β, 19, 23-

tetrahydroxyurs-12-en-28-oic acid, 3α, 19α, 23, 24-tetrahydroxyurs-12-en-28-oic acid [2]

had been found in the fruits of P. guajava.

R1

R3O

H

COOHH

HR2

Number Chemical name R1 R2 R3

PG-1 Asiatic acid OH OH H

PG-3 Corosolic acid OH H H

PG-5

3β-O-cis-coumaroyl-

2α-hydroxy-urs-12-en-28-oic acid

OH H

OH

O

PG-7

3β-O-trans-coumaroyl-

2α-hydroxy-urs-12-en-28-oic acid

OH H

O

HOPG-9 Ursolic acid H H H

2


R2O

H

COOHH

H

R1

Number Chemical name R1 R2

PG-2 Maslinic acid OH

PG-4

3β-O-cis-coumaroyl-2α-hydroxy-olean-12-en-28-oic

acid

OH

OH

O

PG-6

3β-O-trans-coumaroyl-2α-hydroxy-olean-12-en-28-oic

acid

OH

O

HOPG-8 Oleanolic acid H H

Fig.1.1. 1 Chemical structures of 9 determined triterpenoids

1.2 Other constituents

Flavonoids: Flavonoids are a large part of nature products. In the past years, morin,

morin-3-O-lyxoside, morin-3-O-arabinoside, quercetin, quercetin-3-O-arabinoside [8],

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kaempferol, guaijaverin, avicularin, myricetin, hyperin, apigenin [9], myricetin-3-O-β-D-

glucoside, quercetin-3-O-β-D-glucuronopyranoside, 1-O-galloyl-β-D-glucose [6] had

been isolated from the leaves of P. guajava.

Essential oil: Essential oils from the leaves of P. guajava were analyzed by GC-MS

qualitatively and quantitatively. Among which sixty compounds of the essential oils were

identified at the rate of 90.56%. The major components were caryophyllene with the

percentage of 18.81%, copaene with the percentage of 11.80%, [1aR-(1aα, 4aα, 7α, 7aβ,

7bα)]-decahydro-1,1,7-trimethyl-4-methylene-1H-cycloprop[e] azulene with the

percentage of 10.27% and eucalyptol with the percentage of 7.36% [10]. The major

constituents identified in white and red guavas were ethyl benzoate, cinnamyl alcohol,

(E)-3-hexenyl acetate and α-bisabolene and β-caryophyllene, [11].

Section2. Pharmacological effects of P. guajava and its

triterpenoids

2.1 Pharmacological effects of P. guajava

2.1.1 Anti-diabetes

DM (which is short for diabetes mellitus) is determined to be a chronic metabolic disease,

it can be classified into two types: type 1 diabetes (insulin-dependent diabetes mellitus or

IDDM) and type 2 diabetes (non-insulin dependent diabetes mellitus or NIDDM). During

a screening of medicinal plants in order to find the most helpful inhibition of protein

tyrosine phosphatase1B, an extract from P. guajava leaves showed significant inhibitory

effect on PTP1B, furthermore its antidiabetic effect on Leprdb/Leprdb mice was

evaluated and showed excellent antidiabetic results [12]. In vivo, oral administration of P.

guajava leaf extract to strep to zotocin-induced diabetes rats significantly decreased the

levels of glycosylated hemoglobin, blood glucose and improved the levels of plasma

insulin and hemoglobin [13] and it is further proved that the ethyl acetate fraction of the

leaves plays an important role in its antidiabetic effects [14].

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2.1.2 Anti-cancer

It had been investigated that a hexane fraction of P. guajava induces anticancar activity

by suppressing AKT/Mammalian target of Rapamycin/Ribosomal p70 S6 kinase in

human prostate cancer cell [15], the acetone extracts of P. guajava branch have cytotoxic

effects on HT-29 cells [16], its budding leaves contain huge amounts of soluble

polyphenolics including catechin , gallic acid , epicatechin , rutin, and quercetin that can

exhibit potential anticancer activity [17, 18]. The antitumor effect of P. guajava extracts

by inhibiting T regulatory cells and resultant augmentation of Th1 cells [19] have also

been discussed.

2.1.3 Antioxidant

The fruits, pulp, jam through measuring free acidity, pH, ash, nitrogen and water contents

[20] and leaves through 2,2-diphenyl-1-picrylhydryzyl colorimetry with detection scheme

at 515 nm [21] shows excellent antioxidant activity. And it has been identified that the

phenolic phytochemical which inhibit preoxidantion reaction in the living body [22].

Improved antioxidant potential was also showed by decreasing lipid preoxidation and a

significant increase in the activity of various antioxidant enzymes such as superoxide

dismutase, catalase, glutathione reductase and glutathione peroxidase [14].

2.1.4 Anti-diarrhoeal and anti-bacterial

The effect of anti-diarrhoeal of P. guajava has long been investigated. In 1988, George et

al. had found the antidiarrhoeal effect of P. guajava by observing mice locomotor activity

of a narcotic-like principle [23]. After that spontaneously contracting guinea-pig ileum

and stimulated guinea-pig ileum preparations were employed and the results showed that

quercetin have a good reaction by playing a morphine-like role [24]. Later, it has been

identified that a dose of 0.2 ml/kg fresh leaf extract have the production of 65% inhibition

of propulsion, which is equiactive with 0.2 mg/kg of morphine sulphate [25]. Recently

researcher has found that the decoction of P. guajava [26] and the methanol extract [27]

has anti-bacterial effects towards infectious diarrhoeal and it is not totally due to

quercetin. Finally, Sanches et al. found that flavonoid mixture showed good activity on

Staphylococcus aureusbacterial [28].

