Post on 07-Mar-2020
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
Approved by_______________________________________________
supervisor
_______________________________________________
_______________________________________________
Date _______________________________________________
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
make its copies freely available for inspection. However, reproduction of the thesis for any
purpose or by any means shall not be allowed without my written permission. Authorization is
sought by contacting the author at
Address:
Telephone:
Fax:
E-mail:
Signature
Date
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
i
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
ii
Determination of triterpenoids in Psidium guajava
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.
iii
Master of Science, University of Macau
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
An HPLC-DAD-ELSD method was developed for simultaneous determination of nine
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.
Master of Science, University of Macau
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
ii
Determination of triterpenoids in Psidium guajava
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
3.3 Method validation ............................................................................................... 28
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
2.3 Method validation ............................................................................................... 36
Section 3: Results and discussion ................................................................................. 37
iii
Master of Science, University of Macau
3.1 Optimization of hydrolysis conditions ................................................................ 37
3.2 Optimization of chromatographic conditions ..................................................... 38
3.3 Method validation ............................................................................................... 39
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
iv
Determination of triterpenoids in Psidium guajava
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
v
Master of Science, University of Macau
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
vi
Determination of triterpenoids in Psidium guajava
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
vii
Master of Science, University of Macau
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.
Determination of triterpenoids in Psidium guajava
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
Master of Science, University of Macau
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
Determination of triterpenoids in Psidium guajava
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],
3
Master of Science, University of Macau
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].
4
Determination of triterpenoids in Psidium guajava
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].
5
Master of Science, University of Macau
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
Determination of triterpenoids in Psidium guajava
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
7
Master of Science, University of Macau
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
Determination of triterpenoids in Psidium guajava
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].
Determination of triterpenoids in Psidium guajava
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
Master of Science, University of Macau
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
Determination of triterpenoids in Psidium guajava
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
Master of Science, University of Macau
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
Determination of triterpenoids in Psidium guajava
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
Master of Science, University of Macau
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
Determination of triterpenoids in Psidium guajava
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
Master of Science, University of Macau
Fig.2.3. 2Influence of temperature, extraction duration, particle size and extraction cycles
on PLE (n=3)
3.3 Method validation
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
Determination of triterpenoids in Psidium guajava
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
Master of Science, University of Macau
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
Determination of triterpenoids in Psidium guajava
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 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.
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
Master of Science, University of Macau
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
Determination of triterpenoids in Psidium guajava
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
An HPLC-DAD-ELSD method was developed for simultaneous determination of nine
triterpenoids in P. guajava. The method was validated in terms of calibration curve,
33
Master of Science, University of Macau
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
Determination of triterpenoids in Psidium 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
Master of Science, University of Macau
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.
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 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
Determination of triterpenoids in Psidium guajava
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%.
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.
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
Master of Science, University of Macau
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
Determination of triterpenoids in Psidium guajava
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
3.3 Method validation
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
Master of Science, University of Macau
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
Determination of triterpenoids in Psidium guajava
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
Master of Science, University of Macau
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
Determination of triterpenoids in Psidium guajava
Chapter4. Conclusion
An HPLC-DAD-ELSD method was developed for simultaneous determination of nine
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
Master of Science, University of Macau
References
[1]. Gutirrez, R.M.P.; Mitchell, S.; Solis, R.V. Psidium guajava: A review of its traditional uses,
phytochemistry and pharmacology. Journal of Ethnopharmacology. 2008, 117 (1), 1-27.
[2]. Shu, J.; Chou, G.; Wang, Z. [Triterpenoid constituents in fruits of Psidum guajava]. Zhongguo
Zhong Yao Za Zhi = Zhongguo Zhongyao Zazhi = China Journal Of Chinese Materia Medica.
2009, 34 (23), 3047-3050.
[3]. Begum, S.; Hassan, S.I.; Siddiqui, B.S.; Shaheen, F.; Ghayur, M.N.; Gilani, A.H. Triterpenoids
from the leaves of Psidium guajava. Phytochemistry. 2002, 61 (4), 399-403.
[4]. Begum, S.; Siddiqui, B.S.; Hassan, S.I. Triterpenoids from Psidium guajava leaves. Natural
Product Letters. 2002, 16 (3), 173-177.
