ISSN2434-2688 Asian Journal of Dietetics

Vol.4 No.2.3, 2022
Contents
Page T Title and authors
Letter to Editor
29-35

A Future Where Asia Works Together to Eradicate Malnutrition
8th Asian Congress of Dietitians (ACD2022)

Letter to Editor
37-40

Becoming A Dietitian in Asian Countries:
Educational Aspects from Young Dietitians’ Perspective
Trang Thu Nguyen, Marita V. De Guzman, Ishak Halim Octawijaya, Varanya
Techasukthavorn

Review
41-52

The Best Compositions of Late Evening Snack for Liver Cirrhosis
Patients
Using Systematic Review and Network Meta-Analysis
Trang Thu Nguyen, Shigeru Yamamoto

Original Research
53-58

Development of the Revised Food Frequency Questionnaire for
Cambodian School-aged Children
Yoko Horiuchi, Kaoru Kusama, and Eri Kai

Original Research
59-68

A Crossover Study on the Effects of Continuous Soymilk Intake on
Lipid Metabolism and Glucose Metabolism in Vietnamese - Lipid
Metabolism Improving Effect –Ai Yamamoto, Tamaki Shintomi, Vu Thi Thu Hien, Le Danh Tuyen, Nguyen
Thi Diep Anh, Le Anh Hoa, Le The Trung, Tran Van Long, Nguyen Van
Tuyen, Nguyen Cam Thach, Shigeru Yamamoto, Nobuo Yoshiike, Makoto
Sugawara, Ryoichi Unno, Miki Tomoe

Original Research
69-74

Phytochemical Content and Antioxidant Activity of Boiled and Fresh
Ayo (Tetrastigma harmandii Planch.) Fruits and Leaves
Tapaoan Sharlyn. Mae. D., Barrion Aimee. Sheree. A, Tuaño Arvin. Paul. P.

Asian Journal of Dietetics 2022

Letter to Editor
A Future Where Asia Works Together to Eradicate Malnutrition
8th Asian Congress of Dietitians (ACD2022)

The 8th Asian Congress of Dietitians andNutritionists (ACD2022) was held at Pacifico
Yokohama, Yokohama, Japan, from 19-21 August2022. Due to the Corona pandemic, many national
and international scientific meetings andcongresses were conducted online; however, it waspossible to hold ACD2022 face-to-face. The AsianCongress of Dietetic (ACD) is held every four
years in one of the member countries of the AsianFederation of Dietetic Associations (AFDA), andthis is the first time it was held in Japan. AFDA
members include the Japan Dietetic Association,
as well as the Dietetic Associations of Taiwan,

Hong Kong, India, Indonesia, South Korea,
Malaysia, the Philippines, Pakistan, Singapore,
Thailand and Australia. The theme of ACD2022
was "Towards a sustainable healthy society for abright Asian future: addressing confusing nutrition
information to solve contemporary issues". The
conference brought together 1,137 dietitians,
nutritionists and other nutritionists from Japan and17 other Asian countries to discuss nutrition
challenges, as well as measures and policies intheir respective countries through 23 lectures,symposia and workshops, and 272 poster
presentations.

29

A Future Where Asia Works Together to Eradicate Malnutrition

International Congress of Miracles begins

The ACD2022 opened with the soaring "I
hereby declare the opening of ACD2022" by Ms
Shihoko Suzuki, Vice-Chairperson of theOrganizing Committee of the 8th Asian Congressof Dietitians (hereinafter referred to as the
ACD2022 Organizing Committee).

Guidance Office, Health Division, Health Bureau,
Ministry of Health, Labor and Welfare, on behalfof Katsunobu Kato, Minister of Health, Labour andWelfare; Yuji Kuroiwa, Governor of Kanagawa
Prefecture; Hirotoshi Shiro, Deputy Mayor of
Yokohama City; Okami Tsukahara, Vice Presidentof the Japanese Society for Nutrition
Improvement; and Yoshihiko Takigawa, Presidentof the Japan National Dietetic Training Institute
Association, who was present to celebrate theopening.

At the opening ceremony, AFDA President
Gordon Cheung, who was unable to visit Japan due
to travel restrictions, sent a video message to theparticipants in the audience, encouraging them toenjoy the ACD2022 program and to develop
friendships with other Asian dietitians. In his
opening remarks as organiser, Professor Teiji
Nakamura, Chair of the ACD2022 Organizing
Committee, said, “Ladies and gentlemen, welcome
to Japan. We have been able to hold this congress.
Nutrition is not only for health, but is also related
to education, economics, gender, etc., and supports
the sustainable development goals (SDGs) that are
moving towards their realization around the world.
Solving the double burden of malnutrition in Asia
will require developing and deploying nutrition
leaders in every corner of every country. Let's havea lively discussion over the next three days with theaim of making the people of Asia healthier andhappier," he said in his opening remarks.

At the awards ceremony held in conjunction
with the opening ceremony, the Dr Chwang LehChii Asian Dietetics Award was presented to
Shigeru Yamamoto of the Japan Dietetic
Association and Chanida Pachotikarn of the Thai
Dietetic Association. The Dr Ching-Hwa Chiu
Asian Outstanding Young Dietitian Award foryoung dietitians under the age of 45 was alsopresented to Ms Frankie Pui-Lam Siu of the Hong
Kong Dietetic Society. Frankie took to the stage
online and said, "My dream is to connect with andlearn from other dietitians in Asia, and to put thatback into advising patients. I believe that we as
dietitians can create great power by outputting ourknowledge and passion".

Guests of honor at the opening ceremony
included Tokue Seino, Director of the Nutrition

30

Asian Journal of Dietetics 2022

The impact of Japan Nutrition

specialists in various institutions in Japan,
including government, healthcare, schools, welfare
facilities, companies, Self-Defense Forces, prisonsand universities, etc. The Japan Dietetic
Association (JDA) has been working to improve
the nutritional quality of the Japanese people, andhas been promoting the existence of Japan as acountry with a long life expectancy. He added: "I
am convinced that nutrition in Japan is sustainable,leaving no one behind and making healthy
longevity possible. I believe that everyone in Asia
agrees with me. Let us work more closely andamicably and contribute to Asia," he summarized,
to which the audience agreed with loud applause.
The speech was concluded by former Speaker ofthe House of Councilors, Akiko Santo, who, alongwith her congratulatory remarks, stated that she
would do all she could to back the cause of
improved nutrition in Asia.

In a special lecture entitled 'Japan Nutrition',
Chairman Nakamura Teiji presented Japan's
nutrition policy to date and the activities of
dietitians. AFDA Honorary President Chwang
Leh-Chii, who chaired the session, expressed his
surprise when he explained that in 1926 there were
only 13 dietitians in Japan, whereas today there are
more than one million dietitians and nutritionists.
He praised the fact that there are one million
dietitians in the country, saying that it is first of all
a great achievement.
Chairperson Nakamura emphasized that Japan
has continued to improve nutrition in acomprehensive manner based on nutrition
education for the people by converting the foodpolicy of the United States into a nutrition policy
in Japan during the period of food shortages afterWorld War II, implementing proper supply anddistribution of food, and placing nutrition
Mutual learning and discussion of Asian “Dietetics”

31

A Future Where Asia Works Together to Eradicate Malnutrition

The three-day programmer consisted of one
special lecture, two keynote lectures, four
educational lectures, nine symposia and sevenworkshops with a total of 23 contents, covering awide range of topics from diabetes, obesity,
infectious diseases, frailty and sarcopenia inclinical and public nutrition to policies and systems
such as disaster support, food service systems andnutritionist training, as well as a workshop where
young nutritionists from different countries
discussed their vision for future nutritionists.

government is trying to train more dietitians, and if
there was one dietitian per 10,000 people, thecurrent obesity rate of 21.8% could be reduced
even further". He added that a cultural approach isalso needed to combat obesity, noting that "inIndonesia, there is a practice where chubby infants
are seen as being “well looked after by theirmothers". Samitti Chotsriluecha, a Thai dietitian,also commented, “I am impressed that Japanesepeople, young and old, walk a lot. I think the factthat sweet food is also favored in Thailand and that
there is little physical exercise is a factor in theincrease in overweight, especially among middleaged women", describing the current situation.

One of the programs, Workshop 2: Dietitian
Activities in Obesity Control, featured speakers
from Indonesia, Thailand and Japan, each of whom
explained their country's policies and current
situation, followed by a discussion involving
audience participants. Miranti G. Sumapradja, anIndonesian dietitian, citing the Indonesian dietetic
habits of heavy use of salt, sugar and coconut milkin cooking and a preference for fried foods, and thecountry's lack of parks and places for outdoor
exercise, said, "There are about 50,000
professionals involved in nutrition, but dietitians
are only 10-15% of them exist, which is very small
for a population of 170 million people. The

While the situation in various Asian countries
was presented in various sessions, Chairman
Nakamura spoke in Workshop 3: "Towards
realising the commitments of the “Tokyo Nutrition
Summit 2021”, saying that “the word 'Dietetics” in
the Asian Congress of Dietetics is difficult totranslate into Japanese, as it includes not only
nutrition, but also culture, economy andpreferences. The time has come to disseminate'Dietetics' from Asia, without relying on standards
and guidelines from the West"32

Asian Journal of Dietetics 2022

Poster presentations and exhibitions to deepen friendships

In the exhibition hall, 272 posters were
presented, with presenters opening their laptops on
the round tables provided and presenting theirfindings to the assembled participants. The posters
were posted online allowing participants to view
them from a distance on their own devices andcommunicate with the presenters via a chat room.
Also in the exhibition hall, Yakult Honsha Co Ltd,
Ajinomoto Co Ltd, Meiji Co Ltd, California
Walnut Association, "Rice Flour Gentle
Swallowing Food" Consortium, Friesland

Campina Institute, Hasegawa Chemical Industry
Co Ltd, Kikkoman Foods Co Ltd, Nipun Co. JapanSports Nutrition Association, and Nutrition
Support Network LLC exhibited and promoted
their initiatives to visitors. In addition, a variety ofother 'hospitality' activities were offered, including
a booth where visitors could easily experience thetea ceremony, a booth where they could relax withyoga, a photo booth where commemorative photos
could be taken, a rest area and a drinks corner.

33

A Future Where Asia Works Together to Eradicate Malnutrition

Signing of the Yokohama Declaration

After three days of lively discussions anddeepening their friendship, the dietitians from thedifferent countries came to the closing ceremonyon the 21st (Sun) with a sense of farewell. In his
closing remarks, Chairman Teiji Nakamura
thanked all those involved and continued asfollows. 'One thing became clear during our
discussions at ACD 2022. Of course economic andfood aid is necessary to eliminate malnutrition in
the world, but at the same time we need to educate
and train professionals. There is a need to provide

solid education for the profession and to increase
public awareness of dietitians. Asia has the highest
population growth and economic growth rates in
the world. This has led to significant changes in
dietary habits. It is necessary to share information
and work together to solve problems across Asia.
It is an achievement to have spent three days
discussing this with each of you. “Making people
healthier and happier through the power ofnutrition” - see you again in India in four years'
time.’

34

Asian Journal of Dietetics 2022

After that, Dr Yasuhiro Kido, Chair of the
Executive Committee of the ACD2022 Organizing
Committee, gave a summary of the number ofparticipants and other details of ACD2022 andpresented the Poster Awards. 13 presenters wererecognized and Mr. Ying Qian Ong of the
Malaysian Dietetic Association spoke on behalf of
the winners and said, "Many people helped tomake this research that has been recognized. I
would like to send this award to everyone who
helped us". The young award winners were allsmiling and happy as they were presented with
their awards on stage, symbolizing the bright
future of nutritionists in Asia.

infrastructure", particularly in Asia. The mutual
understanding and friendship with other countries
fostered by ACD 2022 will be used as a driving
force to develop new initiatives.

Furthermore, as an outcome of the congress,
the ' Yokohama Declaration' was announced andsigned on stage by the representatives of eachcountry. Finally, a video documenting the threedays and a promotional video of the next hostcountry, India, were shown. ACD2022 was thendeclared closed by Ms Toshiko Saito, ViceChairperson of the ACD2022 Organizing
Committee.
As stated in the ' Yokohama
Declaration', nutrition is fundamental to achieving
the SDGs. Eliminating malnutrition is alsoessential for the health and well-being of all. At the
AFDA General Assembly held during the ACD
2022 session, it was decided that Japan will be thechair country of AFDA from now until 2026 andthat Dr. Teiji Nakamura President of the Japan
Dietetic Association, will assume the position of
AFDA President. As committed to at the Tokyo
Nutrition Summit 2021, the Japan Dietetic
Association will promote the "Training anddeployment of food and nutrition professionals tobuild a sustainable nutrition improvement

35

Asian Journal of Dietetics 2022

Letter to editor
Becoming A Dietitian in Asian Countries:
Educational Aspects from Young Dietitians’ Perspective
Trang Thu Nguyen1*, Marita V. De Guzman2, Ishak Halim Octawijaya3, Varanya Techasukthavorn4
1
Nutrition and Food Culture Research Center, Jumonji University, Saitama, Japan
2
Nutritionist-Dietitians’ Association of the Philippines, Makati, Philippines
3
School of Nutrition and Dietetics, Faculty of Health and Social Services, Kanagawa University of
Human Services, Yokosuka, Japan & Graduate School of Comprehensive Human Sciences,
University of Tsukuba, Tsukuba, Japan
4
Department of Nutrition and Dietetics, Faculty of Allied Health Sciences, Chulalongkorn
University, Bangkok, Thailand
Dear Editor:

simulation, case studies, and role-playing) at colleges oruniversities, as well as taking professional practice(practical training/internships) programs in healthcare orservice facilities. All countries grant pupils diplomas,
bachelor's, or master's degrees by fulfillment of theeducation curriculum, which might be followed byadditional professional practice, passing national
examinations, and official administrations before they
are granted their dietitian license.
A consensus on the core concepts for the dietetics
discipline and dietetic competency standards among thecountries has been acknowledged. In addition to oncampus courses, professional practice in healthcare
facilities, public health centers, or food service facilities
is an essential part of a dietitian education program toequip prospective dietitians with comprehensive skills
for their professional tasks in the future. Naturally, thenumber of training hours in real settings needs anextended consideration since the skills acquired fromthose practices are proportional to the amount of practicetaken. According to International Standard for Educationof Dietitians-Nutritionists by ICDA, the minimum levelof education of a dietitian-nutritionist is a bachelor's
degree and a period of supervised professional practiceof at least 500 hours and meets the international
competency standards for dietitians-nutritionists (2). In
western countries like the British Dietetic Association,
students are expected to undertake not less than 1000
hours of practice-based learning with not less than 350
hours in the clinical setting and not more than 500 hours
in one setting (1).
Despite international standardization attempts,
qualifications and education standards to become adietitian differ in each country. We have realized thatthese standards may differ due to the specific conditionsof the dietetic organizations in the context of thecountry’s education and health systems and the cultural,social, and historical experiences of the country. The
varied qualifications and education standards in Asian
countries are summarized in table 1.
Among all Asian countries, the Philippines has thehighest education units in their Bachelor's program in
Nutrition and Dietetics as they follow the United Statesof America’s standard. For the Bachelor's program in thePhilippines, a total of 165 units which is a combinationof general education and professional courses should becompleted by the student to be able to graduate in the
program. This program includes 3-year didactic teachingand 1-year practical training or practicum in hospitals,
food service, and the community. Schools or universities

Dietitians, who are referred to as Registered
Dietitians (RD) or Registered Nutritionist Dietitians(RND) in some countries, are qualified regulated healthprofessionals dealing with human nutrition and dietetics
issues faced at any life stage. They assess, diagnose andtreat dietary and nutritional problems at an individual andwider public health level (BDA Curriculum 2020) (1).
They also apply the science of food and nutrition topromote health, prevent and treat disease to optimize thehealth of individuals, groups, communities, andpopulations (International Confederation of Dietetic
Associations (ICDA, 2014) (2). The subjects of dietitians'
practice range from hospital patients, school children,
elderly in long-term care facilities, or even professional
athletes. Thus, dietitians have to complete an education
curriculum and satisfy the requirements to prove they arecapable of accomplishing the professional tasks asregulated by national authorities.
From August 19th to 21st, 2022, young dietitians
from six Asian countries (Japan, Taiwan, Thailand,
Philippines, Indonesia, and Vietnam) gathered at The 8th
Asian Congress of Dietetics (ACD 2022) in Yokohama,Japan. For the first time, we shared our ideas about
dietitians’ education and practices in our countries. Given
limited opportunities to gather and share our thoughts,
everyone was eager to bring up a broad range of topics,
such as the legal system related to dietitians, strategies tofight obesity, and malnutrition treatments in clinicalsettings. We have been meeting online twice a monthfrom April to August, preparing the topic we wanted toshare with Asian dietitians during ACD 2022. An
important topic during our meetings was the education
programs compulsory to be dietitians in each country.
Through the online meetings and congress, we sought
how Asian countries could build a mutual understanding
as a foundation to develop a better education program fordietitians in the future. Here is a glance at our perspective.
Practice hours and internshipAccreditation standards for dietetics education
programs across the world, require curriculum to meetthe foundational knowledge, skills and attributes neededfor dietetic practice. The curriculum commonly
incorporates a range of credits acquired by attending*To whom correspondence should be addressed:
trangnn27@gmail.com
lectures, experimental/practical courses (including37

Educational Aspects from Young Dietitians’ Perspective
offering BSND program or equivalent is flexible when it
comes to their curriculum however when it comes toTable 1: A summary of education programs, practice regimes, and Registered Dietician (RD) eligibility
among some Asian countries
Countries

Education
programs

Credits+
Practice hours(At university)

Practicehours(Outside
university)

Internship(Aftergraduation)

RD eligibility

Renewal RD
qualification

Japan

Diploma, BSc,
MSc, PhD

810hr (18cr.)