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2.1.5 Anti-inflammatory and anti-allergic

In vitro, it has been proved that P. guajava ethyl acetate extract has the inhibition of Fc

epsilon RI-dependent signaling events and inflammatory cytokine production in mast

cells [29]. In vivo, Rattus norvegicus was employed as model and the result showed that

the ethanolic extract has an excellent anti-inflammatory effect [30].

2.2 Pharmacological effects of triterpenoids in P. guajava

Asiatic acid, maslinic acid, corosolic acid, oleanolic acid and ursolic acid are the most

popular triterpenoids owing to their multiple pharmacological effects and they have all

been isolated from P. guajava as we have discussed in Section 1.

2.2.1 Asiatic acid

It has been reported that asiatic acid has anti-cancer effects against human breast cancer

cells by inducing apoptosis and cell cycle rest [31] and against colon cancer cells through

mitochondrial death cascade [32], protect neurons from C(2)-ceramide-induced cell death

by antagonizing mitochondria-dependent apoptosis [33] and it has been further proved by

a mouse model in vivo [34], effect of liver protection has been investigated and the

mechanism lies in induction of Smad-dependent inhibition of Smad-mediated

fibrogenesis [35], via redox-regulated leukotriene C(4) synthase expression pathway [36],

anti-inflammatory effects via increasing the activities of activities of catalase,

glutathione peroxidase and superoxide dismutase in the liver [37], and anti-type 1-

diabetes effects through influences on beta-cell massin diabetic rodent mouse models

[38].

2.2.2 Maslinic acid

The neuroprotection of maslinic acid has been widely investigated in recent years, with

the possible mechanism of suppressing inducible nitric oxide synthase activation

[39], increasing the expression of astrocytic glutamate transporters [40] and reducing

neuroinflammation by inhibiting NF-κB signal transducer pathway [41]. It also has the

activities of anti-cancer against human colon–cancer cell via the mitochondrial apoptotic

6


pathway [42], and the effect of antidiabetes by enhanced the glial glutamate transporter

[43].

2.2.3 Corosolic acid

It had been found that CA (10 mg/kg) could significantly reduce the level of blood

glucose of KK-Ay mice, the mechanism of which involved, at least in part, an increase of

glucose transporter isoform 4 translocation in muscle [44], improving glucose

metabolism through reducing insulin resistance [45], inhibiting hydrolysis of sucrose [46]

and inhibiting alpha-glycosidase [47, 48].

The anti-cancer activities and the mechanisms involved of CA have been extensively

investigated since 1998, when Ahn et al. found that CA could dose-dependently inhibit

protein kinase C [49]. CA can suppress the M2 polarization of macrophages and tumor

cell proliferation by inhibiting both signal transducer and activator of transcription-3 and

nuclear factor-kappa B activation [50]. It can also suppress human epidermal growth

factor receptor expression, which in turn promote apoptotic cell death and cell cycle

arrest of gastric cancer cells [51], and mediate activated protein kinase activation which

lead to inhibition of mammalian target of rapamycin, providing a possible mechanism of

inhibition of cancer cell growth and the induction of apoptosis [52]. Therefore, CA might

be a potential lead compound for the treatment of diabetes and cancer.

2.2.4 Oleanolic acid and ursolic acid

It was investigated that both oleanolic acid and ursolic acid have the effects of anti-cancer

in vitro by ways of down-regulating the expressions of apoptosis antagonistic proteins,

Bcl-2, survivin and Bcl-xL [53], potential anti-cancer agents to cause apoptosis in liver

cancer cell lines of HepG2, Hep3B, Huh7 and HA22T [54], the tests on human colon

carcinoma cell line HCT15 had shown that the effect of UA is stronger than the effect of

OA. The possible mechanism may be that both of them have an inhibitory effect on

tumor cell proliferation by inhibiting cell-cycle arrest [55]. Their anti-cancer effects also

had evaluated in vivo [52]. And both of them have anti-diabetes effects, which have been

tested in the kidney of diabetic mice [56], and can enhance glucose uptake by acting as

insulin sensitizers and as insulin mimics [57]. Other effects such as antioxidative and

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antiglycative [58], antimycobacterial [59], anti-inflammatory [60] and antimutagenic [61]

have also been testified.

Section3. Analytical methods for determination of chemical

constituents in P. guajava

3.1 Thin Layer Chromatography

As a simple and time-saving method to obtain accurate result, TLC is appropriate for the

detection of column chromatography for the separation of triterpenoids [62], furthermore,

Planar Chromatography (HPTLC) was used as a analytical method to qualify content of

quercetin in P. guajava [63].

3.2 High Performance Liquid Chromatography.

High performance liquid chromatography (HPLC) was widely used as a main analytical

method for analysis of Chinese medicine due to its excellent stability, precision, accuracy

and reliability. Up to now, Identification of flavonoids and flavonoid glycosides of P.

guajava leaves was carried out by means of high-performance liquid chromatography

coupled with ultraviolet (HPLC-UV) and mass spectrometry [64], but no HPLC method

has been reported about the qualitative and quantitative analysis of triterpenoids of P.

guajava.

