Method for Determining Non-Toxic Potential to Human Cells

Then 0.5 mL of 10 % TCA and 2 mL of distilled water were added, and finally 0.4 mL of 0.1 % AlCl 3 was added. The optical absorbance was determined at 700 nm. The higher the optical absorbance, the stronger the reducing power. Calculate the IC 50 value , which is the amount of sample that increases the optical absorbance by 0.50 [36][37].

c. Method for determining antioxidant capacity on oil-water model

The oil-water emulsion system was prepared with: 10% Olive oil, 85% water and 0.5% Tween 40. The mixture was homogenized at a speed of 10,000 rpm for 5 minutes. Exactly 2 mL of the extract was mixed well with 10 mL of the oil-water emulsion system contained in a 50 mL plastic tube with a lid, placed in a thermostatic cabinet at 50 o C, the oxidation process of boron was observed daily. The hydroperoxide content was determined on the boron extract according to the method of Bligh and Dyer in 1959. The hydroperoxide content was determined according to the method of Richards and Hultin in

2002 Results of calculating hydroperoxide content from the standard curve of Cumene hydroperoxide (HPO) concentration from 0-120 nmol/mL [36] [38] [39].

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In addition, there are several other methods for determining antioxidant capacity [35]:

- TEAC Trolox equivalent antioxidant capacity method: Determination of antioxidant activity compared to the antioxidant capacity of Trolox

- ORAC oxygen radical absorbance capacity method: Determines the ability to absorb free radicals containing active oxygen

- TRAP method (total radical-trapping antioxidant potential)

- FRAP method ferric reducing-antioxidant power : Antioxidant power by ferric reducing method

- Conjugated Diene method: Investigation of conjugated double bonds

The above methods are highly specific, however, the methods are complicated, difficult to perform, the chemicals used are expensive, difficult to provide, and the laboratory is not fully equipped with equipment for analysis.

1.2.3.7. Toxicity test method

Toxicity testing to know the risk of adverse effects on human health when exposed, adverse effects on the environment as well as organisms where the products are used. Helps establish dosage in clinical trials to provide initial information

Commonly used methods for research on herbal subjects are as follows:

- Abnormal toxicity test: Abnormal toxicity or acute toxicity is a sign of adverse effects or death of an organism immediately after exposure to a toxic substance. Acute toxicity occurs due to exposure to a single or multiple factors in a short period of time and acute effects are effects that occur within a few days or even the first few hours after exposure to a toxic substance, usually the time to cause acute toxicity must be less than two weeks. On the other hand, because chronic effects only appear after repeated exposure to a toxic substance, in many cases, continuous exposure is required for months. Meanwhile, the agent causing acute toxicity is rapidly absorbed into the body and immediately produces toxic effects on the body, but there are also cases where acute exposure reduces toxicity. Abnormal toxicity test is a method to determine the standard of a certain type of drug. It is the standard for the safety of the drug. In the abnormal toxicity test, it is clearly stated: What is the test animal, the condition of the animal, the weight of the animal, number of animals, test dose, route of administration, duration of observation, absence of disease and no deaths Purpose: To provide information for classifying the toxicity of the drug, predicting symptoms and planning treatment for acute poisoning; establishing dose levels for further toxicity and efficacy testing and the safety margin of the investigational drug [40][41].

- Subchronic toxicity test: Long-term toxicity test is only conducted after having information on acute toxicity in animals and the test sample is intended for long-term use or exposure in humans. Long-term toxicity test is to determine the tolerance of experimental animals when using the test sample repeatedly. Information needed to determine if there are toxic manifestations after long-term use, including:

Dose level that does not or does not cause significant changes in function, organs or some vital signs that can be observed in experimental animals;

Toxicity observed in animals and reversibility if any [40].

The procedure for testing for unusual toxicity is carried out according to the instructions in Appendix 13.5.

– Vietnamese Pharmacopoeia.

1.2.3.8. Method for determining non-toxicity to human cells

The development of a new herb or food from initial idea to commercialization.

Developing a product is a complex process that can take 12 ÷ 15 years and is expensive. Ideas for a target can come from a variety of sources including academic and clinical research and from the commercial sector. Building a supporting evidence base before selecting a target for a discovery program can take many years. Once a target has been selected, the pharmaceutical industry and, more recently, some academic centers have developed a number of initial processes to identify molecules with suitable properties to generate acceptable product types. This evaluation will identify the initial target, through to assay development, high-throughput screening, optimization, and ultimately selection for clinical development [42].

Method for determining non-toxicity to human cells to evaluate the non-toxicity of the product to human cells when using the product, the method is performed on HEK-293 - human embryonic kidney cells.