180hr (4cr.)

N/A

Accredited course +Practice + Exam

N/A

Taiwan

Diploma andBSc

20 Credits

500hr (7cr.)

2 years(hospital)

Accredited course +practice hours + Exam

120hr. credits
every 6 years

Thailand

BSc and MSc

BSc (>135cr.)
525hr

900hr

1-2 years(if not finish
900hr)

Accredited course +Internship + Exam

N/APhilippines

BSND, MSc,
PhD

BSc (136 units
lecture)+ (40 units lab)

1200hr/ 1
year (24units)

N/A

Accredited course +Practice hours + Exam
Mutual Reciprocity in
US as RD: RND +ExamCPD units(45), Member
of NDAP

IndonesiaBSc272-408hr (69cr.)

317-453hr(7-10cr.)

1723hr(38cr.)

Accredited course +Internship + Exam

Every 5 years

VietnamBSc and MSc

Credits: 35
At hospital:
N/A
N/A
N/A
450hr
254hr
BSc: Bachelor of Science; CPD: Continuing Professional Development; MSc: Master of Science; NDAP: NutritionistDietitians’ Association of the Philippines; RD: Registered Dietician; RND: Registered Nutritionist Dietitian.
professional courses, they should implement asprescribed in the program set by the Commission on
Higher Education.
In Figure 1, the practice hour is divided into oncampus training (laboratory or alternate supervised

experiences such as simulation, case studies, and roleplaying inside the university) and at training sites (inprofessional work settings such as hospitals, catering
companies, community centers…). Compared to theinternational standard, Taiwan and Thailand have met the

Figure 1. Practice hours during the Bachelor of Dietician/Registered Dietician
program in some Asian countries and others

38

Asian Journal of Dietetics 2022
standard with both the practice hours on-campus and at
the training sites ranging from 500 hours to 900 hours.
The Philippines follows the standards of theAccreditation Council for Education in Nutrition andDietetics (ACEND - Academy of Nutrition and Dietetics)
which is 1200 hours for the practicum program (3). On
the contrary, Japan spends more time on on-campus
training and only 180 hours practicing in hospitals orcompanies. Both Indonesia and Vietnam have practicehours lower than 500 hours in either category. In
comparison to western countries like the US, UK, andAustralia where dieticians are required to have at least
800-1000 hours of on-site training, most Asian dietetics
programs seem to spend less time on it (1, 3-4).
Not only the duration of training but different
countries also have different standards of training
protocols. In Thailand, Vietnam, and Indonesia, their oncampus training may focus only on basic sciencelaboratory training due to the limitations of university
facilities. It found some difficulties to develop and set up
the simulator lab like the training sites. Therefore, theymostly spent more practice hours in the real training sites.
However, in Japan, Taiwan, and the Philippines, withwell-established dietetic-related laboratories (e.g. foodservice laboratory, counseling clinics, and patient
simulators), they have more opportunities to practiceboth on-campus and at the hospital training sites.
With a shorter practice time, some countries like
Taiwan and Indonesia require 2 years or nearly 2000
hours of after-graduation professional practice or (below
mentioned as internships to contrast it with theprofessional practice during diplomas or undergraduate
programs) in the hospital if one would like to work in theclinical setting. While the internship can help dieticiansachieve competency and continue their learning, it canalso affect their livelihood as some internships are unpaid.
On the day of the workshop, we had the chance to discuss
whether to spend more time on practice in the university
or reduce the burden of practice in undergraduate training
and complete the training from the internship. In 2003,
the Institute of Medicine (IOM) proposed that healthcare
professionals could no longer assume that they canpractice effectively using the training received in theirundergraduate education, as human memory could nolonger continue to retain the whole knowledge-base on
effective healthcare and was becoming increasingly
unreliable in keeping pace with the ever-expanding
healthcare research (5). The IOM recommended that for
health professionals to meet the needs of the 21st-century
health setting education reform was needed, however,they recognized an “overly crowded curricula” as achallenge. Therefore with these in consideration, we
would like to suggest a manageable number of practicehours around 800-900 hours in professional work settings
along with a one-year internship after graduation. With
this, dieticians will be able to assure the international
competency standard and evolve to be adaptable, work in
new emerging areas of practice, and demonstrate criticalthinking skills to deal with increasing medical
complexity and an aging population.
Registered Dietician (RD)/ Registered Nutritionist
Dietitian (RND)

Regardless of the difference in curricula orrequirements of internships, almost every country, except
Vietnam as they are still establishing its dietetics system,
demands its dietitian candidates take and pass the
National Board exam to be a Registered Dietician(RD)/Registered Nutritionist Dietitian (RND). The exam
in most countries includes assessing the 3 main parts:
basic nutrition knowledge, medical nutrition therapy,
food service and management, and other supporting
roles; community nutrition; and research methodology.
After the board examination, the RD/RND can continue
education through a Master's degree or may upskill in
their chosen field of expertise (Renal Dietitian, Diabetes
Dietitian, Pediatrics Dietitian, Sports Nutritionist, etc.).
Registered Nutritionist-Dietitian in the Philippines may
also take the Registered Dietitian examination in the US
and may practice there once passed. This is from the
Mutual Reciprocity of both countries.
Renewal of license was also discussed in theworkshop. Japan and Thailand have no renewal oflicenses while the Philippines, Indonesia, and Taiwan
have specific guidelines when it comes to the renewal oflicenses (Table 1). However, since the scope of practiceand work of dietitians is continuously changing,developing, and diverse, it is necessary to encourage allnational RD licenses to have a process of renewal. The
renewal process may help dietitians update theirknowledge regularly. It can increase continuing
professional development skills and lifelong learning
skills in parallel with practicing competencies.
Conclusion
Lastly, it was a great opportunity for the group ofyoung Asian dietitians to join the workshop together in
ACD 2022 to discuss each country's perspective. One of
the selected topics, the dietetic legal system focusing on
the eligibility to become a dietitian has been elaborated
on as mentioned in this article. Although most countries
have their own scopes of practice and national standards,
they seem to have a similar core practice concurrent with
the US, and ICDA standards. There would be more
impact on our Asian countries if we could bring thecollaborations to establish the standards of the dietitiansystem. This may go further with beneficial outcomeswith the dietetic student exchange programs, working
across Asian countries, and fruitful collaboration in thenear future. These attempts are surely in line with the“Yokohama Declaration on Dietetics” which has beensigned by the participating countries by the end of ACD
2022, declaring in points 5 and 6 as follows.
“Education and training of dietitians as leaders in
the nutritional improvement and the establishment anddevelopment of a dietetic system in line withinternational standards are also important.”“In order to achieve the above objectives, further Asianand international cooperation and collaboration will benecessary in the future.”

39

Educational Aspects from Young Dietitians’ Perspective
REFERENCE

3) Accreditation Council for Education in Nutrition
and Dietetics of the Academy of Nutrition andDietetics. ACEND Accreditation Standards for
Nutrition and Dietetics Coordinated Programs (CP).
2016.
4) Dietitians Association of Australia. National
Competency Standards for Dietitians in Australia.
2015.
5) Institute of Medicine. Health Professions
Education: A Bridge to Quality. Washington, DC:
The National Academies Press. 2003.

1)

British Dietetic Association (The Association of
UK Dietitians). A Curriculum Framework for the
pre-registration education and training of dietitians.
2020.
2) International
ConfederationofDietetic
Associations.
International
Standards
forDietitians-Nutritionists. 2014.

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Asian Journal of Dietetics 2022

Review
The Best Compositions of Late Evening Snack for Liver Cirrhosis Patients
Using Systematic Review and Network Meta-Analysis
Trang Thu Nguyen1*, Shigeru Yamamoto1
1

Nutrition and Food Culture Research Center, Jumonji University, Saitama, Japan

ABSTRACT
Background and purpose. Liver Cirrhosis (LC) is ranked the 11th in the world and the 10thin Vietnam among common causes of death. Malnutrition frequently imposes a burden on
patients with LC, and it is an independent predictor of lower survival. However, in Vietnam,
the nutrition management in LC patients is insufficient. Moreover, glycogen storage isdecreased in LC patients so if they do not have a late evening snack (LES), they will feel alack of energy when they wake up the next day. However, a nutrient composition for LES
has not been unified yet, there is a lack of evidence in comparing LES compositions witheach other. Method. Network meta-analysis (NMA) was conducted to compare theeffectiveness of different compositions of LES from direct and indirect evidence. The
evidence was the published articles with intervention trials using different types of LES. All
the papers searched from 5 databases (PubMed, Cochrane library, Google Scholar,
ScienceDirect, Medical Online) until March 1st, 2022 that met the inclusion criteria wereselected. The outcome parameters were albumin, pre-albumin, nitrogen balance, respiratory
quotient, AST, ALT, total bilirubin, ammonia, cholinesterase, and BCAA/Tyr. Results. Afterscreening and full-text assessing 270 papers, 15 final papers with 4 types of LES (high CHO,
Protein-CHO, BCAA-CHO, and coconut milk-CHO) were included in the meta-analysis.
One of the most important parameters is albumin, an effective indicator in the managementof cirrhosis and its complications. And according to the meta-analysis, Protein-CHO has thehighest probability of improving it, followed by high CHO, coconut milk-CHO, and then
BCAA-CHO. Other parameters (such as Pre-Alb, nitrogen balance, ALT, AST…) also
indicated the same results. On the other hand, BCAA-CHO was not effective on most of theparameters but only on BCAA/Tyr. There was no direct comparison between BCAA andprotein. Conclusion. The recommendable LES compositions should provide at least 200kcal
with 50g of carbohydrate. About the nitrogen source, protein is maybe more effective than
BCAA in terms of improving protein synthesis and energy metabolism as well as liver
parenchyma damage. However, since there is no direct comparison between protein and
BCAA as LES, a future study is needed to confirm this finding.
Key Words: Cirrhosis, late evening snack, protein, network meta-analysis.
INTRODUCTION
Liver cirrhosis (LC) is one of the leading causes ofmortality worldwide, it was the 11th most common causeof death each year in the world with 2.1% of total deaths(1). According to Institute for Health Metrics and
Evaluation (IHME), in 2019, LC ranked 7th among the
top 10 most common causes of death in Vietnam with a
47.3% increment from 2009 to 2019 (2). The etiologies
of LC are most commonly alcohol, hepatitis B, hepatitis
C, and non-alcoholic fatty liver disease or sometimes
autoimmune hepatitis. Alcoholic liver disease andhepatitis B are the most common causes in most parts ofAsia (3). Cirrhosis is defined as the histologicaldevelopment of regenerative nodules surrounded by
fibrous bands in response to chronic liver injury, whichleads to portal hypertension and end-stage liver disease.
In the asymptomatic phase of the disease, usuallyreferred to as compensated cirrhosis, patients may have agood quality of life, and the disease may progress
undetected for several years. The decompensation phaseis regularly marked by ascites, gastrointestinal bleeding

due to esophageal varices, hepatic encephalopathy (HE),and jaundice (4).
Malnutrition is frequently a burden in patients with
LC; it is usually related to the clinical stage of chronic
liver disease, increasing from 20% in patients with wellcompensated disease to more than 60% in patients withadvanced cirrhosis (5). Malnutrition and muscle massloss (sarcopenia) are associated with a higher rate ofcomplications such as susceptibility to infections, hepatic
encephalopathy (HE), and ascites, as well as being
independent predictors of lower survival in cirrhosis andin patients undergoing liver transplantation (6). Various
mechanisms are considered to contribute to malnutritionin cirrhosis such as poor oral intake, increased intestinal
protein loss, decrease protein synthesis, disturbances insubstrateutilization,
hyper-metabolism,
andmalabsorption (7). According to Trang Thu Nguyen et al.
study in 2020, the prevalence of malnutrition in LC
patients was 60% by subjective global assessment(SGA), an international questionnaire for nutritionassessment (8). According to ESPEN's clinical nutrition
practice guideline for LC patients in 2019, cirrhotic
patients should ingest an increased amount of energy (3035 kcal/kg/d of energy and 1.2-1.5 g/kg/d of protein) (5).

*To whom correspondence should be addressed:

trangnn27@gmail.com
41

Best Late Evening Snack for Liver Cirrhosis Patients
Nonetheless, in Vietnam’s clinical setting, a restricted
protein diet is still recommended for LC patients (9). As
a result, the energy intake and protein intake ofVietnamese LC patients were lower than theirrecommendation, 1445.9 ± 727.7kcal/day and 1.0 ±0.5g/IBWkg/day, respectively (8).
In addition, several guidelines have recommended
short periods of starvation with 4 - 6 meals a day,especially a late evening snack (LES) is highly
recommended as LC is characterized by a state ofaccelerated starvation, with an early shift from glucose tolipid and amino acids utilization for energy during thepostabsorptive state (fasting), which can also lead to adecrement in respiratory quotients of LC patients (10).
Some systematic reviews and meta-analyses have provedthat LES intervention helped to improve liverbiochemical parameters for albumin, ammonia,
respiratory quotients, and liver enzymes including
aspartate aminotransferase (AST) and alanine
aminotransferase (ALT) (11, 12). Moreover, increasing
the number of meals can also help to increase the amountof dietary intake in LC patients. However, 77.5% of
Vietnamese LC patients did not have LES and only
22.5% had the snack but the compositions of the snacks
were inconsistent (8).
Having said that, it is also necessary to find the mostsuitable compositions of LES for Vietnamese patientsfrom taste-wise to economically. In a systematic review,
Tsien et al. recommended a 200kcal with 50g
carbohydrate (CHO) late evening snack to minimize
gluconeogenesis and preserve muscle mass (12).
However, there has been a trend of using branched-chainamino acids (BCAA) in LES and some studies suggested
it was more effective than CHO-LES (13-14). In
addition, there were a number of studies that used a snack
including both protein and carbohydrates, which showed
improvement in LC patients as well (15-17). It seems that
there have been a variety of compositions of LES used
across different studies and to date, there is no trial
comparing the potential effects of different LES in
cirrhotic patients, thus defining effective compositions
among CHO, BCAA, protein, no LES…are necessary.
To attain a better understanding on this issue, theavailable published trials are estimated by updated
Bayesian network meta-analysis in order to investigate
the efficacy of improving various parameters of livercirrhosis patients between the use of different LES
compositions. The results are expected to provide areference for clinical practice.

balance (NB), respiratory quotient (npRQ), alanine
aminotransferase (ALT), aspartate aminotransferase(AST), total bilirubin (TB), ammonia (NH3),
cholinesterase (Che), and BCAA/Tyr (BTR); (v) Studies
lasting ≥ 1week. All the published articles with original
data (except conference abstract and case report) in any
language up to 01 March 2022 met the listed criteria were
included.
Studies including patients in acute phase or hepatic
encephalopathy and/or a specific health condition such as
diabetes, stroke, chronic obstructive pulmonary disease,chronic kidney disease, other critical illnesses, and recent
transplants were excluded. Studies using other nutritional
interventions other than LES or patients who took othernutritional supplements before the study were also
excluded.
Search strategy. The following five electronic
databases were searched until March 2022: (i) PubMed,(ii) Cochrane Library, (iii) Google Scholar, (iv)
ScienceDirect, and (v) Medical Online using thefollowing terms: ("Liver Cirrhos*"[Mesh] OR"Cirrhos*" OR "Hepatic cirrhos*" OR "Liver Fibros*"
OR "Hepatic Fibros*" OR "end-stage liver disease" OR"Advanced liver disease") AND ("Snacks"[Mesh] OR"Late evening snack" OR "nocturnal nutritional
supplementation" OR "nocturnal snack" OR "evening
snack" OR "nocturnal meal" OR "bedtime snack" OR"late night snack" OR "midnight snack" OR "late evening
meal"). In addition to electronic database searches, crossreferencing was conducted by examining the reference
lists of previous review articles as well as each included
study for potential articles that met the inclusion criteria.
Study selection. All retrieved articles were combinedin Endnote20 and Microsoft Excel (V.2016; Microsoft
Corporation; 2016) to remove duplicates. After screening
the titles and abstracts according to the prespecified
criteria, the full texts of articles that potentially met theeligibility criteria were reviewed. Reasons for excluded
studies were recorded using the following categories: (i)inappropriate population, (ii) inappropriate intervention,(iii) inappropriate comparison(s), (iv) inappropriate
outcome(s), (v) inappropriate study design and (vi) other.
Data extraction. Microsoft Excel (V.2016; Microsoft
Corporation; 2016) was used to develop comprehensive
electronic codebooks. The major categories of variables
coded included (i) study characteristics (author, journal,
year of publication, design, etc.), (ii) participantcharacteristics (age, gender, liver cirrhosis etiology, LC
severity, etc.), (iii) intervention characteristics (type,
length, frequency, nutrients compositions, duration, etc.)
and (iv) data for outcomes (sample sizes, baseline andpost LES means and SD, etc.). When relevant
information on design or outcomes was unclear, or whensome needed data was unavailable directly from thestudy, the original authors sought eligible data by email.
Risk of bias and quality assessment. The Revised
Cochrane risk-of-bias tool for randomized trials (RoB 2)(19)
was used to assess the methodological quality of therandomized controlled trials (RCTs), and the Quality
Assessment Tool for Quantitative Studies (19) assessed
the quality of controlled pre-post studies andnonrandomized experimental studies (non-RCTs). In
both quality assessment tools, each domain will be

