Study on biological activities of herbs’ decoction combining Scutellaria barbata, Hedyotis diffusa and Ehretia asperula Текст научной статьи по специальности «Биологические науки»

ФАРМАКОЛОГИЯ, КЛИНИЧЕСКАЯ ФАРМАКОЛОГИЯ

PHARMACOLOGY

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DOI: 10.18413/2658-6533-2024-10-4-0-5 УДК 615.2

Study on biological activities of herbs' decoction combining Scutellaria barbata, Hedyotis diffusa and Ehretia asperula

Hang N. Thi , Tai P. Anh , Dang T.D. Khanh , Tran B.N. Huyen ,

Duy D.N. Anh , Hien N. Thanh

Ho Chi Minh City University of Education, 280 An Duong Vuong St., Ward 4, District 5, Ho Chi Minh City, 30308, Vietnam Corresponding author: Tai P. Anh ([email protected])

Abstract

Background: Cancer is a leading cause of death worldwide. Nowadays, cancer is often treated with different methods, such as surgery, radiation therapy, and chemotherapy. However, these methods have lots of side effects for patients, so natural products are often combined with them to diminish those side effects. According to oriental medicine, there are many folk remedies to support cancer treatment. Each different type of cancer has corresponding remedies. In particular, to support the liver cancer treatment, some medicinal herbs were used, such as Scutellaria barbata, Hedyotis diffusa, Ehretia asperula. The aim of the study: Determining the biological activities of herbal decoctions combining Scutellaria barbata, Hedyotis diffusa, and Ehretia asperula, which were tested on two different weight ratio recipes (denoted R1 and R2). Materials and methods: Mixing Scutellaria barbata, Hedyotis diffusa, and Ehretia asperula into dry mixtures and then collecting the decoction. Testing antioxidant activity by the reducing power assay and the DPPH free radical scavenging assay. Evaluating antibacterial activity by the agar well diffusion assay and cytotoxic ability by the sulforhodamine B assay. Results: R1 decoction, which had a higher ratio of E. asperula, showed greater antioxidant, antibacterial, and cytotoxic capacity than R2 decoction. In the reducing power assay, R1 decoction's optical density value was 2.80, while the other was 2.23 at 0.7% concentration. In the DPPH free radical scavenging assay, the EC50 values of R1 and R2 decoctions were 0.022% and 0.035%, respectively. Both R1 and R2 decoctions had antimicrobial activities against Bacillus subtilis at 100% concentration, as shown by the diameters of inhibition growth zones of 11.33 mm and 7.40 mm, respectively. In the Sulforhodamine B assay, R1 decoction's IC50 was 0.47%, and R2 decoction's IC50 was 0.66%. Conclusion: In the same condition, R1 decoction had better tested biological activities than R2 decoction.

Keywords: DPPH free radical scavenging; reducing power; cytotoxicity

For citation: Thi HN, Anh TP, Khanh DTD, et al. Study on biological activities of herbs' decoction combining Scutellaria barbata, Hedyotis diffusa and Ehretia asperula Research Results in Biomedicine. 2024;10(4):553-562. Russian. DOI: 10.18413/2658-6533-2024-10-4-0-5

Introduction. Nowadays, natural products are widely used in disease treatments all over the world for various reasons, such as solving side effects in chemotherapy, lowering medical service fees, and so on. Vietnam has had extremely rich sources of medicinal herbs for a long time. These herbs have been mixed into various remedies to cure diseases such as diabetes, diarrhea, and so on, especially cancer. This shows the potential of herbs in the pharmaceutical industry. S. barbata has been proven to have antioxidant capacity [1], antiinflammatory activity [2], and cytotoxicity in four different cell lines [3]. Similarity, H. diffusa has also been shown to have antioxidant and hepatoprotective capacity against H2O2 toxicity [4], cytotoxicity on six different cell lines [5], and its ability to enhance the activity of CIK cells - natural anti-cancer cells [6]. In 2020, Chinese scientists demonstrated the ability of the combination of S. barbata and H. diffusa to inhibit the proliferation and migration of tumor cells [7]. E. asperula has received much attention from domestic researchers. Rosmarinic acid, an acid with antimicrobial, anti-inflammatory, antioxidant, and anti-tumor capacities, has been extracted from this herb [8]. In addition, its stems also showed cytotoxicity on three test cell lines [9], and its leaves demonstrated cytotoxicity on three cell lines and antioxidant capacity [10]. The potency of oriental remedies comes not only from the active ingredients of each herb but also from their effects when combined with other herbs. For example, to treat cancer, a remedy combines a host herb that has cancer cytotoxic ability with an increasing immunity or antioxidant herb. In this study, S. barbata, H. diffusa, and E. asper-ula were mixed into remedies, and then their biological activities were determined. The results provide the basic data for later clinical trials.