Sample preparation has considered being the bottleneck of the analysis of Chinese

medicine. Recently pressurized liquid extraction has been developing very fast and has

been utilized in the fields of environmental analysis [65], food analysis [66, 67] and

medicinal plant research [66, 68]. As a main extraction apparatus, Accelerate solvent

extractor (named by Dionex for pressurized liquid extraction) has been widely used and

thoroughly explored [69-71] in different species of Chinese medicines in our lab, so

pressurized liquid extraction is chosen as a main extractor.

Due to diode array detector’s convenience and sensitivity for the detection of compounds

with good ultraviolet absorption, while high performance liquid chromatography coupled

with evaporative light scattering detector has been adopted as an efficient tool for

quantitative analysis of compounds such as triterpenoids which have poor ultraviolet

8


9

absorption. So in recent years, HPLC coupled with DAD and ELSD method has been

applied to analyze multiple constituents weather they has a good or poor ultraviolet

absorption of Chinese medicine [72-75].


Chapter2. Simultaneous determination of nine

triterpenoids in P. guajava

There were many strategies for the quality control of Chinese medicines, and high

performance liquid chromatography is the most commonly used [76] owing to its

excellent stability, accuracy and sensitivity. In present study, an HPLC-DAD-ELSD

method was developed for simultaneous determination of nine triterpenoids in P. guajava.

Pressurized liquid extraction was used as the main extraction method because of its

advantages of time-saving, simply-operating and complete-extraction. So we used it as

our main extraction instrument.

Section 1 Materials and instruments

1.1 Materials and reagents.

Methanol, acetonitrile and formic acid (HPLC grade) were purchased from Merck

(Darmstadt, Germany). The ultra-pure water was purified using a Millipore Milli Q-Plus

system (Millipore, Bedford, MA, USA). The reference compound of CA was isolated by

Jinan University, Guangzhou, China. The samples of P.guajava were purchased in local

herbal stores or collected in Guangdong province, China. The details of all samples are

shown in Table 2.1.1. The voucher specimens were deposited in Institute of Chinese

Medical Sciences, University of Macau, Macao SAR, China.

Table 2.1. 1Details of 15 different Psidium guajava

Numbera Location Collector

PGL-1 Zhanjiang Cai

PGL-2 Qingping1 Zhang

PGL-3 Conghua Wen

21


PGL-4 Qingping2 Niu

PGL-5 Shunde Zhou

PGL-6 Gaoming Zhou

PGL-7 Macau1 Chen

PGL-8 Guangzhou Zhang

PGL-9 Foshan Zhou

PGF-1 Macau1 Chen

PGF-2 Gaoming Zhou

PGF-3 Macau2 Chen

PGF-4 Macau3 Chen

PGF-5 Guanngzhou Zhang

PGF-6 Zhuhai Chen

1.2 Instruments

An Agilent HPLC system (Agilent Series 1200, Agilent Technologies, USA), which

constituted with a quaternary solvent delivery system, on-line degasser, auto-sampler,

column compartment, diode array detector and a evaporative light scattering detector

(Alltech 3300, Grace, USA) was used for simultaneous determination of nine

triterpenoids in P. guajava. The data was processed by Chemstation B3.0 (Agilent). A

Cosmosil MS-II 5C18 (4.6 mm × 250 mm, 5 μm) column was used for separation of the

analytes.

Pressurized liquid extraction was performed on a Dionex ASE 200 (Dionex Corp.,

Sunnyvale, USA) System.

Section2: Methods

2.1 Sample preparation

Sample preparation was conducted on a Dionex ASE 200 system under optimized

conditions. Dried powder of P. guajava (0.50g) was mixed with diatomaceous earth with

a weight proportion of 1:1 and placed into a 11ml stainless steel extraction cell, then the

22


extraction was carried out under the following circumstance: 100% Methanol; particle

size: 120-140 mesh; temperature, 100 ℃; statistic extraction time, 10min; static cycle, 1

cycle; pressure, 1500 p.s.i.; flush volume, 40%. Then the extract was transferred to a

25ml volumetric which was made up to its volume with 100% methanol, and filtered

through a 0.45µm Econofilter (Agilent Technologies, USA) before injecting into HPLC.

2.2 HPLC analysis

A Cosmosil MS-II 5C18 (4.6 mm × 250 mm, 5 μm) column was used for separation and

analysis. A gradient mobile phase consisting of 0.1% formic acid in water (A) and

methanol (B) was used for separation, the program is as following: 0-18 min, 70% B; 18-

20 min, 70%-83% B; 20-60 min, 83% B; afterwards there is a washing column with

100% B for 5 min and then return to the initial 70% B with 5 min post run time. The

inject volume was 10 µL and the column temperature was maintained at 25 . In order to

detect each analyte at its maximum wavelength absorption and avoid baseline drift at 210

nm, a wavelength conversion method was adopted. The program was: 0-22.25 min, 210

nm; 22.26-30.00 min, 254 nm; 30.01-38.00 min, 210 nm; 38.01-51.00 min, 310 nm;

51.01-60.00 min, 210 nm. And during optimization of ELSD parameters we found a

circumstance that have a highest signal-to-noise ratio, i.e. S/N, which turned out to be at

the temperature of 40 using the nitrogen flow rate of 1.6 L/min, the gain ratio was set at

8 which is clearly enough to determine the compounds.

2.3 Method validation

The developed method was validated in terms of calibration curve, sensitivity, precision,

accuracy and stability.