METHODS
The study was conducted according to the Preferred
Reporting Items for Systematic Reviews and MetaAnalyses (PRISMA) extension statement for reporting
network meta-analyses of health care interventions (18).
Eligibility criteria. The eligibility criteria are detailed
below following the participants, intervention, controls,
outcomes, and study design (PICOS) framework: (i)Participants: adults (age >18 years) with cirrhosis, maleand female; (ii) Interventions: any types of late evening
snack (LES) that provides about 200kcal; (iii)
Comparisons: studies that compared 1 or more LES
compositions; (iv) Outcomes: At least one of theseparameters: albumin (Alb), pre-albumin, nitrogen
42

Asian Journal of Dietetics 2022
considered as strong, moderate, or weak and studies will
be classified as high, moderate, and low risk of bias.
Statistical analysis. Network meta-analysis (NMA)
was conducted to estimate the effectiveness of different
types of LES compositions on various parameters. The
results of the comparative effects are presented as themean differences (MDs) or standardized meandifferences (SMDs) and 95% confidence intervals (CIs).
We also estimated the ranking probabilities of theintervention effect using the surface under the cumulative
ranking curve (SUCRA) (20). The larger the SUCRA
value, the better the ranking of the intervention effect.
The consistency between the direct and indirect evidence
was evaluated using inconsistency tests to assess thevalidity of the transitivity assumption. Publication biases
or small sample effects were examined using acomparison-adjusted funnel plot.
In addition, a pairwise meta-analysis using therandom-effects model was performed when the data was
not sufficient for a NMA (Pre-albumin and Nitrogenbalance), and the I2 statistic and p values were calculated
as a measure of the statistical heterogeneity (21), with I2≥ 50% indicating substantial heterogeneity.
Stata version 17 was used to conduct the analyses.
The “metan” package was used for the pairwise metaanalysis, and the “network” package was used to conduct
the NMA. Statistical significance was set as a P value <0.05 in all analyses.
RESULTS
Study selection
A total of 267 articles were retrieved by following the
pre-designed literature retrieval strategy. By further
searching the references included in the articles, 3
additional articles were obtained. After reading the titles
and abstracts, 43 studies were selected for further review.
Finally, 15 studies met the inclusion criteria (13-17, 2231). The detailed process of the search strategy is
described in Figure 1.

Figure 1. Flow chart of studies evaluating LES for cirrhosis through the selection process

43Best Late Evening Snack for Liver Cirrhosis Patients
Six studies compared CHO and no LES with 347
participants. Three studies compared BCAA-CHO andno LES with 89 subjects. Three studies comparedProtein-CHO and no LES with 138 patients. Two studies
compared BCAA-CHO and CHO with 59 subjects. One
study with 35 participants compared Coconut milk and
CHO.
The primary outcomes were albumin (Alb), pre-albumin,
nitrogen balance (NB), respiratory quotient (RQ), alanine
aminotransferase (ALT), aspartate aminotransferase(AST), total bilirubin (TB), ammonia (NH3),
cholinesterase (Che), and BCAA/Tyr (BTR). The risk ofbias assessments showed that most of these studies wereof moderate quality. No publication biases or small
sample effects were found.

Table 1 summarizes the basic characteristics of the
included studies. There were 5 LES compositions
according to these eligible studies: (i) CHO (CHO
provides >80% energy); (ii) BCAA-CHO (Protein 14%,BCAA 12%, CHO 59%, Lipid 15%); (iii) Protein-CHO(Protein 20-45%, CHO 50%?, Lipid ?); (iv) Coconut
milk-CHO (CHO 70%, Protein 2.5%, Lipid 27.5%), and(v) No LES. The included studies were publishedbetween 2006 to 2021, most of the studies were fromJapan (7 studies) and China (6 studies), while Egypt andIndonesia each had 1 study. A total of 695 participants
were included in this review and the average age of theparticipants was 59.6 years. The mean duration of theintervention was 8.6 weeks.

Table 1. Characteristics of the 15 included study
Comparison

Study
Hou W.
2021(22)
YamanakaOkumura H.
2010 (23)(i) CHO(CHO >80%)
Vs(v) No LES(ii) BCAA-CHO(P: 14% P, BCAA 12%;
CHO 59%; L 15%)
Vs(v) No LES(iii) Protein-CHO(P: 20-45%)
Vs(v) No LES(ii) BCAA-CHO(P: 14% P, BCAA 12%;
CHO 59%; L 15%)Vs(i) CHO (CHO>80%)(iv) Coconut milk - CHO(CHO 70%, P 2.5%, L
27.5% )vs(i) CHO (CHO 100%)

Li P. 2021(25)YamanakaOkumura H.
2006 (29)
Dong J. 2016(30)
Yu HW. 2012(31)
Takeshita S.
2009 (26)
Y. Harima.
2010 (27)Maki H. 2019(28)
Ferial. 2014(15)
Xu J. 2015(17)
Chen T.2014(16)
Nakaya Y.
2007 (14)Tatsuki I.
2010 (13)Suwito Indra.
2015 (24)

Locationn

Gender(M/F)

Age

Duration

Outcome

China

86

68/18

51.5 ± 11.8

6 months

ALT, AST, TB, Alb, PreAlb, Che, NH3, BTR

Japan

39

28/11

68.1± 9.7

6 months

BMI, AST, ALT, TB,
NH3, Alb

China

86

62/24

55.4±4.9

3 months

Alb, Pre-Alb

Japan

21

47/0

63 ± 10

1 week

AST, ALT, TP, Alb, TB,
Che, RQ

China

105

-

50.8±8.5

3 months

RQ, TP, Alb, Pre-Alb, Che

China

10

-

42.6±9.7

2 weeks

RQ

Japan

56

40/16

69.1±8.2

2 weeks

Japan

23

19/4

64.5±9.5

5 weeks

Japan

10

5/5

73.1 ± 8.9

1 month

Egypt

30

19/11

-

15 days

China

83

28/11

68.1± 9.7

4 weeks

AST, ALT, TB, NH3, Alb

China

25

14/11

49.4±12.7

6 weeks

Alb, Pre-Alb

Japan

38

20/18

67±8

3 months

Alb, AST, TB, NB, RQ

Japan

21

11/10

66.2±8.2

8 weeks

Alb, AST, ALT, TB, BTR,
NH3, Che

Indonesia

35

8/27

54.3±10.6

1 month

Pre-Alb, Alb

44

Alb, AST, ALT, TB, BRT,
NH3, Che
RQ, BRT, Alb, Pre-Alb,
ALT, TB, Che, NH3
Alb, AST, ALT, Che,
NH3, TB
NH3, NB, TB, ALT, AST.
AlbAsian Journal of Dietetics 2022
Results from network meta-analysis
Albumin
Human serum albumin is a critical plasma protein
produced by the liver. Advanced cirrhosis ischaracterized by reduced albumin concentration as wellas impaired albumin function (32).
Data from 13 studies with all five LES compositions
were included in the Alb analysis (13-17, 22-29). The
network map for Alb in is shown in figure 2. The mostcommon group was the no LES group followed by the
CHO group. The most common comparison was CHO
versus no LES. The inconsistency test exhibited noinconsistencies in the global analysis, indicating that thedirect comparison and indirect comparison results wereconsistent (χ2 = 0.85; p = 0.3556).

Figure 3 shows the results of the effects of the LES
on Alb. Compared with no LES, Protein-CHO (0.62
g/dL, 95% CI: 0.05 to 1.18) resulted in a significant
increment in Alb. There were no significant differences
between the other compositions in terms of theeffectiveness in increasing Alb.

CAA vs No LESCvs No LES

Coconut milk vs No LES
Controlrotein vs No
LES
Coconut milk vs CAA
rotein vs CAA

Coconut milk vs C
rotein vs C
rotein vs Coconut milk

Figure 3. Interval plot for mean difference inAlbumin (g/dL) from NMA*BCAA=BCAA-CHO; Protein=Protein-CHO; Coconut
milk=Coconut milk-CHO
The ranking of treatments for Albumin is shown intable 2. As can be seen, Protein-CHO had the highest
probability of being ranked as the best LES composition
with 77% of the surface under the cumulative ranking
curve (SUCRA). This was followed by CHO andCoconut milk-CHO and then BCAA-CHO.

Figure 2. Network map for Albumin.
The nodes (circles) represent the different LES
compositions while the edges (lines) represent theavailable direct comparisons between pairs of LES.
Both nodes and edges are weighted by the numberof studies involved in each treatment andcomparison, respectively.

Table 2. The surface under the cumulative ranking curve (SUCRA) of different LES compositions in various parameters
LES

Alb

RQ

AST

SUCRA
ALT
TB

NH3

80%

70%

100%

30%

BTR

Che

Protein + CHO(P 20-45%, CHO 50%?, L 10%?)77%

CHO (CHO>80%)

60%

90%

40%

60%

30%

90%

50%

30%

BCAA + CHO(P 14% P, BCAA 12%; CHO 59%; L
15%)

46%

60%

40%

40%

40%

60%

90%

40%

Coconut milk + CHO(CHO 70%, P 2.5%, L 27.5% )55%

No LES

10%

0%

30%

30%

30%

20%

0%

30%

45

100%

Best Late Evening Snack for Liver Cirrhosis Patients
Respiratory quotient (RQ)
In patients with liver cirrhosis, glycogen storage inthe liver is reduced. Therefore, glucose supply is
impaired from nighttime to the early morning fastingstate. In this situation, low RQ has been frequently
reported.
According to the network map (figure 4), there were
3 LES compositions (CHO, BCAA-CHO, and no LES)
from 5 studies (14, 27, 29-31). The overall test forinconsistency was not statistically significant (χ2=3.05,
p=0.08).

From the results of mean difference from NMA,
when compared to no LES, CHO and, BCAA-CHO bothsignificantly increased RQ, 0.06, 95%CI: 0.01-0.11;
0.04, 95%CI: 0.02-0.06, respectively.
From SUCRA, CHO (90% area) had a higher
probability of improving RQ of liver cirrhosis patients
than BCAA-CHO (60% area).

Figure 5. Interval plot for mean difference in RQ from NMA

Figure 4. Network map for RQ
Alanine Aminotransferase (ALT)
ALT is an enzyme that is concentrated primarily inthe liver. In liver cirrhosis, liver cells are damaged which
can lead to the leak of this enzyme into the bloodstream
and cause an abnormal increment of ALT.

The interval plot (Figure 7) indicates that in comparison
with no LES, all the compositions tended to decrease ALT;
however, there was no statistical significance in any of thecomparing pairs.
According to the ranking of SUCRA (table 2), ProteinCHO had the highest probability of being the best in improving
ALT, followed by CHO and BCAA-CHO.

The network map (Figure 6) demonstrates that ALT
data was formed by 8 studies with four compositions(CHO, BCAA-CHO, Protein-CHO, and no LES) (13, 15,
22-23, 26-29). The overall test for inconsistency was notstatistically significant (χ2=0.3, p=0.586).

Figure 6. Network map for ALT

Figure 7. Interval plot for mean difference in ALT (U/L) from NMA

46

Asian Journal of Dietetics 2022
While there was a decrement in AST, no significant
difference was found in the mean difference of AST in
all of the comparisons (Figure 9).
Table 2 indicates that Protein-CHO also had thehighest probability of being the best in improving AST
as well with 80% SUCRA, followed by CHO and BCAACHO with both having 40% of the area.

Aspartate transaminase (AST)
Similar to ALT, AST is also an enzyme that isconcentrated primarily in the liver and usually elevates inliver diseases.
There were also 8 studies and four compositions(CHO, BCAA-CHO, Protein-CHO, and no LES) that
contributed to forming the network map for AST (Figure8) (13-15, 22-23, 26, 28-29). The inconsistency test
showed no inconsistencies in the global analysis(χ2=0.72, p=0.3951).

Figure 8 Network map for AST

Figure 9. Interval plot for mean difference in AST (U/L) from NMA
Figure 11 demonstrates that when comparing to no
LES (-1.39mg/dL, 95% CI: -2.32 to -0.46), to BCAACHO (-1.32mg/dL, 95% CI: -2.45 to -0.2), and to CHO(-1.38mg/dL, 95% CI: -2.5 to -0.26), Protein-CHO
showed great decrement in TB. There was no significant
difference in other comparisons.
Protein-CHO with 100% of SUCRA had the highest
probability of being the best in decreasing TB comparedto others, followed by BCAA-CHO and CHO.

Total bilirubin (TB)
The liver normally removes bilirubin from theblood and disposes of it in the stool. But in liver cirrhosis,
bilirubin builds up in the blood and can cause jaundice.
The network map was formed from the data of 10
studies including four compositions (CHO, BCAA-CHO,
Protein-CHO, and no LES) (Figure 10) (13-15, 17, 2223, 26-29). There were no inconsistencies in theinconsistencies analysis (χ2=0.00, p=0.9742).

Figure 10. Network map for T

Figure 11. Interval plot for mean difference in T (mg/dL) from NMA

47

Best Late Evening Snack for Liver Cirrhosis Patients
Ammonia (NH3)Ammonia (NH3) is a waste product of the digestionof protein. In normal people, ammonia is processed into
urea in the liver. In liver cirrhosis, it builds up in the
bloodstream which leads to an increased serum NH3.
Data from 4 studies including 4 compositions(CHO, BCAA-CHO, Protein-CHO, and no LES) waspresented as the network map for NH3 in figure 12 (13,26-28). According to the global inconsistencies test, there
were no inconsistencies (χ2=0.15, p=0.7009).

Figure 12. Network map for N

The interval plot for the standardized mean difference inNH3 (Figure 13) shows that only the comparison
between CHO and no LES had a considerable decreasein NH3 (-0.72, 95% CI: -1.41 to -0.03). CHO also ranks
the best in having the most effective area of improving
the NH3 status in LC patients (90% SUCRA).

Figure 13. Interval plot for mean difference in N 3 from NMA

3

Branched-chain amino acids to Tyrosine ratio (BTR)
It has been reported that BCAA decreases in severecases of liver cirrhosis patients so BTR(BCAA/Tyrosine) is decreased(6).
Six studies with 3 compositions (CHO, BCAACHO, and no LES) provided the data to create a network
map for the BTR parameter (Figure 14) (13-14, 22, 2828). There were no inconsistencies in the inconsistencies
global analysis (χ2=2.6, p=0.107).

From the results of the mean difference of BTR,BCAA-CHO significantly increased BTR compared to
no LES (0.63, 95%CI: 0.19 to 1.07) (Figure 15). In
addition, the results from table 2 also indicate that
BCAA-CHO had a higher probability of increasing BTR
than CHO (SUCRA 90%, 50%, respectively).

CAA vs No LESC

Figure 14. Network map for TR

vs No LES

Figure 15. Interval plot for mean difference in TR
from NMA

48

Asian Journal of Dietetics 2022
Cholinesterase (Che)
Serum cholinesterase (ChE) is an enzyme
synthesized by hepatocytes and its serum levels reflect
the synthetic function of the liver. In liver cirrhosis, theChe level is decreased.
9 studies reported on Che involving 4 compositions(CHO, BCAA-CHO, Protein-CHO, and no LES). The
data was used to make the network of data for Che(Figure 16) (13, 17, 22-23, 26-30). No inconsistencies
were found by inconsistencies global analysis (χ2=0.25,
p=0.6179).

Figure 17 illustrates that when comparing to no LES(3.72, 95% CI: 2.8 to 3.74), to BCAA-CHO (2.98, 95%
CI: 2.38 to 3.57), and to CHO (3.22, 95% CI: 2.71 to3.73), Protein-CHO was more effective in increasing
Che. There was no significant difference in othercomparisons.
According to table 2, Protein-CHO had the highest
probability of increasing Che with 100% SUCRA.
BCAA-CHO was ranked second with 40% area and CHO
was third with 30% area.

Figure 17. Interval plot for mean difference in Che from NMA

Figure 16. Network map for Che
Results from the pairwise meta-analysis

Table 3. Results of the pairwise meta-analysis
Studiesn

MD (95%CI)p

I2

CHO vs. No LES

4245

5.45 (-2.5, 13.4)

0.18

96.6%

Protein-CHO vs. No LES

2

108

11.6 (6.37, 16.82)

0.00

96.7%

BCAA-CHO vs. No LES

123-1.26 (-5.35, 2.83)

0.55

-

Coconut milk-CHO vs. No LES

1

17-0.4 (-12.16, 11.36)

0.95

-

CHO vs. No LES

1

19-0.04 (-2.2, 2.12)

0.97

-

Protein-CHO vs. No LES

115

5.47 (1.95, 8.99)

0.00

-

BCAA-CHO vs. No LES

119

1.56 (0.02, 3.1)

0.05

-

Pre-Albumin

Nitrogen Balance

Pre-albumin (Pre-Alb)
Pre-albumin is the precursor to albumin. It is also
made by the liver. So in liver cirrhosis, the level of preAlb also decreases.
Pre-Alb was reported in eight out of the 15 studies.
Four studies reported comparison between CHO and no
LES (22, 24-25, 30); two studies reported comparison
between Protein-Cho and no LES (16-17); comparisons

between Coconut milk-CHO with no LES and BCAACHO with no LES each had 1 study (24, 27).
There was statistical heterogeneity among these trials(I2 = 97%). Using the random-effects model, results
indicate that only Protein-CHO had a significant increasein Pre-Alb from baseline (MD = 11.6, 95% CI: 6.37 to
16.82, p = 0.00) (Table 3).