[5]. Begum, S.; Hassan, S.I.; Siddiqui, B.S. Two new triterpenoids from the fresh leaves of Psidium
guajava. Planta Medica. 2002, 68 (12), 1149-1152.
[6]. Fu, H.; Luo, Y.; Zhang, D. Studies on chemical constituents of leaves of Psidium guajava. China
Journal Of Chinese Materia Medica. 2009, 34 (5), 577-579.
[7]. Shao, M.; Wang, Y.; Huang, X.J.; Fan, C.L.; Zhang, Q.W.; Zhang, X.Q.; Ye, W.C. Four new
triterpenoids from the leaves of Psidium guajava. Journal Of Asian Natural Products Research.
2012, 14 (4), 348-354.
[8]. Rattanachaikunsopon, P.; Phumkhachorn, P. Bacteriostatic effect of flavonoids isolated from
leaves of Psidium guajava on fish pathogens. Fitoterapia. 2007, 78 (6), 434-436.
[9]. Wang, H.; Du, Y.J.; Song, H.C. α-Glucosidase and α-amylase inhibitory activities of guava leaves.
Food Chemistry. 2007, 123 (1), 6-13.
[10]. Li, J.; Chen, F.; Luo, J. GC-MS analysis of essential oil from the leaves of Psidium guajava.
Journal Of Chinese Medicinal Materials. 1999, 22 (2), 78-80.
[11]. Thuaytong, W.; Anprung, P. Bioactive compounds and prebiotic activity in Thailand-grown red
and white guava fruit (Psidium guajava L.). Food Science And Technology International. 2011, 17
(3), 205-212.
[12]. Oh, W.K.; Lee, C.H.; Lee, M.S.; Bae, E.Y.; Sohn, C.B.; Oh, H.; Kim, B.Y.; Ahn, J.S. Antidiabetic
effects of extracts from Psidium guajava. Journal of Ethnopharmacology. 2005, 96 (3), 411-415.
[13]. Subramanian, S.; Banu, H.H.; Bai, R.M.R.; Shanmugavalli, R. Biochemical evaluation of
antihyperglycemic and antioxidant nature of Psidium guajava leaves extract in streptozotocin-
induced experimental diabetes in rats. Pharmaceutical Biology. 2009, 47 (4), 298-303.
44
Determination of triterpenoids in Psidium guajava
[14]. Soman, S.; Rauf, A.A.; Indira, M.; Rajamanickam, C. Antioxidant and Antiglycative Potential of
Ethyl Acetate Fraction of Psidium guajava Leaf Extract in Streptozotocin-Induced Diabetic Rats.
Plant Foods for Human Nutrition. 2010, 65 (4), 386-391.
[15]. Ryu, N.H.; Park, K.R.; Kim, S.M.; Yun, H.M.; Nam, D.; Lee, S.G.; Jang, H.J.; Ahn, K.S.; Kim,
S.H.; Shim, B.S.; Choi, S.H.; Mosaddik, A.; Cho, S.K. A Hexane Fraction of Guava Leaves
(Psidium guajava L.) Induces Anticancer Activity by Suppressing AKT/Mammalian Target of
Rapamycin/Ribosomal p70 S6 Kinase in Human Prostate Cancer Cells. Journal of Medicinal Food.
2012, 15 (3), 231-241.
[16]. Lee, S.B.; Park, H.R. Anticancer activity of guava (Psidium guajava L.) branch extracts against
HT-29 human colon cancer cells. Journal of Medicinal Plants Research. 2010, 4 (10), 891-896.
[17]. Chen, K.C.; Hsieh, C.L.; Huang, K.D.; Ker, Y.B.; Chyau, C.C.; Peng, R.Y. Anticancer Activity of
Rhamnoallosan against DU-145 Cells Is Kinetically Complementary to Coexisting Polyphenolics
in Psidium guajava Budding Leaves. Journal of Agricultural and Food Chemistry. 2009, 57 (14),
6114-6122.
[18]. Chen, K.C.; Hsieh, C.L.; Peng, C.C.; Hsieh-Li, H.M.; Chiang, H.S.; Huang, K.D.; Peng, R.Y.
Brain derived metastatic prostate cancer DU-145 cells are effectively inhibited in vitro by guava
(Psidium gujava L.) leaf extracts. Nutrition and Cancer-an International Journal. 2007, 58 (1), 93-
106.