1.2.4. Overview of health food production process

1.2.4.1. Overview of health protection foods

a. Concept of health protection food

Pursuant to Clause 1, Article 3, Decree 15/2018/ND-CP stipulates: "Health Supplement, Dietary Supplement are products used to supplement the daily diet to maintain, enhance, improve the functions of the human body, and reduce the risk of disease"

Health protection foods contain one, more or a mixture of the following substances:

- Vitamins, minerals, amino acids, boron acids, enzymes, probiotics and other bioactive substances;

- Substances of natural origin, including animals, minerals and plants in the form of extracts, isolations, concentrates or metabolites;

- Synthetic sources of the above mentioned ingredients

Health protection foods are presented in processed forms such as capsules, pills, tablets, granules, powders, liquids and other dosage forms, divided into small dose units for use.

b. Benefits of health protection foods

- Quickly replenish nutrients and functional substances that the body needs.

may not be adequately supplied in the daily diet.

- Can temporarily replace meals when normal eating conditions are not available.

such as when in an environment lacking food or unable to eat due to illness

- The products are all in refined form, very convenient, easy to use and preserve.

- There are many products to choose from to suit each person's physical condition.

- Easy to buy and use without needing a doctor's prescription

- When using functional foods, users will have the knowledge to take care of their health, change their habits to have a more suitable diet and a healthy lifestyle that is more beneficial to their health.

- Regular abundant supply, extensive network

1.2.4.2. Overview of main raw materials for producing health protection foods of the topic

- Eurycoma longifolia root powder: Produce extract for direct use and supplement to health food production line.

- Myrtle buds: Have the effect of stabilizing blood sugar for a long time, supporting blood fat reduction, anti-oxidation, treating diabetes. Previous studies have also shown that myrtle buds have many biological effects such as:

- Anti-inflammatory [43];

- Antibacterial [44];

- Anti-cancer [45] [46];

- Enhances and stimulates digestion [46].

Gotu kola: A relatively common vegetable, with many biological activities that are important to human health, often harvested both leaves and vines. Gotu kola is used as food and medicine in folk medicine. Gotu kola has a slightly bitter, sweet taste, slightly cool properties, has the effect of clearing heat and detoxifying, dispersing blood stasis and relieving pain, promoting blood circulation and producing new fluid, and promoting urination. Gotu kola is often used as a tonic, antiseptic, to treat hemoptysis, diarrhea, vaginal discharge, leukorrhea, pimples, and prickly heat. Gotu kola contains alkaloids hydrocotulin and glycosides, asiaticoside and centellosid, which have an effect on connective tissues, helping tissues to regenerate quickly, thereby healing wounds quickly and forming new skin [47].

In 2009, the research group of Frederico Pittella et al. reported that gotu kola extract had significant activity against murine melanoma (B16F1), human breast cancer (MDA MB-231) and glioma (C6) mouse cell lines, with values

IC 50 were 698 μg/mL, 648 μg/mL and 1000 μg/mL, respectively [48].

1.2.4.3. Health food production process

Food in general and health protection foods in particular, to be recognized and commercialized, must comply with the regulations on food product quality according to the Food Safety Law 55/2010/QH12 [49]; Decree No. 15/2018 detailing the implementation of a number of articles of the Food Safety Law [50]. In addition, the content of supplements in the product must comply with the provisions of Circular No. 43/2014/TT-BYT - regulations on the management of functional foods [51]; Circular 18/2019/TT-BYT - guidelines for good manufacturing practices GMP in the production and trading of health protection foods [52]. Therefore, the production process of health protection foods basically includes the following contents:

- Survey of additional content of biologically active ingredients;

- Building production technology process;

- Check product quality;

- Develop a draft product announcement to submit to the specialized state management agency for appraisal and approval.

a. Survey on the content of additional bioactive compounds

Adding a quantity of bioactive compounds extracted from nature to food will affect the quality indicators of the product, chemical composition, sensory, etc., especially with the bitter aftertaste of Euryale, the processing method aims to minimize the loss and denaturation of active ingredients. Based on the survey of the content of active compounds that need to be added, proceed to develop a production process for health protection foods.

b. Building a production process for health protection food products

Research and develop the production process of some health food products on a laboratory scale with the main ingredients listed in section 1.2.4.2 and some other additional ingredients.

c. Product quality control

Checking the quality of health food products to ensure that the products meet quality standards with the determined dosage of supplements, evaluating the sensory preferences of users, and at the same time, ensuring food safety standards.

according to current regulations.

This is the basis for manufacturers to build a quality declaration of health food products and is one of the important criteria for bringing health food products to the market.

d. Product quality declaration

In case of declaring self-produced products, based on Article 10, Circular 43/2014/TT-BYT , individuals and organizations need to ensure the following requirements:

- The main ingredients that create the product's effects must be listed first with their full name and content. Other ingredients are listed next in decreasing order of mass;

- The content of vitamins and minerals in food calculated according to the manufacturer's recommended daily dose must reach at least 15% RNI;

- The maximum content of vitamins and minerals in food calculated according to the manufacturer's recommended daily dose must not exceed the maximum tolerance threshold of vitamins and minerals prescribed;

- The vitamin and mineral content of the product must be stated on the label in numbers and must be declared as a percentage % of RNI, based on the recommended daily dose of the product or based on a serving size.