For calibration curve construction, known amounts of nine triterpenoids were dissolved

with absolute methanol and the solution was consecutively diluted to obtain five gradient

stock solutions. Then the stock solution ware filtered through a 0.45µm Econofilter

(Agilent Technologies, USA) prior to the HPLC analysis. Each concentration was

analyzed triplicate. Then the calibration curves of PG-4, PG-5, PG-6 and PG-7 were

constructed by directly plotting the peak area versus the concentration of every analytes,

and the calibration curves of PG-1, PG-2, PG-3, PG-8 and PG-9 were constructed by

23


plotting the logarithmic of peak area versus the logarithmic of the concentration of every

analytes.

The sensitivity study was achieved by analyzing the limit of detection (LOD) and limit of

quantification (LOQ) which were determined at a signal-to-noise (S/N) ratio above 3 and

10, respectively.

The precision of the method was determined by intra-day and inter-day repeatability. The

intra-day repeatability was evaluated by extracting and analyzing sample PGL-2 (P.

guajava from Qingping) under the optimized extraction and chromatographic conditions

in three duplicates a day. For inter-day repeatability, the measurement was conducted one

time a day for three consecutive days.

The accuracy of the assay was evaluated by spiking recovery test. Known amounts of the

investigated triterpenoids was added to a certain amount (0.25g) of PGL-8 (P. guajava

from Foshan), then the mixture was extracted and analyzed under the conditions that have

been optimized. Triplicates were carried out in order to compare their R.S.D. The

recovery was calculated with the following equation: Recovery (%) = (amount detected-

amount original)/amount spiked ×100%.

The stability was tested by analyzing the sample of PGL-2 (P. guajava from Qingping) at

0, 2, 4, 6, 8, 10, 12, and 24 h, peak areas of CA ware recorded and compared using R.S.D.

Section3: Results and discussion

3.1 HPLC conditions

24

The optimization of HPLC conditions was performed using sample PGL-2 (P. guajava

from Qingping). Several columns of C18 and C8 from different companies were

compared and different gradient elution (acetonitrile-water and methanol-water) were

tested. In order to avoid the peak tailing and increase the symmetry thus obtain a better

resolution, different kinds of acid with different concentrations (0.1-1%) were used as

modifier. Besides, as to get a higher signal and a lower noise, the ELSD was also be

optimized using univariate approach with the parameters of temperature (35, 40, 45 and

50 and nitrogen flow rate (1.3, 1.4, 1.5, 1.6, 1.7 and 1.8 L/min). Finally, we found that


cosmosil 5C18 column (4.6 × 250 mm, 5 μm) can get a best separation, and the

separation was performed with a gradient mobile phase of methanol (B) and 0.1% formic

acid in water (A) at the rate of 1 min/ml. The gradient condition is: 0-18min, 70% B; 18-

20min, 70-83% B; 20-60min, 83% B. The column temperature was maintained at 25oC

and the injection volume is 10µl. In order to avoid the baseline drift, a wavelength

conversion program was used, the condition is: 0-22.25 min, 210 nm; 22.26-30.00 min,

254 nm; 30.01-38.00 min, 210 nm; 38.01-51.00 min, 310 nm; 51.01-60.00 min, 210 nm.

And at the temperature of 40 and nitrogen flow rate of 1.6 L/min the chromatography

has the highest signal-to-noise. The typical chromatograms are shown in Fig. 2.3.1.

C

25


min0 10 20 30 40 50

mV

200

250

300

350

m in10 2 0 3 0 4 0 5 0

m V

1 5 0

2 0 0

2 5 0

3 0 0

3 5 0

D PG-3

PG-4

PG-5

PG-6

PG-7

PG-9

PG-8

PG-1 PG-2

E

PG-3

F

26


m in10 2 0 3 0 4 0 5 0

m A U

5 6 0

5 7 0

5 8 0

5 9 0

6 0 0

6 1 0

6 2 0

6 3 0

1.32

6

G

Fig.2.3. 1 Typical chromatograms of simultaneous determination of 9 triterpenoids

A: Reference of nine triterpenoids with ELSD; B: Reference of PG4, PG5, PG6 and PG7

in 310nm with DAD; C: Reference of nine triterpenoids with wavelength conversion

with DAD; D: Leaf sample (PGL-8) with ELSD; E: Leaf sample (PGL-8) with

wavelength conversion with DAD. F: Fruit sample (PGF-1) with ELSD; G: Fruit sample

(PGF-1) wavelength conversion with DAD.

3.2 Optimization of PLE procedure

This optimization was carried out using PGL-2 (P. guajava from Qingping). The

parameters including temperature (80, 90, 100 and 110 ), extraction duration (5, 10, 15

and 20min), particle size (80-100, 100-120, 120-140 and 140-160 mesh), and extraction

cycle (1, 2 and 3) were optimized using univariate approach. The total peak areas of 9

triterpenoids were used as markers to evaluate the extraction efficiency and the contents

of 9 triterpenoids showed the same trend as the change of the optimized parameters.

The effects of optimizing parameters on extraction efficiency were shown in Fig. 2.3.2.

The results suggested that particle size was the major factor that affects the extraction.

Taking time-saving into our consideration as well as comparing the results of exhausted

extraction, the best conditions of PLE extraction would be as the following: particle size,

120-140 mesh; temperature, 100 ℃ ; static extraction duration, 10min; number of

extraction times, 1 cycle.