[19]. Seo, N.; Ito, T.; Wang, N.; Yao, X.; Tokura, Y.; Furukawa, F.; Takigawa, M.; Kitanaka, S. Anti-
allergic Psidium guajava extracts exert an antitumor effect by inhibition of T regulatory cells and
resultant augmentation of Th1 cells. Anticancer Research. 2005, 25 (6A), 3763-3770.
[20]. Marquina, V.; Araujo, L.; Ruz, J.; Rodrguez-Malaver, A.; Vit, P. Composition and antioxidant
capacity of the guava (Psidium guajava L.) fruit, pulp and jam. Archivos Latinoamericanos De
Nutricin. 2008, 58 (1), 98-102.
[21]. Qian, H.; Nihorimbere, V. Antioxidant power of phytochemicals from Psidium guajava leaf.
Journal Of Zhejiang University. Science. 2004, 5 (6), 676-683.
[22]. Sanda, K.A.; Grema, H.A.; Geidam, Y.A.; Bukar-Kolo, Y.M. Pharmacological Aspects of
Psidium guajava: An Update. International Journal of Pharmacology. 2010, 7 (3), 316-324.
[23]. Lutterodt, G.D.; Maleque, A. Effects on mice locomotor activity of a narcotic-like principle from
Psidium guajava leaves. Journal of Ethnopharmacology. 1988, 24 (2-3), 219-231.
[24]. Lutterodt, G.D. Inhibition of gastrointestinal release of acetylchoune byquercetin as a possible
mode of action of Psidium guajava leaf extracts in the treatment of acute diarrhoeal disease.
Journal of Ethnopharmacology. 1989, 25 (3), 235-247.
45
Master of Science, University of Macau
[25]. Lutterodt, G.D. Inhibition of microlax-asterisk-induced experimental diarrhea with narcotic-like
extracts of Psidium-guajava leaf in rats. Journal of Ethnopharmacology. 1992, 37 (2), 151-157.
[26]. Birdi, T.; Daswani, P.; Brijesh, S.; Tetali, P.; Natu, A.; Antia, N. Newer insights into the
mechanism of action of Psidium guajava L. leaves in infectious diarrhoea. Bmc Complementary
and Alternative Medicine. 2010, 10 11.
[27]. Goncalves, F.A.; Neto, M.A.; Bezerra, J.N.S.; Macrae, A.; de Sousa, O.V.; Fonteles, A.A.; Vieira,
R. Antibacterial activity of guava, Psidium guajava Linnaeus, leaf extracts on diarrhea-causing
enteric bacteria isolated from seabob shrimp, Xiphopenaeus kroyeri (Heller). Revista Do Instituto
De Medicina Tropical De Sao Paulo. 2008, 50 (1), 11-15.
[28]. Sanches, N.R.; Cortez, D.A.G.; Schiavini, M.S.; Nakamura, C.V.; Dias, B.P. An evaluation of
antibacterial activities of Psidium guajava (L.). Brazilian Archives of Biology and Technology.
2005, 48 (3), 429-436.
[29]. Han, E.H.; Hwang, Y.P.; Kim, H.G.; Park, J.H.; Choi, J.H.; Im, J.H.; Khanal, T.; Park, B.H.; Yang,
J.H.; Choi, J.M.; Chun, S.S.; Seo, J.K.; Chung, Y.C.; Jeong, H.G. Ethyl acetate extract of Psidium
guajava inhibits IgE-mediated allergic responses by blocking Fc epsilon RI signaling. Food and
Chemical Toxicology. 2011, 49 (1), 100-108.
[30]. Dutta, S.; Das, S. A study of the anti-inflammatory effect of the leaves of Psidium guajava Linn.
on experimental animal models. Pharmacognosy Research. 2010, 2 (5), 313-317.
[31]. Hsu, Y.; Kuo, P.; Lin, L.; Lin, C. Asiatic acid, a triterpene, induces apoptosis and cell cycle arrest
through activation of extracellular signal-regulated kinase and p38 mitogen-activated protein
kinase pathways in human breast cancer cells. The Journal Of Pharmacology And Experimental
Therapeutics. 2005, 313 (1), 333-344.
[32]. Tang, X.L.; Yang, X.Y.; Jung, H.J.; Kim, S.Y.; Jung, S.Y.; Choi, D.Y.; Park, W.C.; Park, H.