49

Best Late Evening Snack for Liver Cirrhosis Patients
Nitrogen balance (NB)
As mentioned before, with the alternative
metabolism of LC patients, especially at night, theprotein was used as a source of energy instead of glucose,which increases nitrogen loss. Thus, a negative nitrogenbalance was reported in LC patients (32).
Two out of 15 papers investigated the effect of LES
on improving NB in LC patients with three different
comparisons (CHO vs. no LES, Protein-CHO vs. no LES,
and BCAA-CHO vs. no LES) (14-15).
Using the random-effects model, both Protein-CHO
and BCAA-CHO showed an increment in NB but only
Protein-CHO had a statistical significant (MD = 5.47,
95% CI: 1.95 to 8.99, p = 0.00) (Table 3).

probability of improving BTR of LC patients than other
LES compositions.
In this systematic review, serum albumin, prealbumin, and nitrogen balance were all significantly
increased by Protein-CHO. These biomarkers reflect thesynthetic metabolism of liver cells. It is reported thatalbumin has an important clinical significance inestimating the prognosis of patients with cirrhosis and abetter assessment of malnutrition (32). The results of
SUCRA of Alb from NMA showed that Protein
combined with CHO and CHO alone had been more
effective than Coconut milk-CHO and BCAA-CHO. It is
worth noting that in the BCAA contained LES, there was
59% of energy from CHO and 14% from Protein as well
(Aminoleban EN; Otsuka Pharmaceutical, Tokyo,
Japan). It raises the question of whether BCAA isnecessary to be provided in LES or not. In the advanced
stage of cirrhosis (decompensated liver cirrhosis), a
decline in serum BCAA was seen but not in the mildcases (compensated liver cirrhosis) (31). Therefore, the
ESPEN guidelines recommend a supplement of BCAA
for severe cases of LC (6). Nevertheless, in the studies
that used BCAA, all level severity of LC was included
and only a few of them offered an iso-nitrogenous regime
in comparison (34). Of note, there was a large withdrawal
rate (15%) across studies, due to poor palatability,
difficulties with compliance with multiple-dose per day,and adverse gastrointestinal symptoms including
diarrhea and abdominal distension (34-36). In addition,in many countries like Vietnam, oral BCAA supplements
are not available because of the high cost (about
4$/snack). It may be feasible to derive benefits of BCAA
from dietary sources rich in BCAA such as chicken
breast, beef, salmon, and red bean… (37).
Regarding respiratory quotient (RQ) and BCAA/Tyr
ratio (BTR), both of the parameters only included CHO
and BCAA-CHO in the comparison as no Protein-CHO
studies reported them. The results of RQ indicated that
LES helps increase RQ and CHO is maybe more
effective than BCAA-CHO. Masahiro et al. reported thatsurvival rate was significantly lower in patients with low
non-protein RQ (<0.85) than in patients with scores
above 0.85 where 0.85 is the point at which substrate forthermogenesis turns from carbohydrate dominant to lipid
dominant (38). This confirms the importance ofcarbohydrates as the main source of energy for LES andit should be at least 50g as the previous recommendations(6, 12). As for the BTR, BCAA-CHO showed advanced
effectiveness in increasing serum BCAA than CHO.
However, as there is a lack of evidence from othersources of protein, we could not confirm whether theprotein can be as effective as BCAA supplements or not.
The levels of ALT and AST were not significantly
different from the baseline when supplying LES in
cirrhotic patients. This finding was similar to thesystematic review of Guo YJ et al (2018), whichsuggested that bedtime snacks may not contribute to liver
parenchyma damage in patients with cirrhosis in a shorttime (39). However, in this review, total bilirubin andcholinesterase were significantly improved after theProtein-CHO snack administration. Moreover, ammonia
was also successfully decreased by CHO snacks. Thus,
with the appropriate compositions of LES which includes
both CHO and protein, not only protein synthesis and

DISCUSSION
Cirrhosis is characterized by a state of accelerated
starvation, with an early shift from glucose to lipid
utilization for energy during the postabsorptive state.
After an overnight fast, lipids account for 75% of the total
calories utilized in cirrhotic patients, reflecting increased
rates of ketogenesis and gluconeogenesis. In addition,
there is an increased consumption of amino acids as asource of energy (protein catabolism) (33). Therefore,
many international guidelines have suggested a lateevening snack for LC patients (6-7). However, according
to a previous study, 77.5% of Vietnamese LC patients did
not have the LES and 22.5% of them had LES but withinappropriate nutrient compositions (8). Thus, it is anurgent matter to give Vietnamese LC patients nutrition
education regarding LES and know the most appropriate
and suitable nutrient compositions concerning
acceptability, taste, and finance.
In the previous systematic review conducted by Tsienet al, the formula and dosages of LES were suggested toprovide at least 200kcal with 50g of carbohydrates;
however, there was no suggestion for the nitrogen source
whether it is protein or BCAA (12). LC patients areencouraged to have a high protein diet to prevent a
negative nitrogen balance, malnutrition, and sarcopenia.
Adequate protein intake has been defined as 1.2 to 1.5
g/kg body weight daily by the ESPEN guidelines (6). In
recent years, instead of protein, there has been a surge ofinterest in the clinical utility of BCAA, mostly insupplemental forms to be included in LES. Some studies
have suggested a BCAA-LES over CHO and othercompositions (13-14). However, the methodology ofthese studies is usually a comparison between BCAA,
regardless of doses and timing, with control or with CHO
and there are no studies directly comparing BCAA withprotein as the nitrogen source for LES.
This is the first systematic review and network metaanalysis to examine the effects of different types of LES
compositions with the purpose to find the most effective
composition. The results revealed that the combination of
CHO and protein seems to have the highest probabilityof improving the nutritional status and liver function of
LC patients. Protein-CHO has a higher probability of
improving Alb, Pre-Alb, nitrogen balance, ALT, AST,
TB, and Che of LC patients than other LES compositions.
While late evening snack with predominately CHO has
the highest probability of improving RQ and NH3.
Lastly, BCAA-CHO contained LES has a higher
50

Asian Journal of Dietetics 2022
energy metabolism can be improved but liver damage canalso be managed.
Several limitations in this meta-analysis and network
meta-analysis should be considered. First, most studies
included in the meta-analysis were single-center studies;
furthermore, the sample size in some of the studies was
small. Second, the subjects in all searched studies were
Asian (most were Japanese). Final, there was no directcomparison between some of the compositions,especially between Protein-CHO and BCAA-CHO.
Therefore, I would like to conduct a large, multicenter
RCT to confirm the effects of Protein-CHO LES on
patients with liver cirrhosis in comparison to an isonitrogenous and iso-caloric BCAA contained LES.

Dietetics 3(1): 7-12. 2021.
9) Huong Thi Le, Nguyet Thi Phuc Tran. Dinh dưỡng
lâm sàng – tiết chế. Nhà xuất bản Y học 2016.
10) Owen OE, Trapp VE, Reichard GA Jr, et al. Natureand quantity of fuels consumed in patients withalcoholic cirrhosis. J Clin Invest 72(5): 1821-1832.
1983.
11) Chen CJ, Wang LC, Kuo HT, Fang YC, Lee HF.
Significant effects of late evening snack on liverfunctions in patients with liver cirrhosis: A metaanalysis of randomized controlled trials. J
Gastroenterol Hepatol 34(7): 1143-1152. 2019.
12) Tsien CD, McCullough AJ, Dasarathy S. Lateevening snack: exploiting a period of anabolic
opportunity in cirrhosis. J Gastroenterol Hepatol
27(3): 430-441. 2012.
13) Ichikawa T, Naota T, Miyaaki H, Miuma S, Isomoto
H, Takeshima F, Nakao K. Effect of an oral branchedchain amino acid-enriched snack in cirrhotic patientswith sleep disturbance. Hepatol Res 40(10): 971978. 2010.
14) Nakaya Y, Okita K, Suzuki K et al. BCAA-enrichedsnack improves nutritional state of cirrhosis.
Nutrition 23: 113–120. 2007.
15) El-Kalla FS, Mansor LO, El-Bassat HA, Mishaal S,
Attia JF. The effect of a late-evening proteincontaining snack on nitrogen balance in cirrhotic
patients. Tanta Med J 42: 47-52. 2014.
16) T. Chen, J. Yu, and J. M. Zhang. Effects ofcompound protein nutrition agent on nutritional
status and liver function of patients with hepatitis B
virus-related cirrhosis. Journal of Practical Liver
Diseases 17(5): 466–469. 2014.
17) J. Xu, H. Y. Sun, M. Wang, and S. Q. Zhang. Effects
of dietary guidance on prognosis of decompensatedcirrhosis patients. China Practical Medical 10(36):277-278. 2015.
18) B. Hutton, G. Salanti, D. M. Caldwell et al. The
PRISMA extension statement for reporting ofsystematic reviews incorporating network metaanalyses of health care interventions: checklist andexplanations. Annals of Internal Medicine 162(11):
777–784. 2015.
19) Sterne J A C, SavoviÄ‡ J, Page M J, Elbers R G,
Blencowe N S, Boutron I et al. RoB 2: a revised tool
for assessing risk of bias in randomised trials. BMJ
366: l4898. 2019.
20) The Effective Public Health Practice Project(EPHPP). Quality Assessment Tool For Quantitative
Studies,
McMaster
University
National
Collaborating Centre for Methods and Tools. 2010.
21) J. P. Higgins, S. G. Thompson, J. J. Deeks, and D.
G. Altman. Measuring inconsistency in metaanalyses. BMJ 327(7414): 557–560. 2003.
22) Hou W, Lv Z, Yang J, Wu J, Wang ZY, Meng QH.
Long-Term Carbohydrate-Containing Late-Evening

CONCLUSION
With the results from the systematic review andnetwork meta-analysis, the recommendable LES
compositions should provide at least 200kcal with 50g ofcarbohydrate. About the nitrogen source, protein ismaybe more effective than BCAA in terms of improving
protein synthesis and energy metabolism as well as liver
parenchyma damage. However, since there is no directcomparison between protein and BCAA as LES, a future
study is needed to confirm this finding.
ACKNOWLEDGEMENTS
We would like to express my gratitude to Prof.
Shigeru Yamamoto, Professor of Jumonji University forhis guidance; and Prof. Eiji Marui, Professor of
University of Human Arts & Sciences, for consulting
regarding statistical analysis.
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2016: deaths by cause, age, sex, by country and byregion, 2000-2016. 2018.
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Dec 2019)
3) Sumeet K. Asrani1, Harshad Devarbhavi, John
Eaton, et al. Burden of liver diseases in the world.
Journal of Hepatology 70(1): 151–171. 2019.
4) Schuppan, D., & Afdhal, N. H. Liver cirrhosis.
Lancet 371(9615): 838–851. 2018.
5) Plauth M, Bernal W, Dasarathy S, et al. ESPEN
guideline on clinical nutrition in liver disease. Clin
Nutr 38(2): 485-521. 2019.
6) European Association for the Study of the Liver.
European association for the study of the liver:
EASL clinical practice guidelines on nutrition inchronic liver disease. J Hepatol 70(1): 172–193.
2019.
7) Maharshi, S., Sharma, B.C., Srivastava, S.
Malnutrition in cirrhosis increases morbidity andmortality. J Gastroenterol Hepatol 30(10): 1507–
1513. 2015.
8) Trang Thu Nguyen, An Tuong Bui, Linh Thuy
Nguyen, et al. Nutritional Status and Nutritional
Practice of Cirrhotic Patients at Hanoi Medical
University Hospital, 2020. Asian Journal of51Best Late Evening Snack for Liver Cirrhosis Patients
Snack Significantly Improves the Ratio of Branched
Chain Amino Acids to Aromatic Amino Acids in
Adults with Liver Cirrhosis due to Hepatitis B.
Biomed Res Int 2021:1074565. 2021.
23) Yamanaka-Okumura, H., Nakamura, T., Miyake, H.,
Takeuchi, H., Katayama, T., Morine, Y., Imura, S.,
Shimada, M., and Takeda, E. Effect of long-termlate-evening snack on health-related quality of life incirrhotic patients. Hepatology Research 40: 470-476.
2010.
24) Indra S, Gani RA, Syam AF, Shatri H. Effect of
Coconut Milk Supplementation to Nutritional Status
Parameters in Liver Cirrhosis Patients. The
Indonesian
Journalof
Gastroenterology,
Hepatology, and Digestive Endoscopy 16(2): 78-85.
2015.
25) Li Pei. The effect of nutritional intervention before
going to bed in the diet nursing of patients with
hepatitis B cirrhosis. Physician Online 27. 2021.
26) Takeshita S, Ichikawa T, Nakao K, Miyaaki H,
Shibata H, Matsuzaki T, et al. A snack enriched withoral branched-chain amino acids prevents a fall inalbumin in patients with liver cirrhosis undergoing
chemoembolization for hepatocellular carcinoma.
Nutr Res 29(2): 89-93. 2009.
27) Harima Y, Yamasaki T, Hamabe S, Saeki I, Okita K,
Terai S, et al. Effect of a late evening snack using
branched-chain amino acid-enriched nutrients inpatients undergoing hepatic arterial infusion
chemotherapy
foradvanced
hepatocellular
carcinoma. Hepatol Res 40(6): 574-584. 2010.
28) Maki H, Yamanaka-Okumura H, Katayama T,
Ozawa Y, Hosoda A, Kurata N, et al. Late evening
snacks with branched-chain amino acids improve the
Fischer ratio with patients liver cirrhosis at fasting in
the next morning. Clin Nutr ESPEN 30: 138-144.
2019.
29) Dong J, Liu Y, Sun L, He M, Huang J, Hu M, et al.
Effect of late night snack on nutritional status andquality of life in cirrhotic patients. Chinese journalof clinical nutrition 24: 342-348. 2016.

30) Yamanaka-Okumura H, Nakamura T, Takeuchi H,
Miyake H, Katayama T, Arai H, et al. Effect of lateevening snack with rice ball on energy metabolism
in liver cirrhosis. Eur J Clin Nutr 60(9): 1067-1072.
2006.
31) H. W. Yu, J. Zhao, K. F. Wang, J. Li, and S. Y. Shi.
Effects of late evening snack on energy metabolism
in cirrhotic patients with different Child-Pughgrades. Infectious Disease Information 25(4): 216–
219. 2012.
32) Spinella R, Sawhney R, Jalan R. Albumin in chronic
liver disease: structure, functions and therapeutic
implications. Hepatol Int 10(1): 124-132. 2016.
33) Arakawa Y, Moriyama M, Arakawa Y. Livercirrhosis and metabolism (sugar, protein, fat andtrace elements). Hepatol Res 30S: 46-58. 2004.
34) Marchesini G, Bianchi G, Merli M, et al. Nutritional
supplementation with branched-chain amino acids inadvanced cirrhosis: a double-blind, randomized trial.
Gastroenterology 124: 1792–1801. 2003.
35) Muto Y, Sato S, Watanabe A, et al. Effects of oral
branched-chain amino acid granules on event-freesurvival in patients with liver cirrhosis. Clin
Gastroenterol Hepatol 3: 705–713. 2005.
36) Park JG, Tak WY, Park SY, et al. Effects ofbranched-chain amino acids (BCAAs) on the
progression of advanced liver disease: a Koreannationwide,
multicenter,retrospective,
observational, cohort study. Medicine (Baltimore)
96: e6580–e6587. 2017.
37) Yao CK, Fung J, Chu NHS, Tan VPY. Dietary
Interventions in Liver Cirrhosis. J Clin Gastroenterol
52(8): 663-673. 2018.
38) Tajika M, Kato M, Mohri H, Miwa Y, Kato T,
Ohnishi H, Moriwaki H. Prognostic value of energy
metabolism in patients with viral liver cirrhosis.
Nutrition 18(3): 229-234. 2002.
39) Guo YJ, Tian ZB, Jiang N, Ding XL, Mao T, Jing X.
Effects of Late Evening Snack on Cirrhotic Patients:
A Systematic Review and Meta-Analysis.
Gastroenterol Res Pract 2018:9189062. 2018.