Materials and methods

Decoction preparation.

Herbs were purchased at the Research Center for planting and processing medicinal plants, Saigon Medicinal Plants, 35/21B5 Tran Dinh Xu, District 1, Ho Chi Minh City. The decoctions were mixed based on two recipes with different weight ratios. The ratios of S.

barbata : H. diffusa : E. asperula were 2 : 2 : 5 (R1) and 2 : 3 : 4 (R2), respectively. 9 g of each recipe was cut into small pieces, then added to 500 mL of distilled water and boiled for 3 hours at 70-80 °C. This primary decoction was centrifuged at 3000 rpm for 10 minutes, after which sediment was removed. Then, the decoction was boiled down at 50-60 °C until its volume reached 9 mL. The final concentration was 100%. It was diluted to various concentrations to conduct the biological activity assays.

Reducing power assay.

The principle of this assay is based on the antioxidants' capacity for reducing potassium hexacyanoferrate (III) to potassium hexacy-anoferrate (II). The reaction of ferric chloride and potassium hexacyanoferrate (II) forms a colored complex. It was measured at 700 nm. The higher measured optical density value shows better antioxidant activity. The procedure was carried out according to the report by Oyaizu [11] and modified [12].

2.5 mL of phosphate buffer and 2.5 mL of potassium ferricyanide (1% w/v) were added to 1 mL of various concentrations of the decoction. This mixture was kept at 50 °C in a water bath for 20 minutes. Then, 2.5 mL of tri-chloroacetic acid (TCA) (10% w/v) was added and centrifuged at 3000 rpm for 10 minutes if necessary. 2.5 mL of the upper layer of solution was mixed with 2.5 mL of distilled water and 0.5 mL of ferric chloride solution (0.1% w/v). The optical density value was measured at 700 nm. The higher the optical density (OD) value of the mixture, the higher the decoction's reducing power.

DPPH free radical scavenging assay.

2,2-diphenyl-1-picrylhydrazyl (DPPH) is a stable free radical that shows maximum ab-sorbance at 517 nm. The antioxidant activity of the decoction was determined by free radical scavenging capacity following the standard protocol of Brand-Williams [13] with some modifications [14].

5 mL of DPPH methanol solution (0.08 mM) was added to 1 mL of various concentrations of the decoction and kept in the dark for 30 minutes. Then, the absorbance was measured at 517 nm. The free radical scavenging capacity (EC%) was calculated by the following

equation: EC% = (Acontrol - Asample)/Acontrol x

100%. Acontrol: absorbance value of DPPH solution with distilled water; Asample: absorbance value of DPPH solution with decoction. Finally, the linear correlation equation was built, and the EC50 value was determined. The lower the EC50 value, the higher the scavenging capacity.

Agar well diffusion assay.

Escherichia coli and Bacillus subtilis were supplied by the Microbiology and Biochemistry Laboratory, Biology Department, Ho Chi Minh City University of Education.

The antimicrobial activity of the decoction was determined following the standard protocol of Hadacek [15] with modifications [16]. The agar plate surface was inoculated by spreading 100 |iL of microbial inoculum (4-5 x 108 CFU/mL) over the entire agar surface. A 6 mm diameter hole was aseptically punched with a sterile cork borer and filled with 100 ^L of various concentrations of the decoction. After 4 hours of keeping in a cool refrigerator compartment, the plates were incubated at 37 °C for 24 hours. Finally, the diameters of the growth inhibition zones were measured.

Suforhodamine B (SRB) assay.

The principle of this method is based on the property of Sulforhodamine B (SRB) to bind cellular proteins under mild acidic conditions. Thus, colorimetric evaluation provides an estimate of total protein mass, which is related to the number of survival cells. The procedure was carried out according to the report of Monks [17] with some modifications [18].