Asiatic acid induces colon cancer cell growth inhibition and apoptosis through mitochondrial
death cascade. Biological & Pharmaceutical Bulletin. 2009, 32 (8), 1399-1405.
[33]. Zhang, X.; Wu, J.; Dou, Y.; Xia, B.; Rong, W.; Rimbach, G.; Lou, Y. Asiatic acid protects
primary neurons against C2-ceramide-induced apoptosis. European Journal Of Pharmacology.
2012, 679 (1-3), 51-59.
[34]. Krishnamurthy, R.G.; Senut, M.C.; Zemke, D.; Min, J.; Frenkel, M.B.; Greenberg, E.J.; Yu, S.W.;
Ahn, N.; Goudreau, J.; Kassab, M.; Panickar, K.S.; Majid, A. Asiatic acid, a pentacyclic triterpene
from Centella asiatica, is neuroprotective in a mouse model of focal cerebral ischemia. Journal Of
Neuroscience Research. 2009, 87 (11), 2541-2550.
46
Determination of triterpenoids in Psidium guajava
[35]. Tang, L.X.; He, R.H.; Yang, G.; Tan, J.J.; Zhou, L.; Meng, X.M.; Huang, X.R.; Lan, H.Y. Asiatic
acid inhibits liver fibrosis by blocking TGF-beta/Smad signaling in vivo and in vitro. Plos One.
2012, 7 (2), e31350-e31350.
[36]. Ma, K.; Zhang, Y.; Zhu, D.; Lou, Y. Protective effects of asiatic acid against D-
galactosamine/lipopolysaccharide-induced hepatotoxicity in hepatocytes and kupffer cells co-
cultured system via redox-regulated leukotriene C4 synthase expression pathway. European
Journal Of Pharmacology. 2009, 603 (1-3), 98-107.
[37]. Huang, S.S.; Chiu, C.S.; Chen, H.J.; Hou, W.C.; Sheu, M.J.; Lin, Y.C.; Shie, P.H.; Huang, G.J.
Antinociceptive activities and the mechanisms of anti-inflammation of asiatic Acid in mice.
Evidence-Based Complementary And Alternative Medicine: Ecam. 2011, 2011 895857-895857.
[38]. Liu, J.; He, T.; Lu, Q.; Shang, J.; Sun, H.; Zhang, L. Asiatic acid preserves beta cell mass and
mitigates hyperglycemia in streptozocin-induced diabetic rats. Diabetes/Metabolism Research And
Reviews. 2010, 26 (6), 448-454.
[39]. Qian, Y.; Guan, T.; Tang, X.; Huang, L.; Huang, M.; Li, Y.; Sun, H. Maslinic acid, a natural
triterpenoid compound from Olea europaea, protects cortical neurons against oxygen-glucose
deprivation-induced injury. European Journal Of Pharmacology. 2011, 670 (1), 148-153.
[40]. Qian, Y.; Guan, T.; Tang, X.; Huang, L.; Huang, M.; Li, Y.; Sun, H.; Yu, R.; Zhang, F. Astrocytic
glutamate transporter-dependent neuroprotection against glutamate toxicity: an in vitro study of
maslinic acid. European Journal Of Pharmacology. 2011, 651 (1-3), 59-65.
[41]. Huang, L.; Guan, T.; Qian, Y.; Huang, M.; Tang, X.; Li, Y.; Sun, H. Anti-inflammatory effects of
maslinic acid, a natural triterpene, in cultured cortical astrocytes via suppression of nuclear factor-
kappa B. European Journal Of Pharmacology. 2011, 672 (1-3), 169-174.
[42]. Reyes Zurita, F.J.; Rufino Palomares, E.E.; Lupiez, J.A.; Cascante, M. Maslinic acid, a natural
triterpene from Olea europaea L., induces apoptosis in HT29 human colon-cancer cells via the
mitochondrial apoptotic pathway. Cancer Letters. 2009, 273 (1), 44-54.
[43]. Guan, T.; Qian, Y.; Tang, X.; Huang, M.; Huang, L.; Li, Y.; Sun, H. Maslinic acid, a natural
inhibitor of glycogen phosphorylase, reduces cerebral ischemic injury in hyperglycemic rats by
GLT-1 up-regulation. Journal Of Neuroscience Research. 2011, 89 (11), 1829-1839.