52

Asian Journal of Dietetics 2022

Original
Development of the Revised Food Frequency Questionnaire for Cambodian
School-aged Children
Yoko Horiuchi1*, Kaoru Kusama2, and Eri Kai3
1

Department of Health and Nutrition, Faculty of Psychological and Physical Science, Aichi Gakuin
University, Nisshin, Aichi 320-0195, Japan2
Department of Food and Health Sciences, Faculty of Health and Human Development, The
University of Nagano, 8-49-7 Miwa, Nagano 380-8525, Japan
3
Foundation for International Development/Relief (FIDR); No. 82F, St. 210 Sangkat Teuk La-ak III
Khan Toul Kork, Phnom Penh 12158, Cambodia
ABSTRACT: Background and purpose. This study aimed to describe the process ofrevising the Food Frequency Questionnaire (FFQ) developed in 2016 for school-aged
children in Cambodia (FFQ-CSC). Methods. First, we updated the food composition
database using the updated version of the ASEAN Food Composition Table; we also
added specific values used in the SMILING food composition tables for Cambodia
and Thailand and in the food composition tables for Singapore, Thailand, and Japan.
The previous portion sizes were modified based on the current reference amounts.
Second, we re-examined the photographs in a picture book and adjusted themaccording to the revised portion sizes. Result. The revised FFQ-CSC was created witha new data entry form in Excel, which allows users to input data more accurately. The
new picture book comprises 64 pages for 53 food items and provides respondents withmore comprehensive information about various portion sizes. We also created amanual explaining how to use the FFQ-CSC. Conclusion. The revised FFQ-CSC withthe updated food pictures can be used to assess the dietary intake of Cambodian
children. With the newly developed manual, we expect this questionnaire to be usedwidely and more frequently. It is important to update and revise this tool regularly.
Furthermore, the revised FFQ-CSC must be validated and its reliability must be
assessed.
Key Words: Cambodia, school-aged children, food frequency questionnaire, revised
version, development

INTRODUCTION
The Food Frequency Questionnaire (FFQ) is one of
the most commonly employed dietary assessment tools
in large epidemiological studies (1). It generallycomprises a list of commonly consumed foods withsome possible options regarding the frequency of theirconsumption (2) and can assess habitual food intake
over a set time period (3). Several versions of the FFQ
have been developed over the last few years (4,5); avalidated FFQ can provide information on food intake
during a certain period (6,7).
In Cambodia, however, before we developed the
FFQ for Cambodian school-aged children (FFQ-CSC) in
2016 to assess their intake of macronutrients andcalcium, there was no FFQ to measure their dietaryintake. The original FFQ-CSC was validated through a
comparison with the 24-hour dietary recall method(hereafter 24-h) developed by the Foundation forInternational Development/Relief (FIDR) in 2017 (8).
Nutritionists usually recommend using or modifying an
existing questionnaire in epidemiological surveys (9).
Communities should use FFQs that were developedwith considerations for how ethnicity, culture, andeconomic status may affect food intake and dietary
habits (10,11). Assigning valid nutritional values to thefood items on the quantitative FFQ requires the use of
an appropriate dietary database (12) and replacement of
the food composition table (FCT) used to calculate
nutrient intake, by making appropriate changes to the*To whom correspondence should be addressed:
yhori@dpc.agu.ac.jp

portion sizes and updating the program code fornutrient calculation (13,14).
Hence, we aimed to modify the FFQ-CSC by using
the updated Association of Southeast Asian Nations(hereafter ASEAN) FCT and other food composition
data as nutritional data. We also assessed and revised
the food items, their frequencies and portion sizes, andthe program code to calculate the nutritional values,
and provided a picture book that allows children tovisualize portion sizes more precisely. In this paper, we
describe the process of revising the FFQ-CSC to matchthe recent dietary trends in Cambodia.
METHODS
Based on the results of our validation study and thecurrent dietary trends, we modified the previous
version of the FFQ-CSC (8) as described below.
Data collection
The nutritional data were updated primarily using
the 2014 ASEAN FCT (15). The original FFQ-CSC
adopted the 2000 ASEAN FCT (16) which was thenrevised in 2014 (15) and SMILING FCT: Cambodia (17)as its nutrition database. To address the missing data in
the ASEAN FCT, we also incorporated other data from
Smile FCT: Cambodia (17) and Thailand (18) and FCTs
from Singapore (19), Thailand (20), and Japan (21). The
nutritional data used in the FFQ-CSC were energy(calories) and nutrients (protein, fat, carbohydrate, andcalcium).
Food Items/Frequency/Portion Sizes
Improvements to the FFQ-CSC made in a previous
study (8), the current portion sizes in the market wereassessed to identify any limitations.

53

Development of the Revised Food Frequency Questionnaire in Cambodia
Program Code for the Nutrient Calculation
We re-created the input sheet on Microsoft Excel
2016 for the revised questionnaire by updating the fooditems. In addition, a pull-down input was adopted forthe frequency and portion size choices.
To assess the quantities of oil and sugar used in thefood items, we enabled a system that automaticallyadds the amount of oil and sugar in proportion to theenergy value, instead of requiring respondents toprovide these quantities.
Picture Book
We provided a picture book containing the updated
representative portion sizes of the food items to help
children select their portion sizes easily. The sizes ofthe dishes and spoons in the photographs wereconsistent so that respondents could imagine moreaccurate portion sizes.

Second, regarding the modified food items andportion sizes, in a previous study, 33 food items wereadded to and 5 food items and beverages were excluded
from the FFQ to account for regional and generational
dietary changes (14). Another study used altered portion
sizes to improve the accuracy of the participants’dietary intakes (13).
Third, the weak correlation between the fat levelsin the FFQ-CSC and 24-h (8) was considered.
Concerning fat intake, because our survey inquired
about respondents’ consumption of stir-fried or deepfried dishes to measure oil intake, it may have beendifficult for children to answer these questions (8). A
study conducted in Tehran (26) showed that the weakcorrelation between liquid oil and solid fat might bedue to respondents’ lack of culinary knowledge. In
addition, people use various types of volume measures,such as teaspoons, tablespoons, and cups, for liquid oils
and solid fats in food preparations. Therefore,
estimating the actual amount of lipids is a challenge.
Further, measuring the nutritional values of ingredientsin mixed dishes when assessing dietary intake is always
a taxing process, and there is a risk of potential loss of
dietary information (27). Thus, there was a need toaddress the question about “oil intake.” Similarly, aquestion about “sugar consumption” in the previous
FFQ-CSC used teaspoons as the measuring unit, which
may have made it difficult for school-aged children to
estimate their sugar intake.
To address these limitations, we developed a new
system in which oil and sugar quantities wereautomatically added per each individual’s calorie
intake. In addition, we considered that a picture bookwould help the respondents visualize the food items,
making it easier to estimate their portion sizes. An
accurate estimation of food portion size remains animportant challenge in the collection of dietary data. In
China, a food atlas with adequate visual references wasdeveloped to improve the accuracy of dietary surveys(28). We should also continue to enhance the foodpictures to help respondents accurately determine their
portion sizes.
Finally, we emphasize that our next step is tovalidate the revised FFQ-CSC to ensure its feasibility
and reproducibility.
Over the past decade, dietary assessment methods
have increasingly made use of online technologies,
with the traditional paper-based questionnaires getting
replaced (29). An online self-administered FFQ has beenvalidated (30). Digital solutions may minimize some ofthe errors associated with paper-based questionnaires,and the problem of missing values can be eliminatedthrough automated pop-up reminders and mandatory
questions. These in turn may ease respondents’cognitive burden when choosing the right portion sizeand potentially reduce errors from inaccurate
estimations of portion sizes (30). Therefore, we aim todevelop an online version of the FFQ-CSC after
evaluating the modified FFQ-CSC.
In conclusion, the revised FFQ-CSC with updated
food pictures can now be used to assess the dietaryintake of Cambodian children. We expect this tool to be
used widely and more frequently in Cambodia.
However, the validity and reliability of the revised
FFQ-CSC must be assessed. Furthermore, we believethat FFQ-CSC should be updated and revised in the
future with changing environmental conditions.

RESULTS
Nutritional Data
Table 1 shows the updated nutritional data on the
final list of food items.
Food Items/Frequency/Portion Sizes
Table 2 shows the modifications to the nutritional
data. Two items (mung bean cake and salt for food) weredeleted, one item was modified (salt for young fruits),
and portion sizes of all except 17 items (e.g., rice
noodles) were revised. The frequency of each item was
unified.
Program Code to Calculate the Nutritional
Values
We generated a new input worksheet on Microsoft
Excel 2016 with pull-down lists for frequencies andportion sizes to enable easy input. The input numbers
corresponding to the frequencies and portion sizes
were converted into the quantities used to calculate thenutritional values. In addition, automatically calculated
amounts of sugar and oil in the food items were
included in the program code (Table 2). Based on thefollowing formula, the amounts of oil and sugar per1000 kcal were calculated to be 21.1 g and 83.9 g,
respectively.
Formula: Adding sugar amount (g) per 1000 kcal =(Averagecarbohydrate
intakeonly
from“sugar”/Average energy intake)/S; S is carbohydrate per
100g of sugar (=99.4). Adding oil amount (g) per 1000kcal = (Average fat intake only from “oil”/Averageenergy intake)/O; O is fat per 100g of oil (=90.8).
Picture Book
A 64-page picture book was created to supplement
the food items listed in the FFQ-CSC. To ensure that thephotographs were as realistic as possible, the bowl orplate containing the food was photographed against abackground of vertical and horizontal coordinates
calibrated in units of length (scale with 2.5 cm × 2.5cm), and the images were color-printed.
DISCUSSION
We updated the FFQ-CSC based on the results of aprevious validation study (8) and the current dietary
trends. First, we referred to various FCTs (15, 17-21) tomodify the nutritional values of the FFQ-CSC. Thismethod had also been employed for the previously
developed FFQs (22-25). In Croatia, the FFQ wasdeveloped using a combination of different food
databases to analyze the meals because their national
food composition database is outdated and impracticalto use (25).

54

noodle with flavor, corn, sweet potato

Morning glory, amaranth, spinach, cucumber,

II. Vegetables

55

All

for young fruits*, fermented fish

paste in the dish, fish sauce, (salt for food), salt

(Soybean oil), (sugar for food and drink), fish

powder, soft drink juice, soft drink tea(mung bean cake), Milo powder, Ovaltine

Rice cake, snack made from potato or corn,

powder, ice cream, condensed milk

Fresh milk, yogurt, yogurt drink, cheese, milk

pork blood

duck egg, beef, pork, beef meat ball, chicken,

snail, shrimp, crab, canned fish, dried shrimp,

bean, roasted peanut, snake head fish, clam,

Fermented soybean, soybean milk, boiled mungplum, rambutan

guava, green mango, ripe papaya, Spanish

Yellow banana, green banana, coconut cream,

Q1–Q58

Q51–58

Q44–50

Q37–43

Q20–36

Q12–19

Q7–11

Q1–6

57

8

7

7

16

8

5

6

1

0

0

0

1

0

0

0

SMILING
FCT
Cambodia2013

Q1–Q53

Q50–53

Q44–49

Q37–43

Q20–36

Q12–19

Q7–11

Q1–6

27

0

4

6

7

7

1

2

ASE
AN FCT
2014

Q. No.

ASEAN
FCT
2000

Q. No.

20

2

0

0

9

1

4

4

SMILING
FCT
Cambodia
2013

2

1

1

0

0

0

0

0

SMILINGFCT Thailand
2013

1

0

1

0

0

0

0

0

Singapore
FCT 2003

Note: FCT: Food Composition Table; (Foods enclosed in brackets have been removed in the updated version); * Modified in the updated version

VII.
Condiments

VI. Snacks anddrinksV. Milk andmilk products

VI. Meat, fish,
egg, and beans

III. Fruits

Boiled rice, rice noodle, sandwich bread, wheat

I. Cereal andcereal products

bean sprout

Name of food item

Food group

Updated version

Previous version

Table 1. Question numbers and composition of nutritional data in the previous and updated versions of the questionnaire

2

1

0

0

1

0

0

0

ThailandFCT 2015

1

0

0

1

0

0

0

0

Japan
FCT
2020

Asian Journal of Dietetics 2022

Portion sizes of rice noodles were adjusted according to a commonly recognized unit of measurement used in the local market
or store.
In the previous version, for boiled rice, the choice of frequency was set as once a day, 2–3 times, 4–5 times, or 6 times or more
per day. However, when revising the questionnaire, we considered potential respondents who never eat boiled rice. Therefore,
we set the frequency choice as that for the other items, with ten response categories: never, once a month, 2–3 times a month, 1–
2 times a week, 3–4 times a week, 5–6 times a week, once a day, 2–3 times a day, 4–5 times a day, or 6 times or more per day.

To improve the FFQ-CSC to assess omission of oil, we implemented a system that automatically adds the amount of oil inproportion to energy value, instead of asking about the intake of “oil.”
The amount is calculated based on the values from a 24-hour dietary recall questionnaire, which was administered in thevalidation study of the FFQ-CSC [8] by using the following formula:
Adding oil amount (g) per 1000 kcal = (Average fat intake only from “oil”/Average energy intake)/O; O is fat per 100g of oil(=90.8).
To improve the FFQ-CSC to assess omission of sugar, we implemented a system that automatically adds the amount of sugar inproportion to energy value, instead of asking about the intake of “sugar for food” and “sugar for drink.”
The amount was calculated based on the values from a 24-hour dietary recall questionnaire, which was administered in thevalidation study of the FFQ-CSC [8] by using the following formula:
Adding sugar amount (g) per 1000 kcal = (Average carbohydrate intake only from “sugar”/Average energy intake)/S; S iscarbohydrate per 100g of sugar (=99.4).

Rice noodle

Oil

56

Sugar

Frequency

Modified in the updated version to reflect its real use in Cambodian cuisine. It refers to the mixed dipping salt used for eating
young fruits, such as mangoes. The nutritional data for "Salt for young fruit" is not available in the FCT. Therefore, it has been
modified using nutritional information provided by food companies.

Removed in the updated version due to the difficulty faced by children in answering the question. Moreover, sodium is not atargeted nutrient in the updated FFQ-CSC.

Removed in the updated version because every participant selected “0” for the frequency question.

Changes made in the updated version

Salt foryoung fruits

Mung bean
cake
Salt

Item

Table 2. Frequency of food consumption in the past three months

Development of the Revised Food Frequency Questionnaire in Cambodia

Asian Journal of Dietetics 2022

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58

Asian Journal of Dietetics, 2022

Original
A Crossover Study on the Effects of Continuous Soymilk Intake on Lipid Metabolism
and Glucose Metabolism in Vietnamese - Lipid Metabolism Improving Effect –Ai Yamamoto1*, Tamaki Shintomi1, Vu Thi Thu Hien2, Le Danh Tuyen2, Nguyen Thi Diep Anh2, Le Anh
Hoa2, Le The Trung3, Tran Van Long3, Nguyen Van Tuyen4, Nguyen Cam Thach4, Shigeru Yamamoto5,
Nobuo Yoshiike6,Makoto Sugawara7, Ryoichi Unno7, Miki Tomoe1
Department of Nutrition, Faculty of Home Economics, Kyushu Women’s University, Fukuoka, Japan
2
Vietnam National Institute of Nutrition, Hanoi, Vietnam
3
Nam Dinh University of Nursing, Nam Dinh, Vietnam
4108 Military Central Hospital, Hanoi, Vietnam
5Department of International Nutrition, Jumonji University Graduate School of Human Life Sciences,
Saitama, Japan
6
Graduate School of Health Sciences, Aomori University of Health and Welfare, Aomori, Japan
7
Research and Development Division, MARUSAN-AI Co., Ltd., Aichi, Japan
1

ABSTRACT Background and purpose. Unprepared soymilk is expected to have a physiologicaleffect due to its high protein content, but there are few clinical trials on soymilk intake in Vietnam
and other Asian countries targeting patients with dyslipidemia. This study was conducted a crossoverstudy to verify the effect of unprepared soymilk intake on improving lipid metabolism in a joint
study with Vietnam National Institute of Nutrition, Nam Dinh University of Nursing, MARUSANAI Co., Ltd., and Kyushu Women's University. Methods. It was conducted with the approval of the
Kyushu Women's University Ethics Review Committee and the Vietnam National Institute of
Nutrition Ethics Review Committee. Thirty-seven type 2 diabetic patients with LDL cholesterolborderline or mild dyslipidemia attending hospital in Nam Dinh City, Vietnam, were included in theanalysis. The study design was a randomized crossover study of 2 groups, and only the intervention
group was given 500 mL/day of unadjusted soymilk (MARUSAN-AI Co., Ltd.) for 8 weeks, andthe washout period was 4 weeks. The primary endpoints were LDL-C, TG and the secondary
endpoints were TC, HDL-C. Result. The amount of change in LDL-C and TG in the intervention
group were significantly reduced at both the 4W-intake and 8W-intake. In addition, the amount ofchange in HDL-C in the intervention group were significantly increased at the 8W-intake.
Conclusion. For Vietnamese with type 2 diabetes with dyslipidemia, it became clear that thecontinuously intake of unprepared soymilk could decrease LDL-C and TG and increase HDL-C.
Keywords: Vietnamese, crossover study, unprepared soymilk, LDL cholesterol, lipid metabolism
INTRODUCTION
In the Socialist Republic of Vietnam (hereinafterreferred to as Vietnam), lifestyle-related diseases suchas diabetes mellitus, obesity and hypertension are
increasing as in developed countries (1-3). The
proportion of people who died of hyper-LDLemia
increased by 37.3% in the 10 years from 2009 to 2019(4), which is thought to be due to increased intake ofanimal foods and fats and lack of exercise (5-7). In
many meta-analyses, soy protein intake has beenreported to reduce serum low-density lipoproteincholesterol (LDL-C) and serum triglyceride (TG) (8,914), and soy globulin is expected to have aphysiological effect. Furthermore, β-conglycinin, themain component of soy globulin, has been shown to
reduce the accumulation of serum triglyceride andvisceral fat (15). In recent years, Zhang et al. (16) havereported a meta-analysis that fasting blood glucose
level, fasting insulin level are significantly reduced bysoy protein intake. However, according to a report by
Le et al. (17) in 2014, the energy intake of Vietnamese
people is about 2,000 kcal/day, and the protein intake
is 61.6 g/day, but the protein intake from soybean
products is 2.8 g/day (less than 5%). Soymilk is one of
the soybean products that can easily ingest soybean

protein. In particular, unprepared soymilk has a highprotein content, so soy protein intake is expected tohave a physiological effect. Recently, Ta NT et al. havereported that soy protein intake significantly reduced
fructosamine (FRA), total cholesterol (TC), and TG in
Vietnamese patients type 2 diabetes mellitus (DM)with hyper-LDLemia in a pre- and post-comparison
within the soy protein intake group (18) . In this way,
it has been suggested that the intake of soy protein may
improve lifestyle-related diseases in Vietnamese
people.
In addition, the soymilk market share in Vietnam
is dominated by prepared soymilk with added sucrose,and the protein content is as low as about 2 g/100mL.
Therefore, it is considered that the physiological effectof soy protein cannot be sufficiently obtained. Also,
there are few clinical trials in Vietnam and other Asian
countries targeting patients with hyperlipidemia due tosoymilk intake alone.
In this study, a joint research with Vietnam
National Institute of Nutrition (NIN), Nam Dinh
University of Nursing (NDUN), MARUSAN-AI Co.,
Ltd., and Kyushu Women's University, weinvestigated the physiological effects of continuous
intaking unprepared soymilk without sucrose (soyprotein content 4.5 g/100mL, carbohydrate content 1.5
g/100mL, MARUSAN-AI Co., Ltd. products) in
Vietnamese patients with type 2 DM with