190 |iL of cell cultures were incubated with 10 ^L of various concentrations of the decoction in 10% dimethylsulfoxide (DMSO) for 3 days on 96-well plates. In contrast, 190 ^L of cell cultures were incubated with 10 |iL of H2O, DMSO, or medium as a negative control. Cells were fixed in 10% TCA for 30 minutes, then stained for 1 hour with SRB at 37 °C. Then, the plates were washed three times with 5% acetic acid and dried at room temperature. Finally, the pellets were solubilized with 10 mM Tris base and shaken for 10 minutes. The

absorbance was measured at 490 nm. The cytotoxic capacity (IC%) was calculated by the following equation: IC% = 100% - (Asample -

Ablank)/(Acontrol - Ablank) x 100%. Acontrol: absorbance value of solution containing cells and medium. Asample: absorbance value of solution containing cells and sample. Ablank: absorbance value of the solution containing only culture medium. Then, the correlation equation was built, and the IC50 was determined. The lower the IC50, the higher the cytotoxic capacity.

Statical analysis. The data of the study were analyzed in the GraphPad Prism 9 statistical program. The values were presented as means ± SD. The correlation equation was determined by the equation with a p-value < 0.05.

Results

Result of antioxidant activity.

In this study, the antioxidant abilities of both R1 and R2 decoctions were determined through reducing power and DPPH free radical scavenging assays. Based on the measured optical density value of the samples, the ferrous ion reducing capacity of herbal decoction at 0.1% concentration was compared in Fig. 1. The absorbance of EA decoction at 700 nm (0.581 ± 0.039) was 3.2 times that of SB decoction (0.180 ± 0.009) as well as the positive control (0.180 ± 0.003), and 6 times that of HD decoction (0.096 ± 0.004). The ferrous ion reducing capacity of R1 and R2 decoctions increases with increasing concentration (p < 0.04) (Fig. 2). The optical density value of R1 decoction is always higher than that of R2 decoction, about 2 times higher, from 0.01% to 0.1% concentration. When the concentrations of R1 and R2 range from 0.5% to 0.7%, the OD value of R1 is 20% higher than that of R2. The positive control (ascorbic acid) regression equation is: y = 0.0123x - 0.0001 (R2 = 0.998). At 0.7% concentration, the capacities of R1 and R2 decoctions are equivalent to 226.024 |ig/mL and 182.772 |ig/mL ascorbic acid, respectively. So R1 had a higher capacity in reducing power assay.

Fig. 1. Ferrous ion reducing power of herbal decoctions at 0.1% concentration Note: Ehretia asperula decoction (EA); Scutellaria barbata decoction (SB); Hedyotis diffusa decoction (HD); positive control (PC) - 16 ^g/mL ascorbic acid

Fig. 2. Ferrous ion reducing power of R1 and R2 decoctions Note: R1: recipe 1 decoction; R2: recipe 2 decoction

At 0.1% concentration, the DPPH free radical scavenging capacity of EA, SB, HD decoctions, and positive control was 88.02 ± 1.366%, 49.52 ± 1.049%, 11.27 ± 0.368%, and 48.22 ± 0.303%, respectively (Fig. 3). The correlation equations' graphs of herbal decoctions were described in Fig. 4. The slope of R1 decoction's graph is higher than that of R2 decoction.

From the correlation equations (p < 0.0001), the EC50 values of EA, SB, HD, R1, and R2 decoctions were calculated (Table 1). The correlation equation and EC50 value of the positive control (ascorbic acid) were estimated as y = 3.1147x + 0.1909 (R2 = 0.998) and 16 |ig/mL, respectively.

Fig. 3. Percentage of DPPH free radical scavenging of herbal decoctions at 0.1% concentration Note: Ehretia asperula decoction (EA); Scutellaria barbata decoction (SB); Hedyotis diffusa decoction (HD); positive control (PC) - 16 ^g/mL ascorbic acid

I

CL ■

Q- 0 I 1 1 ............................

Q 0.00 0.02 0.04 0.06

Concentration (%)

Fig. 4. Percentage of DPPH free radical scavenging of R1 and R2 decoctions Note: recipe 1 decoction (R1); recipe 2 decoction (R2)

Table 1

Regression equations, R-squared, and EC50 of t ecoctions in DPPH assay

Decoction Regression equations R-squared EC50

EA Y = 3210*X + 9.823 0.923 0,013%

SB Y = 460.1 *X + 2.931 0.984 0.102%

HD Y= 53.78*X + 25.38 0.984 0.458%

R1 Y = 2280*X - 1.270 0.985 0.022%

R2 Y = 1365*X + 2.775 0.991 0.035%

Note: Ehretia asperula decoction (EA); Scutellaria barbata decoction (SB); Hedyotis diffusa decoction (HD); recipe 1 decoction (R1); recipe 2 decoction (R2)

Result of antibacterial activity. but incapacity against E. coli (Table 2). R1 de-

Both recipes showed antibacterial capac- coction has a larger inhibition zone when comity against B. subtilis at 100% concentration, pared to R2 decoction.