27


Fig.2.3. 2Influence of temperature, extraction duration, particle size and extraction cycles

on PLE (n=3)


3.3.1 Calibration curves

For the calibration cuves, PG-4, PG-5, PG-6 and PG-7 were constructed by directly

plotting the peak area versus the concentration of each analyte, and the calibration curves

of PG-1, PG-2, PG-3, PG-8 and PG-9 were constructed by plotting the logarithmic of

peak area versus the logarithmic of the concentration of each analyte, the results were

shown in Table. 2.3.1.

3.3.2 Sensitivity

Fir the sensitivity test, the stock solutions were diluted until S/N was about 3 and 10, the

results were shown in Table. 2.3.1.

Table 2.3. 1linear regression data, LOD and LOQ of the 9 triterpenoids

Analytes Retation Time Calibration curve

Test range (µg)

R2 LOD(ng) LOQ(ng)

PG-1 22.495 y = 1.39x + 4.46 30.25-484 0.9993 33.36 111.21

28


PG-2 34.946 y = 1.52x + 4.76 34.25-548 0.9992 29.87 99.56

PG-3 36.418 y = 1.50x + 4.60 77.5-1240 0.9993 29.51 98.35

PG-4 40.182 y = 8,165.82x -0.51 2.81-22.5 1.0000 3.01 10.05

PG-5 42.482 y = 10,232.97x - 5.44 5.16-82.5 0.9998 2.54 8.47

PG-6 46.753 y = 13,596.12x - 3.55 4.69-75 0.9998 1.84 6.14

PG-7 49.802 y = 14,543.73x - 11.33

5.39-86.25 0.9999 2.29 7.65

PG-8 54.487 y = 1.57x + 4.24 73.75-1180 0.9993 108.46 361.52

PG-9 56.274 y = 1.59x + 4.44 47.5-760 0.9992 97.60 325.34

3.3.3 Precision

The precision of the method was determined by intra-day and inter-day repeatability, the

results were shown in Table 2.3.2.

Table 2.3. 2Intra- and inter- day precision of the 9 triterpenoids

Triterpenoids Intra-day (n=3) Inter-day (n=3)

Content R.S.D (%) Content R.S.D (%)

PG1 4.30±0.06 1.35 4.48±0.20 4.50

PG2 3.6±0.10 2.84 3.64±0.01 0.39

PG3 19.27±0.14 0.70 19.4±0.86 4.44

PG4 0.7±0.03 4.97 0.70±0.01 1.81

PG5 1.25±0.04 2.94 1.22±0.03 2.18

PG6 0.77±0.03 4.20 0.77±0.02 2.70

PG7 1.63±0.05 3.25 1.60±0.03 1.90

PG8 N.D. N.A. N.D. N.A.

29


PG9 4.26±0.05 1.21 4.35±0.17 3.96

3.3.4 Accuracy

The accuracy of the assay was evaluated by spiking recovery test, the results were

summarized in Table 2.3.3.

Table 2.3. 3Recoveries of the 9 triterpenoids in P. guajava

Triterpenoids Original

(mg) Spike (mg) Found (mg)

Recovery

(%) R.S.D. (%)

PG1 1.54

1.23 2.76 99.19 1.42

1.60 3.25 106.87 1.02

1.89 3.54 105.82 1.57

PG2 1.33

1.01 2.32 98.02 1.64

1.38 2.74 102.17 3.37

1.60 2.90 98.12 2.60

PG3 6.81

5.50 12.45 102.55 1.67

6.80 13.70 101.32 1.24

8.16 14.85 98.5 3.57

PG4 0.32

0.22 0.55 104.54 1.04

0.33 0.66 103.03 1.97

0.40 0.73 102.5 4.42

PG5 0.67

0.52 1.16 94.23 0.22

0.62 1.27 96.77 4.33

0.81 1.46 97.53 2.49

PG 6 0.51 0.35 0.85 97.14 3.80

30


0.52 1.05 103.84 2.74

0.62 1.16 104.84 8.50

PG7 1.18

0.80 1.94 95 0.70

1.18 2.39 102.5 1.15

1.42 2.62 102.82 0.24

PG8 0.46

4.27 4.48 104.92 3.20

4.98 5.32 106.83 1.87

5.55 5.85 105.41 3.51

PG9 1.77

1.50 3.30 102 0.73

1.79 3.58 101.69 1.32

2.10 3.77 95.24 0.76

3.3.5 Stability



The results indicated that the sample was stable within at least 24 hours.

3.4 Sample determination

The identification of the 9 triterpenoids was carried out by comparing their retention time

and UV spectra with references under the same HPLC conditions. The developed HPLC-

DAD-ELSD method was applied to analyze 9 triterpenoids in 15 P. guajava samples.

Among them, PG1, PG2, PG3, PG8, PG 9 were analyzed using ELSD whereas PG 4, PG

5, PG 6, PG 7 were analyzed using DAD because they have UV absorption. The results

were shown in Table 2.3.5.

From Table 2.3.5 it was found that triterpenoids mainly exists in leaves not fruits of P.

guajava. Taking corosolic acid as a marker because a HPLC-PAD method was used to

quickly determine its content in P. guajava (as is shown in section 2), we can see that

31


ELSD has a lower LOD and LOQ, which make it more appropriate for the determination

of triterpenoids that has a weaker UV absorption.