[44]. Miura, T.; Itoh, Y.; Kaneko, T.; Ueda, N.; Ishida, T.; Fukushima, M.; Matsuyama, F.; Seino, Y.
Corosolic acid induces GLUT4 translocation in genetically type 2 diabetic mice. Biological &
Pharmaceutical Bulletin. 2004, 27 (7), 1103-1105.
[45]. Miura, T.; Ueda, N.; Yamada, K.; Fukushima, M.; Ishida, T.; Kaneko, T.; Matsuyama, F.; Seino,
Y. Antidiabetic effects of corosolic acid in KK-Ay diabetic mice. Biological & Pharmaceutical
Bulletin. 2006, 29 (3), 585-587.
47
Master of Science, University of Macau
[46]. Takagi, S.; Miura, T.; Ishibashi, C.; Kawata, T.; Ishihara, E.; Gu, Y.; Ishida, T. Effect of corosolic
acid on the hydrolysis of disaccharides. Journal Of Nutritional Science And Vitaminology. 2008,
54 (3), 266-268.
[47]. Hou, W.; Li, Y.; Zhang, Q.; Wei, X.; Peng, A.; Chen, L.; Wei, Y. Triterpene acids isolated from
Lagerstroemia speciosa leaves as alpha-glucosidase inhibitors. Phytotherapy Research: PTR. 2009,
23 (5), 614-618.
[48]. Benalla, W.; Bellahcen, S.; Bnouham, M. Antidiabetic medicinal plants as a source of alpha
glucosidase inhibitors. Current Diabetes Reviews. 2010, 6 (4), 247-254.
[49]. Ahn, K.S.; Hahm, M.S.; Park, E.J.; Lee, H.K.; Kim, I.H. Corosolic acid isolated from the fruit of
Crataegus pinnatifida var. psilosa is a protein kinase C inhibitor as well as a cytotoxic agent.
Planta Medica. 1998, 64 (5), 468-470.
[50]. Fujiwara, Y.; Komohara, Y.; Ikeda, T.; Takeya, M. Corosolic acid inhibits glioblastoma cell
proliferation by suppressing the activation of signal transducer and activator of transcription-3 and
nuclear factor-kappa B in tumor cells and tumor-associated macrophages. Cancer Science. 2011,
102 (1), 206-211.
[51]. Lee, M.S.; Cha, E.Y.; Thuong, P.T.; Kim, J.Y.; Ahn, M.S.; Sul, J.Y. Down-regulation of human
epidermal growth factor receptor 2/neu oncogene by corosolic acid induces cell cycle arrest and
apoptosis in NCI-N87 human gastric cancer cells. Biological & Pharmaceutical Bulletin. 2010, 33
(6), 931-937.
[52]. Lee, M.S.; Lee, C.M.; Cha, E.Y.; Thuong, P.T.; Bae, K.; Song, I.S.; Noh, S.M.; Sul, J.Y.
Activation of AMP-activated protein kinase on human gastric cancer cells by apoptosis induced by
corosolic acid isolated from Weigela subsessilis. Phytotherapy Research. 2010, 24 (12), 1857-
1861.
[53]. Shan, J.Z.; Xuan, Y.Y.; Ruan, S.Q.; Sun, M. Proliferation-inhibiting and apoptosis-inducing
effects of ursolic acid and oleanolic acid on multi-drug resistance cancer cells in vitro. Chinese
Journal Of Integrative Medicine. 2011, 17 (8), 607-611.
[54]. Yan, S.L.; Huang, C.Y.; Wu, S.T.; Yin, M.C. Oleanolic acid and ursolic acid induce apoptosis in
four human liver cancer cell lines. Toxicology In Vitro: An International Journal Published In
Association With BIBRA. 2010, 24 (3), 842-848.
[55]. Li, J.; Guo, W.J.; Yang, Q.Y. Effects of ursolic acid and oleanolic acid on human colon carcinoma
cell line HCT15. World Journal Of Gastroenterology. 2002, 8 (3), 493-495.
[56]. Wang, Z.H.; Hsu, C.C.; Huang, C.N.; Yin, M.C. Anti-glycative effects of oleanolic acid and
ursolic acid in kidney of diabetic mice. European Journal Of Pharmacology. 2010, 628 (1-3), 255-
260.
48
Determination of triterpenoids in Psidium guajava
[57]. Lee, M.S.; Thuong, P.T. Stimulation of glucose uptake by triterpenoids from Weigela subsessilis.