*To whom correspondence should be addressed:

a-yama@kwuc.ac.jp

59Effects of continuous soymilk intake on lipid metabolism
dyslipidemia. In order to examine whether the lipid
metabolism is improved by daily intaking 22.5 g of soyprotein contained in 500 mL (250 mL x 2 times) ofunprepared soymilk for 8 weeks, a crossover study
was performed in two groups, the unprepared soymilk
intake group (intervention group) and the preparedsoymilk non-intake group (control group). A dietary
survey confirmed the effects of diet and consumptionof the unprepared soymilk on energy and nutrient
intake. For evaluating the effect of continuous intakeof raw soymilk on improving lipid metabolism andglucose metabolism, changes in LDL-C, TG, and FRA
from before to 4 weeks and 8 weeks after intake wereused as primary endpoints. Secondary endpoints werechanges in TC, high-density lipoprotein cholesterol(HDL-C), and fasting blood glucose (FPG) levels frombefore to 4 weeks and 8 weeks after intake.
MATERIALS AND METHODS
1. Ethical considerations
This study was conducted in accordance with the
Declaration of Helsinki-Ethical Principles of Medical
Research Including the Human Body (World Medical
Association) and "Ethical Guidelines for Medical and
Health Research Including the Human Body"(Ministry of Health, Labor and Welfare, Japan). The
ethics review was approved by the Kyushu Women's
University Ethics Review Committee (approval
number H30-14) and the Vietnam National Institute of
Nutrition Ethics Review Committee. After that,
informed consent was given to the subjects, andconsent was obtained. The research period is from
August 2020 to March 2022.

period. 3) Person who can drink soymilk continuously
for 8 weeks. 4) A person who has given written consent
to participate in this study. Exclusion criteria: 1)
Person who are DM and have been used insulin fortreatment. 2) Person who are allergic to soybeans. 3)
Person with serious disease. 4) Person who has aserious medical history or history of gastrectomy. 5)
Person taking internal medicine or formula milk forDM. 6) Excessive alcoholic drinkers or excessive
smokers. 7) Any other person who are deemed
inappropriate to participate in this study.
The sample size was calculated based on theformula [1] proposed by Hassard.

N=2xGroup AGroup B
Soymilk intake statusrecordMeasurement items
1) Dietary survey2) Anthropometry3) Blood tests

●
●
●

th

4 weekWashout
periodth

8 week

4 weeks

2nd Periodth

12 weekth

16 weekControl group

Intervention group
Control group
Group A

●
●
●

●
●
●

●
●
●

Figure 1. Study schedule and measurement items

60th

20 week

Intervention group
Group B

●
●
●

[1]

Study design and schedule
The study design was a crossover study and was
randomly assigned to two groups, group A and group
B. The study schedule is shown in Figure 1. In the first
period, only Group B ingested unprepared soymilk(MARUSAN-AI Co., Ltd., made in Thailand) 500
mL/day (250 mL x 2 times) for 8 weeks, and thewashout period was 4 weeks. In the second period,
only Group A ingested 500 mL/day (250 mL x 2 times)of unprepared soymilk for 8 weeks.
The nutritional composition of unpreparedsoymilk is shown in Table 1. The period before intake
of unprepared soymilk hereinafter referred to as"before intake", and 4 weeks and 8 weeks after intake
of unprepared soymilk hereinafter referred to as "4Wintake" and "8W-intake", respectively. The
measurement
items
were
dietary
survey,anthropometry, and blood tests, and the test was
conducted once for each period for a total of 6 times.
3.

Subjects
The sample size of the subjects was set toforty-four. For type 2 DM patients with borderline ormild LDL cholesterolemia who attending hospital in
Nam Dinh City, Vietnam, a screening test was
conducted to determine eligibility in 45 patients who
gave their consent, and those who met all the following
inclusion criteria 1) to 4) and did not meet any of the
exclusion criteria 1) to 7) were selected. Inclusion
criteria: 1) Patients who are type 2 DM diagnosed inpast 5 years with FBG > 7 mmol/L, are using oral drug
for management DM, had no severe complications andno advisory of dietetics for controlling DM, havehyperlipidemic with high LDL-C (boundary range:
3.10-3.59 mmol/L, mild range: 3.60-4.10 mmol/L) but
not yet taking drugs to control hyperlipidemic. 2)
Those who can participate in the test during the test

BeforeIntake (0W)( 1  2 ) 2Zα = 1.96, Zβ = 0.84,  is the standard deviation of
LDL-C concentration, 1 is the change in LDL-C
concentration from baseline to 8 weeks of taking
soymilk, 2 is change of LDL-C concentration frombaseline to 8 weeks of no taking soymilk. We also
assume that  is approximately 0.245 mmol/L (basedon results of our previous research in Vietnamese).
Therefore, sample size as calculated as 37 subjects.
After adding 20% of dropt out, the suggested sample
size is 44 (subjects).

2.

1st Period( Z  Z  ) 2 . 2●●●

Asian Journal of Dietetics, 2022

Table 1. Nutritional composition of unprepared soymilk (test beverage)
Nutrients

Unprepared soymilk (per 100mL)

Energy (kcal)

44

Protein (g)4.5

Lipid (g)2.2

Carbohydrate (g)1.5

Sucrose (g)0.4Dietary fiber (g)0.3Anthropometry
Weight and height were measured while
participant standing, wearing light clothing and no
shoes. Body fat percentage was measured by TANITA
scale (model BC- 541N, TANITA Corporation). Waist
circumference was measured by the minimum
circumference between the umbilical cord and the iliac
crest, and hip circumference was measured by thewidest circumference of the buttocks.

calculation of nutrient intake was based on the
Vietnamese Food Composition Table published in
2017.

4.

5.

Blood tests
The subjects fasted from 9 pm the day before
blood tests, visited the hospital by 9 am on the day ofblood tests (water intake was possible), and had blooddrawn. The test items are serum total protein (TP),serum albumin (Alb), FPG, TC, LDL-C, HDL-C, TG,
alanineaminotransferase(ALT),
aspartateaminotransferase (AST), creatinine (Cre), and FRA.

Statistical analysis
All values are shown as mean ± standard
deviation. Using Microsoft® Excel 2016 for Windows,
an unpaired t-test were performed on the carry-overeffects in Group A and Group B prior to the analysis
of a crossover study. The significance level was set to10%. Statistical analysis software IBM SPSS Statistics
20 (IBM Japan Headquarters, Inc.) was used toanalyze the attributes and endpoints of the subjects.
Analysis of change between two groups of the same
subjects used paired t-test and change between two
groups of different subjects performed unpaired t-test.
All tests were two-sided test, and the significance level
was set to 5%.

Dietary survey
The 24-hour dietary recall method was used forthe dietary survey, and a total of 6 surveys wereconducted at 0th week, 4th week, 8th week, 12th week,
16th week, and 20th week. Regarding the meals on theprevious day, we conducted an interview survey on themeals (type and amount of food) from the time of
waking up to the time of bedtime. To answer the intake
accurately, we prepared photographs of the actual sizeof standard vessels (bowl, cup, spoon) used in ordinary
households, and conducted an interview survey while
showing them. In addition, “Calorie Smile” software(Quest-Computer Co., Ltd.,) which also includes
photo data of common dishes, was used during thesurvey to improve the accuracy of the survey. The

RESULTS
1. Subject selection and attributes
Of the 45 subjects who gave their consent, 42
subjects who met the selection criteria were randomly
assigned to each of the two groups, Group A andGroup B, by 21. After the start of the study, 5 subjects
dropped out due to non-participation in the study (3subjects) or taking medication for the treatment ofdyslipidemia (2 subjects), and finally 19 subjects ingroup A and 18 subjects in group B, a total of 37
subjects, were included in the analysis (Figure 2). The
attributes of the subjects are shown in Table 2. There
were no significant differences in age, height, weight,
body mass index (BMI), and body fat percentage
between the two groups, Group A and Group B.

7.

6.

61

Effects of continuous soymilk intake on lipid metabolism
Figure 2. Flow chart from selection of subjects to analysis

Table 2. Subject attributes
Group A(n=19)
Mean ± SD

Group B
(n=18)
Mean ± SD

Mean ± SD

Age (years)

61.8 ± 6.9

64.7 ± 6.3

63.2 ± 6.8

0.21

Height (cm)155.4 ± 7.7

155.4 ± 6.6

155.4 ± 7.2

1.00

Weight (kg)All (n=37)

p value†

58.1 ± 11.6

57.7 ± 7.6

57.9 ± 9.8

0.91

2

BMI (kg/m )23.9 ± 3.7

23.8 ± 2.5

23.9 ± 3.2

0.93

Body fat percentage (%)

30.9 ± 7.3

30.4 ± 7.2

30.7 ± 7.3

0.83

†

: Unpaired t-test, n=37

2. Verification of carry-over effect
As a result of verifying the carry-over effect of
the crossover study, LDL-C and TG, which are theprimary endpoints of lipid metabolism, had no carryover effect. However, TC, which is a secondary
endpoint, was excluded because it had a carry-overeffect. In addition, a carry-over effect was observed in
FRA, which is the primary endpoint of glucose
metabolism, so it was not possible to analyze glucose
metabolism.

group was 2.96 ± 1.12 mmol/L before intake, 2.46 ±0.80 mmol/L at 4W-intake, and 2.38 ± 0.65 mmol/L at
8W-intake. The Δ4W-intake in TG intake was 0.24 ±0.60 mmol/L in the control group and −0.49 ± 0.64
mmol/L in the intervention group, and the intervention
group significantly decreased (p<0.001). The Δ8Wintake was 0.06 ± 0.64 mmol/L in the control groupand −0.58 ± 0.79 mmol/L in the intervention group,and the intervention group significantly decreased(p<0.001).
In HDL-C, the control group was 1.14 ± 0.18
mmol/L before intake, 1.14 ± 0.17 mmol/L at 4Wintake, and 1.16 ± 0.21 mmol/L at 8W-intake. Theintervention group had 1.17 ± 0.25 mmol/L before
intake, 1.21 ± 0.19 mmol/L at 4W-intake, and 1.28 ±0.19 mmol/L at 8W-intake. There was no significant
difference between the two groups in the Δ4W-intake.
The Δ8W-intake in HDL-C was 0.02 ± 0.16 mmol/Lin the control group and 0.11 ± 0.19 mmol/L in theintervention group, and the intervention group
significantly increased (p=0.048).
In addition, the results of the stratified analysis
was shown in Table 3. In Δ8W-intake of LDL-C, there
was no significant difference between the inner groupand outer group in both the control group and theintervention group. In TG, there was no significant
difference between the inner group and the outer value
group in the control group, but the outer value group
showed a significantly lower value in the intervention
group (p<0.001). In HDL-C, there was no significant
difference between the inner group and outer group in
the control group, but the outer value group showed asignificantly higher value in the intervention group(p=0.002).

3. The amount of change in LDL-C, TG, and
HDL-C at the 4W- and 8W-intake
Table 3 shows the amount of change in LDL-C
and TG as the primary endpoints and HDL-C as thesecondary endpoint at 4W- and 8W-intake. The
amount of change from before intake to 4 and 8 weeks
of intake period was defined as Δ4W-intake and Δ8Wintake, respectively.
In LDL-C, the control group was 3.43 ± 0.27
mmol/L before intake, 3.66 ± 0.41 mmol/L at 4Wintake, and 3.75 ± 0.53 mmol/L at 8W-intake. Theintervention group was 3.46 ± 0.26 mmol/L before
intake, 3.09 ± 0.36 mmol/L at 4W-intake, and 2.85 ±0.32 mmol/L at 8W-intake. The Δ4W-intake in LDLC was 0.23 ± 0.35 mmol/L in the control group and−0.37 ± 0.30 mmol/L in the intervention group, and theintervention group significantly decreased (p<0.001).
The Δ8W-intake was 0.32 ± 0.46 mmol/L in thecontrol group and -0.60 ± 0.28 mmol/L in theintervention group, which was significantly lower in
the intervention group (p<0.001).
In TG, the control group was 2.95 ± 1.07 mmol/L
before intake, 3.19 ± 1.26 mmol/L at 4W-intake, and3.01 ± 1.08 mmol/L at 8W-intake. The intervention

62

Asian Journal of Dietetics, 2022

Table. 3. The amount of change in LDL-C, TG, and HDL-C at the 4W- and 8W-intake and stratified analysis of
the Δ8W-intake between the inner and outer groups of reference value†

Δ4W-intake
Items (unit)(Standard value)
LDL-C (mmol/L)(< 3.4 mmol/L)

BeforeintakeGroup
Control
group

Mean
SD

Intervention Mean
group
SD

4W-intake 8W-intake

Amount of†† Amount of††change p valuechange p value

3.43
0.27

3.66

3.75

0.41

0.53

0.23
0.35

3.46

3.09

2.85

-0.37

0.26

0.36

0.32

0.30

0.32
<0.001

Mean
Control
Inner group
groupSD< 3.4 mmol/L
Mean
Intervention(n=17)
group
SD
Mean
Control
Outer group
group
SD≥3.4 mmol/L
Mean
Intervention(n=20)
groupSD(1.7-2.25 mmol/L)

3.24
0.15

3.45
0.32

3.52
0.43

0.27
0.44

3.23

2.91

2.68

-0.56

0.11
3.51

0.31
3.75

0.28
3.90

0.29
0.26

0.24

0.40

0.53

0.47

3.62
0.20

3.26
0.34

3.07
0.28

-0.62

Mean

2.95

3.19

3.01

0.24

SD
Intervention Mean
group
SD

1.07
2.96

1.26
2.46

1.08
2.38

0.60
-0.49

1.12

0.80

0.65

0.64

Control
group

Control group: 0.57
Intervention group: 0.36

0.26
0.06
<0.0010.64-0.58<0.0010.79†††

p value

Mean
Control
Inner group
group
SD< 2.25 mmol/L
Mean(n=14) Intervention
group
SD

1.98

2.03

2.17

0.20

0.34
1.85

0.38
1.81

0.59
1.86

0.64
0.01

0.19

0.23

0.16

0.24

Mean

3.55

3.52

-0.02

SD

0.91

3.90
1.07

0.99

0.63

Intervention Mean
(n=23)group
SD
HDL-C (mmol/L)
Mean
Controlgroup(1.03-1.55 mmol/L)
SD

3.63

2.86

2.69

-0.94

0.90

0.63
1.16

0.80

1.14

0.76
1.14

0.18

0.17

0.21

0.14

Intervention Mean
group
SD

1.17
0.25

1.21

1.28
0.19

0.05
0.20

Control
group

0.19

Control group: 0.33
Intervention group: <0.001

0.01

0.02
0.37

0.16
0.11

0.048

0.19
p value†††

Subgroup#
Mean
SD

1.20
0.17

1.20
0.16

1.22
0.21

0.02
0.17

Intervention Mean
(n=27)
group
SD

1.28
0.13

1.25
0.19

1.33
0.18

0.05
0.14

Mean
Control
Outer group
group
SD< 1.03mmol/LIntervention Mean
(n=10)
group
SD

0.98

1.00

1.00

0.02

0.08
0.87

0.10
1.10

0.10
1.14

0.11
0.27

0.26

0.12

0.13

0.23

Inner group≥1.03 mmol/L<0.0010.28

Subgroup#

Outer group≥2.25 mmol/L

0.46
-0.60
p value†††

Subgroup#

TG (mmol/L)†

Δ8W-intake

Control
group†

Control group: 0.89
Intervention group: 0.002† : The amount of change from before intake to 4 and 8 weeks of intake period was defined as Δ4W-intake and: The amount of change from before intake to 4 and 8 weeks of intake period was defined as Δ4W-intake and††Δ8W-intake,: Pairedt-test,n=37,
controlgroup
vs intervention
group. †††: Unpaired t-test, n=37,
Δ8W-intake,respectively.
respectively. ††: Pairedt -test,n=37,
controlgroup vsintervention
group.
control
groupvsintervention
group.
#:
Inner
group
and
outer
group
mean
an
inner
and outer group the reference†††: Unpaired
t-test, n=37, control group vs intervention group. #: Inner group and outer group mean an inner and outer groupvalue,
respectively.
the reference value, respectively.