In case Vietnam does not have RNI level and maximum tolerance threshold, it shall apply according to the regulations of CODEX or relevant international organizations.

- Health claims announcement:

Health claims must be true to the nature of the product, only declare the effects of the main active ingredient or declare the combined effects of the active ingredients when there is scientific evidence to prove it and not declare the effects by listing the effects of the ingredients;

The announcement of health recommendations, dosage, users and appropriate usage must be consistent and appropriate with the documents in the dossier;

When the content of vitamins, minerals, and biological active ingredients is lower than the level proven in scientific documents, the product's uses cannot be declared;

When the content of vitamins, minerals and biological active substances reaches the level specified in the document.

Scientifically recommended data must be published for use but must indicate the appropriate subjects and dosage;

When the content of the constituent ingredients does not have an RNI level, scientific documents proving the uses of that ingredient and recommended dosage must be provided when declaring.

- Target users:

The object must be in accordance with the declared uses and accepted by the competent state agency through the Certificate of Declaration of Compliance with Food Safety Regulations;

Objects must be warned not to be used if any.

1.3. Overview of the research situation of honey in and outside the country

For many years, research results on Tongkat Ali in our country and around the world have been published, especially its roots. The studies mainly refer to the chemical composition of isolated substances, survey of biological activity of extracts and some applications in pharmaceuticals and health foods.

1.3.1. Research on chemical composition

There have been many domestic and international research projects on the isolation and determination of chemical composition of many compounds that have been published since the 1960s of the last century. Some of the following research results can be mentioned:

In 1968, the research group of Le Van Thoi and Nguyen Ngoc Suong isolated the compounds β-sitosterol, campesterol, 2,6-dimethoxybenzoquinone and some bitter compounds such as eurycomalactone from the ether extract of the oil extracted from the bark and leaves of the Eurycoma longifolia plant using column chromatography on alumina [53].

In 1982, a research group from Hiroshima University of Medicine and Pharmacy, Japan isolated two highly oxidized quassinoid compounds named eurycomanone and eurycomanol from the roots of the Indonesian-originated Eurycoma longifolia plant [54].

In 1986, the research group of KLChan and colleagues found new compounds belonging to the quassinoid group, namely 3,4-dihyroeurycomalactone, 5,6-dehyroeurycomalactone, 6-hydroxy-5,6-dehydroeurycomalactone and an alkaloid group named 10-hydroxycantin-6-one, yellow crystals from the ether oil extract of the root of the plant. In addition, a coumarin compound called scopoletin was also discovered from the chloroform extract of the root [55].

In 1989, the research group of KL Chan and colleagues isolated a quassinoid glycoside named eurycomanol-2-O- -D-glucopyranoside extracted from n-butanol extract of Euryale root [56].

In 1982, the research group of Nguyen Ngoc Suong and colleagues isolated two compounds laurycolactone A and laurycolactone B, which are quassinoids with a basic C18 skeleton [57].

Next in 1991, the research group of KL Chan and colleagues isolated the compound 13β,18-dihydroeurycomanol, crystallized in methanol from the root extract with petroleum ether [58].

Kadono et al. published the results of research on the cytotoxic and antipyretic components of the roots, and isolated four alkaloids belonging to the canthin-6-one group, namely 9-methoxycanthin-6-one, 9-methoxycanthin-6-one-N-oxide, 9-hydroxycanthin-6-one and 9-hydroxycanthin-6-one-N-oxide and -carboline-1-propionic acid in 1991 [59].

Also in 1991, the research group of H Tada and colleagues isolated the compound paskbumin A eurycomanon and two new compounds also having the quassinoid skeleton, pasakbumin B, pasakbumin C from the methanol extract [60]. The research group from the University of Medicine and Pharmacy, Tokyo - Japan, in the process of studying the biological activity of the Eurycoma longifolia plant, isolated two new compounds with the squallan skeleton. These are also two stereoisomers of each other, one is eurylene, the other is teurylene, both are colorless crystals, with the molecular formula C 34 H 58 O 8 [61].

In 1993, the research group of H Itokawa and colleagues isolated a

The compound containing the squallane skeleton is called longilen peroxide, which is a colorless compound with the molecular formula C 30 H 52 O 8 [62].

In 2014, the research team of Seon Ju Park et al. isolated and

The structures of five compounds belonging to the quassinoid group were identified: Eurylactone E, eurylactone F, eurylactone G, eurycomalide D, and eurycomalide E [63].

In 2015, the research group of Le Thi Huyen and colleagues isolated 3 substances belonging to the quassinoid group from the methanol extract of the roots of Tongkat Ali: Pasakbumin-C, 13 α ,21-epoxyeurycomanone and eurylactone A [64].

In 2017, the research team of Nguyen Huu Tung and colleagues found a

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