Phytotherapy Research. 2010, 24 (1), 49-53.
[58]. Yin, M.C.; Chan, K.C. Nonenzymatic antioxidative and antiglycative effects of oleanolic acid and
ursolic acid. Journal of Agricultural and Food Chemistry. 2007, 55 (17), 7177-7181.
[59]. Tanachatchairatana, T.; Bremner, J.B.; Chokchaisiri, R.; Suksamrarn, A. Antimycobacterial
activity of cinnamate-based esters of the triterpenes betulinic, oleanolic and ursolic acids.
Chemical & Pharmaceutical Bulletin. 2008, 56 (2), 194-198.
[60]. Santos Rosa, C.; Garca Gimenez, M.D.; Saenz Rodriguez, M.T.; De la Puerta Vazquez, R.
Antihistaminic and antieicosanoid effects of oleanolic and ursolic acid fraction from Helichrysum
picardii. Die Pharmazie. 2007, 62 (6), 459-462.
[61]. Aparecida Resende, F.; de Andrade Barcala, C.A.M.; da Silva Faria, M.C.; Kato, F.H.; Cunha,
W.R.; Tavares, D.C. Antimutagenicity of ursolic acid and oleanolic acid against doxorubicin-
induced clastogenesis in Balb/c mice. Life Sciences. 2006, 79 (13), 1268-1273.
[62]. Begum, S.; Hassan, S.I.; Ali, S.N.; Siddiqui, B.S. Chemical constituents from the leaves of
Psidium guajava. Natural Product Research. 2004, 18 (2), 135-140.
[63]. El Sohafy, S.M.; Metwalli, A.M.; Harraz, F.M.; Omar, A.A. Quantification of flavonoids of
Psidium guajava L. preparations by Planar Chromatography (HPTLC). Pharmacognosy Magazine.
2009, 5 (17), 61-66.
[64]. Liang, Q.R.; Qian, H.; Yao, W.R. Identification of flavonoids and their glycosides by high-
performance liquid chromatography with electrospray ionization mass spectrometry and with
diode array ultraviolet detection. European Journal of Mass Spectrometry. 2005, 11 (1), 93-101.
[65]. Burkhardt, M.R.; ReVello, R.C.; Smith, S.G.; Zaugg, S.D. Pressurized liquid extraction using
water/isopropanol coupled with solid-phase extraction cleanup for industrial and anthropogenic
waste-indicator compounds in sediment. Analytica Chimica Acta. 2005, 534 (1), 89-100.
[66]. Mustafa, A.; Turner, C. Pressurized liquid extraction as a green approach in food and herbal plants
extraction: A review. Analytica Chimica Acta. 703 (1), 8-18.
[67]. Delgado Zamarreo, M.M.; Bustamante Rangel, M.; Snchez Prez, A.; Carabias Martnez, R.
Pressurized liquid extraction prior to liquid chromatography with electrochemical detection for the
analysis of vitamin E isomers in seeds and nuts. Journal of Chromatography A. 2004, 1056 (1),
249-252.
[68]. Lee, H.J.; Kim, C.Y. Simultaneous determination of nine lignans using pressurized liquid
extraction and HPLC-DAD in the fruits of Schisandra chinensis. Food Chemistry. 120 (4), 1224-
1228.
49
Master of Science, University of Macau
[69]. Wan, J.B.; Lai, C.M.; Li, S.P.; Lee, M.Y.; Kong, L.Y.; Wang, Y.T. Simultaneous determination of
nine saponins from Panax notoginseng using HPLC and pressurized liquid extraction. Journal of
Pharmaceutical and Biomedical Analysis. 2006, 41 (1), 274-279.
[70]. Chen, X.J.; Zhao, J.; Meng, Q.; Li, S.P.; Wang, Y.T. Simultaneous determination of five
flavonoids in licorice using pressurized liquid extraction and capillary electrochromatography
coupled with peak suppression diode array detection. Journal of Chromatography A. 2009, 1216
(43), 7329-7335.