63

Effects of continuous soymilk intake on lipid metabolism

4. The amount of change in Cre, TP, Alb, ALT, and
AST at the 4W- and 8W-intake
Table 4 shows the amount of change in Cre, TP,

Alb, ALT, and AST, which were conducted as a safety
confirmation test. No significant difference was foundbetween the two groups in any of the items.

Table 4. The amount of change in Cre, TP, Alb, ALT, and AST at the 4W- and 8W-intake

Items (unit)(Standard value)
Cre (mmol/L)(62 - 120 mmol/L)

Group
Control
group

Mean
SD

Intervention Meangroup
SD
TP (mmol/L)(3.9 - 6.4 mmol/L)

Alb (g/L)(35 - 52 g/L)

AST (U/L)(< 50 U/L)

ALT (U/L)(< 50 U/L)

Mean
SDIntervention Mean
group
SD
Mean
Controlgroup
SDIntervention Mean
group
SD
Mean
Controlgroup
SDIntervention Mean
group
SD
Mean
Controlgroup
SDIntervention Meangroup
SD
Control
group

Beforeintake83.03

4Wintake
82.94

8Wintake
82.82

16.37
82.36
16.85
76.36
4.24
76.88
4.11
41.69
2.13
41.70
2.10
27.14
12.17
37.81
78.21
16.29
6.42
17.87
13.72

17.14
82.35
17.89
76.59
3.90
77.52
4.64
41.58
1.87
41.94
2.75
28.85
19.39
28.61
31.17
15.84
5.92
16.16
7.02

17.94
84.29
18.23
76.54
4.38
77.06
4.06
41.66
2.10
41.67
1.87
29.42
31.21
33.25
54.75
16.41
7.37
16.14
8.16

Δ4W-intake†Δ8W-intake†Amount†† Amount††of change p value of change p value
-0.13
-0.21
7.20
-0.01
4.83
0.23
3.00
0.64
3.11
-0.10
1.76
0.24
2.18
1.71
11.37
-9.21
47.61
-0.45
5.70
-1.71
8.37

0.94

0.60

0.47

0.25

0.45

9.42
1.94
6.33
0.18
3.89
0.18
3.43
-0.03
2.03
-0.03
1.92
2.29
21.85
-4.56
24.47
0.12
7.72
-1.73
8.07

0.25

1.0

0.99

0.37

0.39

†

† : The amount of change from before intake to 4 and 8 weeks of intake period was defined as Δ4W-intake and: The amount of change from before intake to 4 and 8 weeks of intake period was defined as Δ4W-intake andΔ8W-intake,
n=37,control
controlgroup
group
intervention
group.
Δ8W-intake,respectively.
respectively. ††††:: Paired tt-test,-test, n=37,
vs vsintervention
group.
5. The amount of change in body weight, BMI,
waist circumference, and hip circumference atthe 4W- and 8W-intake
Table 5 shows the amount of change in bodyweight, BMI, waist circumference, and hip
circumference at the 4W- and 8W-intake. There was asignificant difference between the two groups in waist
circumference and hip circumference. The Δ8Wintake of waist circumference was 0.83 ± 1.99 cm inthe control group and -0.58 ± 2.23 cm in theintervention group, showed significantly lower values
in the intervention group (p=0.010). The Δ8W-intake
of hip circumference was 0.86 ± 1.49 cm in the control
group and -0.05 ± 1.57 cm in the intervention group,
showed significantly lower values in the intervention
group (p=0.042).

64

Asian Journal of Dietetics, 2022

Table 5. The amount of change in body weight, BMI, waist circumference, and hip circumference at the 4W- and8W-intake
Items (unit)
Weight (kg)BMI (kg / cm2 )Waist circumference(cm)

Hip circumference
(cm)

Group

Beforeintake57.2

Δ4W-intake†Δ8W-intake†Amount of†† Amount of
4W-intake 8W-intake
p valuep value††changechange
57.6
57.4
0.38
0.17

Control
group

Mean
SD

9.6

9.7

9.8

0.68

Intervention
group

Mean

57.4

57.2

57.3

-0.18

SD

9.9

9.7

9.7

0.86

1.15

Control
group

Mean

23.8

24.0

23.9

0.17

0.08

SD

3.2

3.2

3.3

0.31

Intervention
group

Mean

23.9

23.8

23.8

-0.10

SD

3.3

3.1

3.1

0.46

0.58

Control
group

Mean

83.8

84.1

84.6

0.38

0.83

SD

7.8

7.9

8.2

1.80

Intervention
group

Mean

83.5

83.2

82.9

-0.30

-0.58

SD

7.9

7.8

7.8

1.71

2.23

Control
group

Mean

90.4

90.6

91.3

0.21

0.86

SD

5.3

5.3

5.7

1.10

Intervention
group

Mean

90.2

90.1

90.2

-0.10

-0.05

SD

5.1

5.1

5.1

1.22

1.57

0.002

0.002

0.13

0.23

0.80
-0.11

0.37
-0.07

1.99

1.49

0.24

0.17

0.010

0.042

†

: The†: The
amountofchangechangefromintake
beforeintake4 and
8 weeks
ofasintake
periodamount offrom before
to 4 and
8 weeksto
of intakeperiodwas definedΔ4W-intake
and was defined as Δ4W-intake and†† ††: Paired t-test, n=37, control group vs intervention group.
Δ8W-intake,respectively.
Δ8W-intake,respectively. : Pairedt -test, n=37, control group vs intervention group.
6. The amount of change in nutrient intake at thesignificantly increased (p=0.004). The Δ8W-intake
4W- and 8W-intake
was -0.7 ± 29.6 g/day in the control group and 17.8 ±Table 6 shows the amount of change in energy,
34.9 g/day in the intervention group, and theprotein, lipid, carbohydrate, and sugars at the 4W- andintervention group significantly increased (p=0.027).
8W-intake. In Δ4W-intake and Δ8W-intake of energy,
In sugars intake, no significant difference waslipid, and carbohydrate, there was no significant
observed between the two groups in the Δ4W-intake.
difference between the intervention group and theIn Δ8W-intake of sugars was 10.2 ± 18.4 g/day in thecontrol group. In protein intake, Δ4W-intake was -1.4
control group and 1.4 ± 18.2 g/day in the intervention± 29.2 g/day in the control group and 21.4 ± 30.1 g/day
group, and the control group significantly increasedin the intervention group, and the intervention group(p=0.040).
Table 6. The amount of change in nutrient intake at the 4W- and 8W-intake

65Effects of continuous soymilk intake on lipid metabolism†

Items (unit)
Energy (kcal)

Protein (g)Lipid (g)Carbohydrate (g)Sugars (g)GroupControl
Mean
group
SD
Intervention Meangroup
SD
Control
Mean
group
SD
Intervention Meangroup
SD
Control
Mean
group
SD
Intervention Meangroup
SD
Control
Mean
group
SD
Intervention Meangroup
SD
Control
Mean
group
SD
Intervention Mean
group
SD

Beforeintake
1479
384
1573
428
66.6
20.2
69.0
24.8
31.5
21.7
37.2
22.2
232.3
68.6
240.9
84.2
11.5
12.2
16.7
16.6

4Wintake
1498
379
1654
306
65.1
26.5
90.4
22.7
35.3
23.0
45.5
17.3
230.1
59.0
220.3
60.1
16.7
19.5
16.6
14.6

8Wintake
1459
375
1593
327
65.9
27.3
86.8
23.0
30.7
18.2
39.8
18.6
230.1
47.8
221.6
58.4
21.7
20.7
18.0
15.0

†

Δ4W-intake
Δ8W-intake
Amount†† Amount††of change p value of change p value
19
-20
466
475
0.61
0.74
80
20
475
494
-1.4
-0.7
29.2
29.6
0.004
0.027
21.4
17.8
30.1
34.9
3.9
-0.8
25.8
26.7
0.52
0.60
8.3
2.6
26.9
22.7
-2.3
-2.2
78.2
79.0
0.40
0.39
-20.6
-19.3
82.0
84.4
5.3
10.2
20.8
18.4
0.21
0.040
-0.1
1.4
18.1
18.2

†

†

: The amount of change from before intake to 4 and 8 weeks of intake period was defined as Δ4W-intake and†† from before intake to 4 and 8 weeks of intake period was defined as Δ4W-intake and: Δ8W-intake,
The amountof change
respectively.
: Paired t -test, n=37, control group vs intervention group.
Δ8W-intake, respectively. ††: unpaired t-test, n=37, control group vs intervention group.
DISCUSSION
In this study, it was confirmed that it can be to
reduce LDL-C and TG by continuously unprepared
The amount of change in LDL-C and TG in thesoymilk intake for 8 weeks in Vietnamese patients
intervention group significantly decreased at both thewith type 2 DM who have borderline or mild LDL
4W- and 8W-intake. In addition, the amount of changecholesterolemia. Regarding glucose metabolism, thein HDL-C in the intervention group significantly
effect of unprepared soymilk intake could not bedecreased at 8W-intake. From the results of theseclarified because FRA, which is the primary endpoint
primary and secondary endpoints, the effect ofof glucose metabolism, had a carry-over effect.
continuous intake of soymilk on improving lipid
However, although no significant change in bodymetabolism was confirmed. Furthermore, as a result of
weight was observed, the amount of change in both
stratified analysis of the Δ8W-intake between the inner
waist circumference and hip circumference wasand outer groups of reference value in LDL-C, TG, andsignificantly reduced at the Δ8W-intake. Waist
HDL-C, respectively, in all items, a significant
circumference is adopted as an essential item in the
improvement effect was observed in the outlier groupdiagnostic criteria for metabolic syndrome in Japan asof the intervention group.
an indicator of visceral fat accumulation. It is known
The American Heart Association (AHA) has
to increase the risk of which visceral fat accumulation
reported the mechanism of action of soy protein oncauses hypertension, hyperglycemia, and lipid
serum lipids, such as thyroid hormone and otherabnormalities through abnormal adipocytokine
metabolic hormones, bile acid excretion promoting
secretion, resulting in cardiovascular disease (25). In
action, and involvement in LDL receptors (19). Also,
addition, waist circumference has been closelyin 1999, the U.S. Food and Drug Administration
associated with insulin resistance and therefore is(FDA) allowed a soy protein health claim, "Ingesting
recommended as one of the diagnostic criteria for
25 g of soy protein per day as part of a diet low in
metabolic syndrome by NCEP-ATP III (National
saturated fatty acids and cholesterol reduces the risk of
Cholesterol Education Program-Adult Treatment
heart disease." (20) In Japan as well, the cholesterolPanel III) (26). In this study, it found that the
reducing effect of soy protein in humans has been
continuous intake of unprepared soymilk 500 mL/day
recognized, and the Consumer Affairs Agency has
for 8 weeks, the waist circumference, which indicatesapproved it as one of the functional substances in Foodthe visceral fat accumulation, was significantly
for Specified Health Uses (FOSHU) (21). In addition,
reduced. Therefore, it is suggested that continuous
it has been reported that triglyceride lowering effect ofintake of soymilk can be expected to reduce insulin
soy protein is due to the decrease in fatty acid synthesis
resistance by improving the secretion ofability in the liver and the suppression of liver fat
adipocytokines, and further research is needed in theaccumulation due to the decrease in blood insulin
future.
concentration (22-24). Although many studies on lipid
Dietary survey revealed that a protein intake wasmetabolism of soy protein have been reported so far,significantly higher in the intervention group than infew studies have been conducted on the effect of
the control group, both in the Δ4W-intake and in thesoymilk on the improvement of lipid metabolism
Δ8W-intake. As 100mL of unprepared soymilk
disorders, and few studies have been conducted in thecontains 4.5g of protein, it is considered that the intake
Asian region.
of 500mL of unprepared soymilk a day significantly

66Asian Journal of Dietetics, 2022
increased protein intake in the intervention group. As
mentioned above, it has been reported that soy protein
reduces LDL-C and TG and increases HDL-C, and in
this study, we were able to obtain similar results fromblood tests. Furthermore, despite an increase in energy
intake of 80 kcal in the 4th week in the intervention
group, the body weight decreased significantly
compared to the control group, and waist
circumference and hip circumference decreased
significantly in the 8th week, respectively. MartinezVillaluenga C et al. has reported that peptides derived
from β-conglycinin, a major component of soy protein,inhibit fatty acid synthase at the molecular level (27).
It has also been reported that administration of soyprotein peptide to rats enhances the thermogenic
capacity of mitochondria in brown adipose tissue (28).
In this way, the results of animal studies have beenreported so far, but in this human study, the continuous
intake of unprepared soymilk significantly reduced
both waist circumference and hip circumference. In
the future, we would like to conduct further research
to clarify the relationship between the intake of
unprepared soymilk and lipid and sugar metabolism.
In conclusion, In this study, it was confirmed thatit can be to reduce LDL-C and TG, and to increase
HDL-C by continuous intake of unprepared soymilk
for 8 weeks in Vietnamese patients with type 2 DM
who have borderline or mild LDL cholesterolemia. In
addition, it was clarified that the improvement effectwas higher in the outer group for TG and HDL-C thanin the inner group of reference value. Furthermore, asa new finding, it was revealed that the continuous
intake of unprepared soymilk significantly reduced
both waist circumference and hip circumference. In
the future, we would like to conduct further research
to clarify the relationship between the intake of
unprepared soymilk and lipid and sugar metabolism.

prevalence and associated factors. Glob Health
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ACKNOWLEDGEMENTS
The authors would like to thank all theparticipants in the study. We are grateful to all staffs inVietnam National Institute of Nutrition (NIN), Nam
Dinh University of Nursing (NDUN), commune healthcenters and hospitals who cooperated in conducting
the survey.
CONFLICTS OF INTEREST (COI)This study will be conducted as a joint research
between NIN, NDUN, Kyushu Women's University
and MARUSAN-AI Co., Ltd. and will be conducted at
a research funding based on a joint research agreement.
MARUSAN-AI Co., Ltd. provided research expenses
and samples for his COI relationship to be disclosed inthis study.

1.

2.

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68

Asian Journal of Dietetics 2022

Original
Phytochemical Content and Antioxidant Activity of Boiled and Fresh Ayo (Tetrastigma
harmandii Planch.) Fruits and Leaves
Tapaoan Sharlyn. Mae. D1., Barrion Aimee. Sheree. A.*, Tuaño Arvin. Paul. P2.
1

Institute of Human Nutrition and Food, College of Human Ecology, University of the Philippines Los Baños
2
Institute of Chemistry, College of Arts and Sciences, University of the Philippines Los Baños
Abstract Background and purpose. One of the underutilized indigenous plants in the Philippines
is Ayo, a vine whose fruits and leaves are used by locals as a souring ingredient in native dishes.
The study determined the effect of boiling on the phenolic, flavonoid, tannin, and anthocyanidin
content of Ayo fruits and leaves. Method. The abilities of the samples to act as antioxidants were
evaluated by radical scavenging activity assay. Results. Boiling significantly decreased (p <0.05) the phytochemicals and antioxidant activity of the samples, except for the anthocyanidin
in the fruits (p > 0.05). Generally, fruit samples retained higher levels of phytochemicals duringcooking than did leaf samples. The changes in the phytochemical content and antioxidant activityof boiled samples might be due to leaching out of the bioactive compounds from their matrix
into the boiling water. Conclusion. The study revealed that Ayo could be a good source ofphytochemicals, and it exhibited a high radical scavenging activity. Greater antioxidants could
be obtained by eating the fruits fresh and raw. When boiling is unavoidable as in the case of theleaves, less water and less cooking time are recommended to retain the optimum benefits of thebioactive compounds present in the plant.
Keywords: Ayo, phenols, tannins, flavonoids, antioxidant activity
INTRODUCTION

capita per year, far below the requirement set by the
WHO (4). Factors that contribute to this trend include
cultural issues, impediments in the supply chain,urbanization resulting in inaccessibility to nutritious
meals, low socio-economic status, and high costs ofproduce. Households that lack resources have limited
dietary choices since they cannot afford the more
popular vegetables in the market (3).
The Food and Agriculture Organization recognizes theurgent need to raise public awareness on the role ofunderutilized indigenous food resources in countering
food and nutrition insecurity. There is also a pressing
demand to diversify the food base as a response to
climate uncertainty. Underutilized crops are tolerant todrought and natural hazards, and resistant to pests anddiseases. They address cultural needs, while being lessdamaging to the environment. Unbeknownst to many,
their nutrient content is much higher than the more
popular species that are commonly produced andconsumed. Thus, they can be tapped to provide
palatable and nutritious foods and increase fruit andvegetable consumption at a minimal cost and effort(5).
Ayo (Tetrastigma harmandii Planch.) is anunderutilized plant that thrives at low to medium
elevations in several provinces of the Philippines. It isa slender vine with branchless, coiled tendrils that isharvested from the wild and used locally as a source offood, medicine, and fiber. Its russet-brown, globose
fruits grow in clusters and contain a juicy, sour flesh
that may be consumed raw or made into preserves. The
leaves are likewise sour and can be utilized as a