Table 2.3. 4Contents of 9 triterpenoids in P. guajava sa

mpl

es

PG1±

R.S

.D

PG2±

R.S

.D

PG3±

R.S

.D

PG4±

R.S

.D

PG5±

R.S

.D

PG6±

R.S

.D

PG7±

R.S

.D

PG8±

R.S

.D

PG9±

R.S

.D

Tota

l

PGL-

1

2.50

±0.0

9

1.35

±0.0

7

7.05

±0.3

4

0.45

±0.1

1.01

±0.0

8

0.75

±0.0

3

1.78

±0.0

1

-

1.85

±0.0

5

17.2

4

PGL-

2

4.35

±0.0

7

3.38

±0.1

2

18±0

.30

0.63

±0.0

2

1.18

±0.0

2

0.81

±0.0

2

1.53

±0.0

6

-

3.80

±0.0

6

34.6

2

PGL-

3

2.75

±0.0

7

2.15

±0.0

7

11.9

1±0.

13

0.46

±0.0

3

0.91

±0.0

2

0.62

±0.0

3

1.41

±0.0

8

-

2.50

±0.1

0

23.4

2

PGL-

4

3.34

±0.0

6

2.68

±0.0

9

14.1

7±0.

18

0.4±

0.01

0.81

±0.0

4

0.52

±0.0

1

1.11

±0.0

3

-

3.15

±0.0

6

26.9

8

PGL-

5

3.26

±0.1

5

11.6

5±0.

24

5.95

±0.0

7

0.41

±0.0

2

0.77

±0.0

3

0.50

±0.0

3

1.21

±0.0

7

-

2.08

±0.1

2

26.5

7

PGL

-6

2.20

±0.0

9

0.91

±0.0

5

4.35

±0.0

4

0.27

±0.0

1

0.55

±0.0

4

0.34

±0.0

1

0.94

±0.0

2

-

1.74

±0.0

9

11.8

1

PGL

-7

3.36

±0.0

5

2.31

±0.0

3

11.9

7±0.

17

0.77

±0.0

4

1.34

±0.0

5

0.80

±0.0

2

1.73

±0.0

6

-

3.85

±0.1

2

27.0

0

32


PGL

-8

3.88

±0.1

0

2.83

±0.0

9

15.2

5±0.

32

0.78

±0.0

2

1.26

±0.0

6

0.74

±0.0

4

1.56

±0.0

3

-

3.93

±0.0

3

31.0

7

PGL

-9

3.08

3±0.

19

2.67

±0.2

0

13.6

2±0.

30

0.63

±0.0

6

1.35

±0.0

5

1.03

±0.0

2

2.21

±0.0

3

-

3.54

±0.1

9

29.2

0

PGF-

1

- - - - - - - - - -

PGF-

2

- - - - - - - - - -

PGF-

3

- - - - - - - - - -

PGF-

4

- - - - - - - - - -

PGF-

5

- - - - - - - - - -

PGF-

6

- - - - - - - - - -

“a”PGL=Leaves of P. guajava, PGF=Fruits of P. guajava. “-” undetectable.

Section 4: Summary


triterpenoids in P. guajava. The method was validated in terms of calibration curve,

33


34

sensitivity, precision, accuracy and stability. And it was proved that they had good

repeatability, accuracy and precision and were reliable for determination of nine

triterpenoids in P. guajava. The developed HPLC-DAD-ELSD method was applied to

analyze 9 triterpenoids in 15 P. guajava samples, and it was found that triterpenoids

mainly exists in leaves not fruits of P. guajava


Chapter3. Hydrolysis of P. guajava

With the existence of esters group of CA, we assume that hydrolysis can increase the

content of CA in P. guajava. A Syncore Polyvap, Analyst and Reactor were used for

extraction and hydrolysis.

Section1. Materials and instruments

1.1 Materials and Reagents

Methanol, acetonitrile and formic acid (HPLC grade) and hydrochloric acid (AR grade)

from Merck (Darmstadt, Germany), methanol (AR grade) from Kaitong (Tianjin, China),

and ethanol and acetone (AR grade) from UNI-CHEM (Hungary) were purchased. The

ultra-pure water was purified using a Millipore Milli Q-Plus system (Millipore, Bedford,

MA, USA). The reference compound of CA was isolated by Jinan University,

Guangzhou, China. The samples of P. guajava were purchased in local herbal stores or

collected in Guangdong province, China. The details of all samples are shown in Table

2.1.1. The voucher specimens were deposited in Institute of Chinese Medical Sciences,

University of Macau, Macao SAR, China.

1.2 Instruments and apparatus

A Waters 2695 HPLC system (Waters, Milford, USA) coupled with a quaternary solvent

delivery system, on line degasser, column compartment, auto sampler and Waters 2996

photodiode array detector were used for a quick quantitative determination of CA in P.

guajava. The data was processed by Empower software. Agilent SB-C18 (4.6 mm × 250

mm, 5 μm) column was used for the separation and analysis.

Hydrolysis was carried out on a Syncore Polyvap, Analyst and Reactor (BUCHI,

Switzerland).

35


Section 2: Methods

2.1 Sample preparation

The dried leaf and fruit powder of P. guajava was accurately weighed (0.50 g), fluxed

with methanol for 5 h under the temperature of 100oC, then the extract solution was

transferred into a 25 ml volumetric flask which was made up to its volume with

extraction solvent. The resultant solution was centrifuged (3000 r/min) for 15 min under

20oC, the supernatant was filtered through a 0.45 μm Econofilter (Agilent Technologies,

USA) prior to injection into the HPLC system.