[71]. Yi, Y.; Zhang, Q.W.; Li, S.L.; Wang, Y.; Ye, W.C.; Zhao, J.; Wang, Y.T. Simultaneous
quantification of major flavonoids in "bawanghua", the edible flower of Hylocereus undatus using
pressurized liquid extraction and high performance liquid chromatography. Food Chemistry. In
press, (0),
[72]. Yu, Q.T.; Qi, L.W.; Li, P.; Yi, L.; Zhao, J.; Bi, Z.M. Determination of seventeen main flavonoids
and saponins in the medicinal plant Huang-qi (Radix Astragali) by HPLC-DAD-ELSD. Journal of
Separation Science. 2007, 30 (9), 1292-1299.
[73]. Qi, L.W.; Yu, Q.T.; Li, P.; Li, S.L.; Wang, Y.X.; Sheng, L.H.; Yi, L. Quality evaluation of Radix
Astragali through a simultaneous determination of six major active isoflavonoids and four main
saponins by high-performance liquid chromatography coupled with diode array and evaporative
light scattering detectors. Journal of Chromatography A. 2006, 1134 (1-2), 162-169.
[74]. Yan, S.K.; Luo, G.A.; Wang, Y.M.; Cheng, Y.Y. Simultaneous determination of nine components
in Qingkailing injection by HPLC/ELSD/DAD and its application to the quality control. Journal of
Pharmaceutical and Biomedical Analysis. 2006, 40 (4), 889-895.
[75]. Wang, H.L.; Yao, W.F.; Zhu, D.N.; Hu, Y.Z., Chemical Fingerprinting by HPLC-DAD-ELSD and
Principal Component Analysis of Polygala japonica from Different Locations in China. 2010. p.
343-348.
[76]. Li, S.P.; Zhao, J.; Yang, B. Strategies for quality control of Chinese medicines. Journal of
Pharmaceutical and Biomedical Analysis. 2011, 55 (4), 802-809.
[77]. M, O. Optimization and validation of an HPLC-UV method for analysis of corosolic, oleanolic,
and ursolic acids in plant material: application to Prunus serotina Ehrh. Acta. Chromatographica.
2008, 20 643-659.
50
Determination of triterpenoids in Psidium guajava
Appendix A: HPLC chromatograms of 9 triterpenoids P.
guajava samples
PGL-1
51
Master of Science, University of Macau
PGL-2
PGL-3
52
Determination of triterpenoids in Psidium guajava
PGL-4
PGL-5
53
Master of Science, University of Macau
PGL-6
PGL-7
54
Determination of triterpenoids in Psidium guajava
PGL-8
PGL-9
55
Master of Science, University of Macau
PGF-1
PGF-2
56
Determination of triterpenoids in Psidium guajava
PGF-3
PGF-4
57
Master of Science, University of Macau
PGF-5
PGF-6
58
Determination of triterpenoids in Psidium guajava
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
PGL-2 without hydrolysis
59
Master of Science, University of Macau
AU
0.00
0.05
0.10
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00
CA
PGL-2 with hydrolysis
AU
0.00
0.05
0.10
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00
CA
PGL-3 without hydrolysis
AU
0.00
0.05
0.10
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00
CA
PGL-3 with hydrolysis
AU
0.00
0.05
0.10
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00
CA
PGL-4 without hydrolysis
60
Determination of triterpenoids in Psidium guajava
AU
0.00
0.05
0.10
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00
PGL-4 with hydrolysis
AU
0.00
0.05
0.10
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00
PGL-5 without hydrolysis
AU
0.00
0.05
0.10
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00
PGL-5 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
CA
PGL-6 without hydrolysis
61
Master of Science, University of Macau
AU
0.00
0.05
0.10
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00
PGL-6 with hydrolysis
AU
0.00
0.05
0.10
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00
PGL-7 without hydrolysis
AU
0.00
0.05
0.10
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00
CA
CA
PGL-7 with hydrolysis
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
Determination of triterpenoids in Psidium guajava
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
PGF-2 without hydrolysis
AU
0.00
0.05
0.10
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00
PGF-2 with hydrolysis
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
PGF-3 without hydrolysis
63
Master of Science, University of Macau
AU
0.00
0.05
0.10
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00
PGF-3 with hydrolysis
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
PGF-4 without hydrolysis
AU
0.00
0.05
0.10
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00
PGF-4 with hydrolysis
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
PGF-5 without hydrolysis
64
Determination of triterpenoids in Psidium guajava
AU
0.00
0.05
0.10
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00
PGF-5 with hydrolysis
65
Master of Science, University of Macau
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.