The importance of daily and adequate fruit andvegetable consumption in the maintenance of health,and the prevention of non-communicable diseases andmalnutrition has been extensively researched,documented, and advocated. Low intake of thesecrops’ accounts for approximately 1.7 million (2.8%)
deaths worldwide, and it is estimated to cause around
14% of gastrointestinal cancer deaths, 11% ofischemic heart disease deaths, and 9% of stroke deaths.
(1). The report of the joint WHO and FAO expert
consultation on diet, nutrition, and the prevention ofchronic diseases that took place in 2003 recommendsa minimum of 400 g or 5 servings of fruits andvegetables per day. Eating a variety of fruits andvegetables regularly as part of a well-balanced dietprovides enough of most micronutrients, dietaryfibers, and essential non-nutrient substances needed by
the body, thereby encouraging weight management,
and reducing morbidity and the risk of mortality (2).
Despite the known health benefits of fruits andvegetable, numerous interventions and dietaryguidelines promoting consumption, many populations
do not meet the recommended amount. The annual
average consumption of vegetables globally is 102 kg
per capita, with the highest level in Asia and the lowest
in Africa and South America (3). In the Philippines, it
is only about 111 g per capita per day or 40 kg per*To whom correspondence should be addressed:

aabarrion1@up.edu.ph69

Phytochemical Content and Antioxidant Activity of Boiled and Fresh Ayo
condiment to add flavor to dishes. Both fruits andleaves are typically eaten by the natives with fish (6).
Ayo is indigenous in the Philippines, but there islimited data about this plant and there is no literaturereporting its nutrient and phytochemical content, andantioxidant capacity. It was therefore imperative toundertake research that would raise awareness andexpand the knowledge about this plant. The findingsof the study will be useful to future researchers whowill be interested in the potential impacts of Ayo on
nutrition, and in the product development of Ayo as ajam, jelly, or commercial souring ingredient. Finally,
the study would contribute to the documentation of thecharacteristics of underutilized, indigenous crops andtheir significant roles in preserving cultures,developing, and sustaining local food systems, andprotecting human well-being and health. This study
was intended to determine the effect of boiling on thephytochemical content and the antioxidant capacity of
Ayo (Tetrastigma harmandii) fruits and leaves.

manner as the samples. The absorbance was measured
at 760 nm against a blank using a UVspectrophotometer. The results were expressed as mg
gallic acid equivalents (GAE) per g of dried sample.
Flavonoid content
The total flavonoid content of the samples wasestimated using the aluminium chloride method (7). A
1 ml aliquot was mixed with 4 ml of distilled water,and subsequently added with 0.30 ml of 10% sodium
nitrite (NaNO2). After five minutes, the mixture wasadded with 0.30 ml of 10% aluminium chloride(AlCl3) solution and 2 ml of 1% sodium hydroxide(NaOH) solution, then it was thoroughly mixed. A setof standard solutions of quercetin (25, 50, 75, 100,
125, 150 µg/ml) was prepared and analyzed using thesame procedure described initially. The absorbancewas measured at 510 nm against a blank using a UVspectrophotometer. The results were expressed as mg
quercetin equivalents per g of dried sample.
Total tannin content
The total tannin content of the samples wasassessed by performing the Folin-Ciocalteu assay (7).
A 1 ml aliquot was transferred to a 10 ml volumetricflask, then added with 7.5 ml of distilled water, 0.5 mlof Folin-Ciocalteu’s phenol reagent, and 1 ml of 35%
sodium carbonate solution. It was diluted to 10 ml with
distilled water and mixed thoroughly. The mixture was
kept at room temperature for 30 minutes. A set ofstandard solutions of catechin (20, 40, 60, 80, 100µg/ml) was prepared and analyzed in the same manneras the samples. The absorbance was measured at 700
nm against a blank using a UV-spectrophotometer.
The results were expressed as mg catechin equivalents
per g of dried sample.
Total anthocyanidin content
The total anthocyanidin content of the samples wasdetermined using the vanillin assay (7). A 1 ml aliquot
was mixed with 2.5 ml of 1% vanillin and 2.5 ml of 9
M hydrochloric acid (HCl). It was incubated for 30
minutes at 30 ˚C. A set of standard solutions ofcatechin (100, 150, 200, 250, 300, 350 µg/ml) was
prepared and analyzed following the same proceduredescribed initially. The absorbance was measured at
500 nm against a blank using a UVspectrophotometer. The results were expressed in mg
catechin equivalents per g of dried sample.
Antioxidant activity
The antioxidant activity of the samples wasevaluated by performing the 1,-diphenyl-2picrylhydrazyl (DPPH) radical scavenging assay (8).
A 1 ml aliquot was diluted with 4 ml of distilled water,then mixed with 1 ml of methanolic solution of DPPH.
The mixture was incubated in the dark for 20 minutes,
after which the absorbance was measured against a
blank at 517 nm. The ability of the samples toscavenge the DPPH radical was calculated using theformula:

MATERIALS AND METHODS

Sample preparation
Ripe fruits and young shoots of Ayo were collectedfrom Brgy. Culing West, Cabatuan, Isabela. They
were transported to the Bio-Assay Laboratory of theInstitute of Human Nutrition and Food at the
University of the Philippines-Los Baños, where thestudy would be conducted. The fruits and the leaves
were initially cleaned by washing them thoroughly
under running water. The rind of the fruits was peeled
off. Using a digital weighing scale, two 200 g fruits
and two 200 g leaves were prepared before cooking. A
portion of the leaves and the fruits were heated toboiling point in a stock pot for three minutes. The freshand boiled fruits and leaves were placed in a moisturedish, then oven-dried at 45 ˚C overnight. The driedmaterials were pulverized into coarse powder using agrinder. The dried, powdered samples were transferred
into a plastic vial and stored in a desiccator. An extract
was obtained by weighing 50 mg of dried, powderedsample and diluting it with absolute methanol to makea 50 ml solution. The solution was swirled for 10
minutes using a vortex mixer, then percolated througha filter paper. The filtrate was decanted into a test tube,
and the residue was discarded.
Total phenolic content
The total phenolic content of the samples wasdetermined according to the Folin-Ciocalteu
method(7). A 1 ml aliquot was mixed with 1 ml offreshly prepared Folin-Ciocalteu’s phenol reagent.
After five minutes, 10 ml of 7% sodium carbonate(Na2CO3) solution was added, followed by 13 ml ofdistilled water. The mixture was shaken thoroughly,
then kept in the dark for 90 minutes at 23 ˚C. A set ofstandard solutions of gallic acid (100, 200, 300, 400,
500 µg/ml) was prepared and analyzed in the same
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑡𝑒𝑠𝑡 𝑠𝑎𝑚𝑝𝑙𝑒 𝑥 100
𝐷𝑃𝑃𝐻 𝑠𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (%) =𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙
Statistical analysis

All functional analyses were carried out in
triplicates (n=3). The results were presented as mean ±70

Asian Journal of Dietetics 2022

standard deviation of three independent observations.
Paired sample t-tests were performed to determine
whether the differences in the averages weresignificant. Significance levels were defined at p <0.05.
RESULTSphytochemicals analyzed, phenols had the greatest
concentration in all the samples. The phenolic content
of the samples ranged from 423.3 mg GAE/g to 551.1
mg GAE/g. The fresh fruits had the highest phenoliccontent, while the boiled leaves had the lowest
phenolic content. After boiling, the phenolic contentsof the leaves and the fruits decreased by 23% and 21%,respectively. A two-sample t-test revealed asignificant difference (p < 0.05) in the phenoliccontents of the fresh samples and the boiled samples.

Total phenolic content
The total phenolic contents of fresh and boiled Ayo
were presented in Figure 1. Among the

Figure 1. Total phenolic contents of fresh and boiled
Ayo

Total tannin content
As shown in Figure 2, the tannin concentration of thedifferent samples ranged from 9.09 mg CE/g to 101.82
mg CE/g. Levels of tannins were found to be highest
in the fresh fruits and lowest in the boiled leaves.
Boiling for three minutes significantly (p < 0.05)
decreased the tannin contents of the leaves and thefruits by 73% and 23%, respectively.

Figure 2. Total tannin contents of fresh and boiled
Ayo

Flavonoid content
The flavonoid contents of the fruits and leaves before
and after cooking were summarized in Figure 3. The
flavonoid concentration of the samples varied from
2.17 QE mg/g to 19.90 QE mg/g. The fresh fruits
possessed the highest flavonoid levels. On the other
hand, the boiled leaves contained the lowest flavonoid
levels. The flavonoid concentration of the leaves andthe fruits significantly decreased (p < 0.05) after
boiling.

71

Phytochemical Content and Antioxidant Activity of Boiled and Fresh Ayo

Figure 3. Total flavonoid contents of fresh andboiled Ayo

Figure 4. Total anthocyanidin contents of freshand boiled Ayo

Total anthocyanidin content
Anthocyanidin had the lowest concentration in all thesamples. The results presented in Figure 4 suggest thatthe highest anthocyanidin content was observed in the
fresh fruits with 9.33 ± 3.82 mg CE/g dry weight,
while the lowest was observed in the boiled leaves
with 2.67 ± 1.44 mg CE/g dry weight. After theapplication of heat, the anthocyanidin content of theleaves and the fruits were reduced by 55% and 44%,
respectively. There was a significant difference (p <0.05) in the anthocyanidin content of the leaves before
and after cooking, whereas there was no significant
difference (p > 0.05) in the anthocyanidin content of
the fresh and the boiled fruits.

Antioxidant activity
The antioxidant activity of the samples was shown in
Figure 5. The greatest radical scavenging activity(94.24 ± 0.08 %) was demonstrated by the fresh fruits,
while the least radical scavenging activity (92.89 ±0.03 %) was exhibited by the boiled leaves. There wasa significant difference (p < 0.05) in the antioxidant
activity of the fresh and boiled samples. The fresh
samples had a greater radical scavenging activity
compared to the boiled samples. The antioxidant
activity may be classified as high (>70%),
intermediate (40-70%) or low (<40%) levels of
inhibition (9). There was a significant difference in theradical scavenging activity between the fresh leaves
and the fresh fruits, and between the boiled leaves andthe boiled fruits. Nevertheless, the antioxidant
capacities of the four samples were classified as high.

72

Asian Journal of Dietetics 2022

Figure 5. Antioxidant activity of fresh and boiled Ayo
DISCUSSION

the loss of these compounds (14). Cooking the samples
prompted the phenolic compounds to move from
uniform distribution in the vacuoles and localize aroundthe cell walls. It also caused the cell-wall components tosoften and rupture, which enabled the water-soluble
phenolic compounds to be released from the matrix andbe dissolved in water (13). According to research on
almond skin, the polyphenols decreased during the
blanching process, while these compounds increased in
the water (15).
Many researches positively correlated the radical
scavenging effect with the total amount of phenolic
compounds (11); (16); (17). Hence, the decline inantioxidant activity of the boiled samples in this study
was a consequence of the leaching process, and structuralchanges or thermal degradation of phenols, flavonoids,tannins, and anthocyanidins (12). The greater reductionof antioxidant activity in boiled leaves than in boiled
fruits could also be due to the larger surface area of theleaves in contact with the water (18). In a study reported,
radical scavenging activity of colored peppers
considerably decreased (p < 0.05) to below 77% of its
initial level after boiling for five minutes. It was further
reduced to 64% when the boiling time was prolonged to
30 minutes (19). The cooking water was also analyzed,
and it was found that there was no significant difference(p > 0.05) between the RSA of the raw pepper samples,
and the sum of the RSA values in cooked tissues andcooking water after five minutes of boiling. The
researchers noted that this was an indication that the
antioxidant compounds could have leached into theboiling water.

Polyphenols are a large group of secondary metabolites
present in significant amounts and widely distributed in
the plant kingdom, with more than 8,000 structures foundin different species. They include simple phenols,
flavonoids, tannins, and anthocyanidins, which were thephytochemicals involved in this study. They aresynthesized through the phenylpropanoid and polyketide
pathways in response to physiological stimuli, stress,
ultraviolet radiation, or aggression by pathogens (10).
The antioxidant properties of polyphenolic compounds
are attributed to the inherent propensity of their hydroxyl
groups to dismutate radicals by donating their hydrogenatoms (11). The free radical scavenging activity of thewhole molecule is defined by the number of hydroxyl
groups it contains (12). Polyphenols are highly reactive
and good substrates for different enzymes including
glycosidases,
esterases,
peroxidases,
andpolyphenoloxidases. They can undergo degradation andpolymerization through enzymatic and non-enzymatic
reactions during post-harvest storage or food processing,which may result in structural changes and a decrease in
their biological activities. The behavior of polyphenols
after processing is influenced by several factors such asconcentration, type of heat treatment applied, location
inside the cell, and interactions with other components ofthe food (13). The difference in the polyphenoliccontents of the fresh and boiled samples in this study
could be explained by the fact that polyphenols are polar
compounds and leaching occurred during boiling.
Polyphenols are also thermo-liable, which means that thegreater the amount of water used in cooking, the greater73

Phytochemical Content and Antioxidant Activity of Boiled and Fresh Ayo
CONCLUSION

Journal of Medicinal Plan Studies. 4,4,282-286. 2016.
8. Spanou, C., Stagos, D., Tousias, L., Angelis, A.,
Aligiannis, N., Skaltsounis,A.,
andKouretas, D. Assessment of antioxidant activityof extracts from unique Greek varieties ofLeguminosae plants using in vitro assays.
Anticancer Research. 27,5, 3403–3410. 2007.
9. Hassimotto, N., Genovese, I., And Lajolo, F.
Antioxidant activity of dietary fruits, vegetables
and commercial frozen fruit pulps. Journal of
Agricultural and Food Chemistry. 49,8, 40764082. 2005.
10. Piccolella, S., and Pacifico, S. Chapter Five Plant-Derived
Polyphenols:
A
Chemopreventive and Chemoprotectant WorthExploring Resource in Toxicology. Advancesin Molecular Toxicology, 9, 161-214. 2015.
11. Aksoy, L., Kolay, E., Agilonu, Y., Aslan, Z.,
Kargioglu, M. Free radical scavenging activity,
total phenolic content, total antioxidant status,
and total oxidant status of endemic Thermopsis
turcica. Saudi Journal of Biological Sciences.
20,3, 235-239. 2013.
12. Galanakis, C. Polyphenols: Properties,
Recovery, and Applications. Sawston, CA:
Woodhead Publishing. 2018.
13. Boekel, M., Fogliano, V., Pellegrini, N.,
Stanton, C., Scholz, G., Lalljie, S., Somoza, V.,
Knorr, D., Jasti, P., And Eisenbrand, G. A
review on the beneficial aspects of foodprocessing. Molecular Nutrition and Food
Research. 54, 9, 1215-1247. 2010.
14. Dolinsky, M., Agostinho, C., Ribeiro, D.,
Rocha, G., Barroso, S., Ferreira, D., Polinati,R., Ciarelli, G., And Fialho, E. Effect ofdifferent cooking methods on the polyphenol
concentration and antioxidant capacity of
selected vegetables. Journal of Culinary
Science and Technology. 14,1, 1-12. 2016.
15. Milbury, P., Chen, C., Dolnikowski, G., And
Blumber, J. Determination offlavonoids
and phenolics and their distribution in almonds.
Journal of Agricultural and Food Chemistry.
54,14, 5027-5033. 2006.
16. Kiselova, Y., Ivanova, D., Chervenkov, T.,
Gerova, D., Galunska, B., and Yankova, T.
Correlation between the in vitro antioxidant
activity and polyphenol content of aqueous
extracts from Bulgarian herbs. Phytotherapy
Research. 20,11, 961-965. 2006.
17. Faller, A. L. K., and Fialho, E. The antioxidant
capacity and polyphenol content of organic andconventional retail vegetables after domestic
cooking. Food Research International. 42, 210215. 2009.
18. Howard, L. A., Wong, A. D., Perry, A. K., andKlein, B. P. B-carotene and ascorbic acidretention in fresh and processed vegetables.
Journal of Food Science. 64, 929–936. 1999.
19. Chuah, A., Lee, Y., Yamaguchi, T., Takamura,
H., Yin, L., And Matoba, T. Effect of cooking
on the antioxidant properties of coloured
peppers. Food Chemistry. 111, 20-28. 2008.

The study determined the effect of boiling on thephenolic, flavonoid, tannin, and anthocyanidin content,
and antioxidant activity of Ayo fruits and leaves. The
highest concentration for each of the four
phytochemicals was consistently observed in the fresh
fruits, whereas the lowest amount was invariably notedin the boiled leaves. There was a significant loss (p <0.05) of phytochemical content in the samples after
boiling, except for the anthocyanidin in fruits. The
antioxidant activity also significantly decreased (p <0.05) in the boiled samples which might be a result of theleaching of the thermo-liable and water-soluble bioactive
compounds into the boiling water. In conclusion,
maximum antioxidants could be obtained by eating fresh
Ayo fruits. However, when boiling is unavoidable as in
the case of the leaves, less water and less cooking time
are suggested to retain the optimum benefits of thebioactive compounds present in the plant. The water used
for boiling could also be consumed in addition to Ayo
because soluble bioactive compounds would be present
in the boiling water. This study provided some basis forthe consumption of Ayo and its possible uses as afunctional food or ingredient. Nevertheless, more studies
on its proximate composition and vitamin content should
be done to obtain comprehensive nutritional properties ofthis plant.
1.

2.

3.

4.

5.

6.

7.

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