2.2 HPLC analysis

An Agilent SB-C18 (4.6 mm × 250 mm, 5 μm) column was used for separation and

analysis. The separation was performed with a constant mobile phase of acetonitrile (B)

and 0.2% formic acid in water (A) (75:25) at a flow rate of 1 ml/min. The chromatogram

was monitored with a Photodiode array detector at the wavelength of 210 nm; the sample

injection volume was 10 μl.


The developed method was validated in terms of calibration curve, sensitivity, precision,

accuracy and stability.

For calibration curve construction, known amounts CA were dissolved with absolute

methanol and the solution was consecutively diluted to obtain five gradient stock

solutions. Then the stock solution ware filtered through a 0.45µm Econofilter (Agilent

Technologies, USA) prior to the HPLC analysis. Each concentration was analyzed

triplicate. Then the calibration curve of CA was constructed by directly plotting the peak

area versus the concentration of each analyte.

The sensitivity study was achieved by analyzing the limit of detection (LOD) and limit of

quantification (LOQ) which was determined at a signal-to-noise (S/N) ratio above 3 and

10, respectively.

36


The precision of the method was determined by intra-day and inter-day repeatability. The

intra-day repeatability was evaluated by extracting and analyzing sample PGL-2 (P.

guajava from Qingping) under the optimized extraction and chromatographic conditions

in three duplicates a day. For inter-day repeatability, the measurement was conducted one

time a day for three consecutive days.

The accuracy of the assay was evaluated by spiking recovery test. Known amounts of the

investigated triterpenoids was added to a certain amount (0.25g) of PGL-2 (P. guajava

from Qingping), then the mixture was extracted and analyzed under the conditions that

have been optimized. Triplicates were carried out in order to compare their R.S.D. The

recovery was calculated with the following equation: Recovery (%) = (amount detected-

amount original)/amount spiked ×100%.



Section 3: Results and discussion

3.1 Optimization of hydrolysis conditions

The hydrochloric acid hydrolysis conditions were optimized by an orthogonal design

experiments, the results were summarized in Table 3.3.1. By comparing the range of

these three factors, it could be found that these three factors had different effects on the

hydrolysis of CA esters. From the K values, the best hydrolysis conditions for CA esters

could be figured out, i.e., samples were treated with 0.5 mol/l hydrochloric acid for 5

hours under the temperature of 100 oC.

Table 3.3. 1Results of the orthogonal design experiments for sample hydrolysis

Temperature

(oC)

Time

(h)

Acid

Concentration

(mol/l)

CA Content

(mg/g)

1 80 3 0.25 5.19

2 80 4 0.5 5.96

3 80 5 1 5.64

37


4 90 3 0.5 5.58

5 90 4 1 6.37

6 90 5 0.25 5.62

7 100 3 1 6.01

8 100 4 0.25 5.77

9 100 5 0.5 7.11

K1 5.50 5.54 5.48

K2 5.81 5.98 6.16

K3 6.24 6.07 5.96

Range 0.69 0.53 0.68

3.2 Optimization of chromatographic conditions

In the previous publications on the analysis of P. guajava, different kinds of acid with

different concentrations (0.1-1%) were used as modifier to reduce peak tailing of the

analytes, thus leading to the improvement of resolution [77]. In present study, formic acid

and acetic acid as modifier, and methanol and acetonitrile as organic phase were tested

for the quick determination of CA. It was found that the baseline separation of CA from

other analytes could be achieved within 11 min, when the mobile phase composed of

acetonitrile and 0.2% formic acid in water with the proportion of 75: 25 at the flow rate

of 1 ml/min.

The resolution of several columns of C18 and C8 from different companies were also

compared, it was found that Agilent SB-C18 (4.6 mm × 250 mm, 5 μm) column was

suitable for the determination of CA (Fig. 3.3.1)

38


Fig.3.3 1Typical chromatograms of quick determination of CA

A: Reference of Corosolic acid; B: Leaf sample (PGL-2); C: Fruit sample (PGF-1); 1:

Corosolic acid


The calibration curve was figured out to be y = 4.36×106 x +1.04×104 with R2 = 0.9999;

the LOQ and LOD were 221.0 and 66.3 ng, respectively; the relative standard deviations

(RSDs) of intra-day and inter-day repeatability were 1.8% and 1.5%, respectively; the

average spike recovery (n = 6) was 100.8%; and the stability study showed that the

sample was stable within at least 24 hours.

3.4 Sample determination

The established quick CA determination method was used to determine seven leaf

samples and five fruits samples of P. guajava with or without hydrochloric acid

hydrolysis, the representative chromatograms were shown in Fig. 3.3.1, the results were

A 1

1 B

AU

0.00

0.05

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.0

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

Minutes

0.10

A 1

AU 0.05

0.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

B1

C

39


summarized in Table 3.3.2. From Table 3.3.2, it was found that CA could be detected in

all seven leaf samples with the range from 3.84 to 9.94 mg/g of the dried leaves. The

highest content was found in the samples from Qingping and Gaoming China, which is

up to almost 1.0%, although more samples should be analyzed to find out the best

location.

It was also excited to find that hydrochloric acid hydrolysis could significantly increase

the content of CA, the increasing rates are from 48% to 125% among all six leaf samples

determined, suggesting that CA co-exist with its esters in the leaves of P. guajava.

Hydrochloric acid hydrolysis might be a cost-effective approach to produce CA from the

leaf of P. guajava.

The developed method was also applied to analyze five fruit samples of P. guajava.

However, CA was undetectable in all five fruit samples with or without hydrochloric acid

hydrolysis under the present chromatographic conditions.

Table 3.3. 2Content of CA in samples of P. guajava with and without hydrolysis

samplea Without hydrolysis With hydrolysis Increase rate (%)

PGL-1 5.03 ± 0.05 9.08 ± 0.05 80.60

PGL-2 9.82 ± 0.24 15.38 ± 0.02 56.60

PGL-3 8.23 ± 0.13 12.76 ± 0.10 55.07

PGL-4 9.94 ± 0.10 14.77 ± 0.49 48.60

PGL-5 3.84 ± 0.18 8.67 ± 0.03 125.80

PGL-6 2.57±0.12 5.69±0.32 121.40

PGL-7 8.54 ± 0.03 14.71 ± 0.06 72.20

PGF-1 - - /

PGF-2 - - /

PGF-3 - - /

PGF-4 - - /

40


PGF-5 - - /

“a”PGL=Leaves of Psidium guajava, PGF=Fruits of Psidium guajava.“-” undetectable;

“/” not applicable

Fig.3.3 2Typical chromatograms of quick determination of CA in P. guajava

A: Leaf sample (PGL-2) without hydrolysis; B: Leaf sample (PGL-2) with hydrolysis. C:

Fruit sample (PGF-1) without hydrolysis; D: Fruit sample (PGF-1)with hydrolysis; 1:

Corosolic acid

AU

0 . 0 0

0 . 0 2

0 . 0 4

0 . 0 6

0 . 0 8

0 . 1 0

M in u t e s0 . 0 0 2 . 0 0 4 . 0 0 6 . 0 0 8 . 0 0 1 0 . 0 0 1 2 . 0 0

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

AU

0.00

0.05

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.0

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

Minutes

A 1

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

B 1

C

D

41


Section 4. Summary

The leaf of P. guajava is the potential resource rich in CA. Hydrochloric acid hydrolysis

could significantly increase the content of CA in leaf samples, and might be a cost-

effective way to produce CA from the leaf of P. guajava.

42


Chapter4. Conclusion


triterpenoids and successfully applied to determine triterpenoids in 15 samples of P.

guajava. The results showed that The determined triterpenoids mainly exists in the leaves

not fruits of P. guajava. An HPLC-PAD method was established to quickly determine

CA in P. guajava and found that the leaf of P. guajava is a potential resource rich in CA,

and hydrochloric acid hydrolysis might be a cost-effective approach to produce CA from

the leaf of P. guajava.

43


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50


Appendix A: HPLC chromatograms of 9 triterpenoids P.

guajava samples

PGL-1

51


PGL-2

PGL-3

52


PGL-4

PGL-5

53


PGL-6

PGL-7

54


PGL-8

PGL-9

55


PGF-1

PGF-2

56


PGF-3

PGF-4

57


PGF-5

PGF-6

58


Appendix B: HPLC chromatogram of hydrolysis

samples of P. guajava AU

0.00

0.05

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00

PGL-1 without hydrolysis

AU

0.00

0.05

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00

PGL-1 with hydrolysis

AU

0.00

0.05

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00

CA

CA

CA


59


AU

0.00

0.05

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00

CA


AU

0.00

0.05

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00

CA


AU

0.00

0.05

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00

CA


AU

0.00

0.05

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00

CA


60


AU

0.00

0.05

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00


AU

0.00

0.05

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00


AU

0.00

0.05

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00


AU

0.00

0.05

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00

CA

CA

CA

CA


61


AU

0.00

0.05

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00


AU

0.00

0.05

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00


AU

0.00

0.05

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00

CA

CA


AU

0.00

0.05

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00

CA

PGF-1 without hydrolysis

62


AU

0.00

0.05

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00

PGF-1 with hydrolysis

AU

0.00

0.05

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00


AU

0.00

0.05

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00


AU

0 .00

0.02

0.04

0.06

0.08

0.10

0.12

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00


63


AU

0.00

0.05

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00


AU

0 .00

0.02

0.04

0.06

0.08

0.10

0.12

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00


AU

0.00

0.05

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00


AU

0 .00

0.02

0.04

0.06

0.08

0.10

0.12

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00


64


AU

0.00

0.05

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00


65


66

Publications

Ying Chen et al. Psidium guajava, a potential resource rich in corosolic acid revealed by

high performance liquid chromatography, Journal of Medicinal Plants Research, 2011,

5(17): 4261-4266.

Liming Lu, Jingchun Zeng and Ying Chen, Quality of reporting in randomized

controlled trials conducted in China on the treatment of cancer pain, Expert review of

anticancer therapy 2011; 11(6):871-877.

JianBo Wan, Ying Chen et al. Saponins from Panax Species: Chemistry, Isolation and

Analysis. in Rani Koh and Isaac Tay: Saponins: Properties, Applications and Health

Benefits. Nova Publishers, 2012 (in press)

Ying Chen et al. Corosolic acid in Psidium guajava (Abstract), 10th CGCM in Shanghai

on 25th-28th, Aug., 2011.

determination of triterpenoids in psidium guajavalibrary.umac.mo/etheses/b25903214_ft.pdfin...

Documents