Table 2

Inhibition growth zones (mm) of R1 and R2 decoctions

Bacteria Recipe 1 Recipe 2

1% 10% 100% 1% 10% 100%

Eschirichia coli — — — — — —

Bacillus subtilis - - 11.33 ± 1.76 — — 7.40 ± 0.92

Result of cytotoxic activity.

The cytotoxic activity of both recipes decoctions at various concentrations was demonstrated through an SRB assay on a Hep-G2 cell line (Fig. 5). The correlation equation was

built, and the IC50 values of R1 and R2 decoctions were calculated. The results were 0.47% and 0.66%, respectively (Table 3). The presented data show that the cytotoxic capacity of the R1 decoction was stronger than the other.

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.с 1 40-

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R1

R2

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Fig. 5. Percentage of cell growth inhibition of R1 and R2 decoctions Note: R1: recipe 1 decoction; R2: recipe 2 decoction

Table 3

Regression equations, R-squared, and IC50 of decoctions in SRB assay

Decoction Regression equations R-squared IC50

Ri Y = i8.89 + 76.25*X — 20.96*XЛ2 0.973 0.470%

R2 Y = i5.02 + 65.44*X — 16.40*XЛ2 0.958 0.66i%

Note: R1: recipe 1 decoction; R2: recipe 2 decoction

Moreover, the morphological changes of HepG2 cells were illustrated in Fig. 7. HepG2 cells have an epithelial-like morphology. In negative controls, the cells that were treated with medium or H2O (Fig. 6a and b), after 3 days, the growth extended outward from the adherent cell colonies. While in Fig. 6c and d, cells attached in small patches of cells or single cells, there were many round cells floating. In Fig. 7e, the cell colonies were smaller than in Fig. 7a and b, which meant the density of cells decreased.

Based on the morphology, the density of cells in R1 and R2 was lower than the positive control because in R1 and R2 there were many round cells floating in the medium, they were dead cells, and the colonies attached to the well surface were smaller than the positive control. While in positive control (0.07 |ig/mL camp-tothecin), the cells growth extended wider than R1 and R2, which were alive cells. In suspension in the medium, there were little round cells floating. In conclusion, at 1% concentration, R1 and R2 had better inhibition capacities than the positive control.

(e)

Fig. 6. HepG2 cells incubated with samples Note: (a) no samples; (b) H2O 5%; (c) 1% R1 decoction; (d) 1% R2 decoction; (e) 0.07 |g/mL camptothecin

Discussion. The antioxidant capacity of R1 and R2 decoctions was proven through ferrous ion reducing power and DPPH free radical scavenging capacity due to the antioxidant activity of each herb. In fact, S. barbata has been proven to contain polysaccharides, and these components have antioxidant capacity through the total antioxidant activity test and the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) free radical scavenging assay. From the concentrations of 0.24 to 0.48 mg/mL, it had similar effects as the positive control in the total antioxidant activity test. In the ABTS assay, its effects increased and reached the positive control's value at a concentration of 1.2 mg/mL [1]. In 2015, Xin Gao et al. demonstrated H. diffusa water extract's antioxidant capacity by DPPH assay with a calculated EC50 value of 0.15 mg/mL [4]. E. as-

perula total extract (ethanol 90%) and ethyl acetate extract's antioxidant activity was determined through the DPPH free radical scavenging assay with EC50 values of 48.5 |g/mL and 46.9 |g/mL respectively [9]. The R1 and R2 decoction's EC50 values in the DPPH assay and OD values at 0.1% concentration in the reducing power assay were compared to the determined component herbs' values in the same assay and condition (Fig. 1 and Table 1). From these data, it could be concluded that the anti-oxidant capacity was ranked in ascending order: H. diffusa, S. barbata, R2, R1, and E. as-perula. This could be due to the ratio of EA in these decoctions: R1 decoction (the ratio of SA:HD:EA was 2:2:5, w/w/w) and R2 decoction (the ratio of SA:HD:EA was 2:3:4, w/w/w). According to the oriental medicine formulation, E. asperula can be considered the main herb in this remedy, while H. diffusa and

S. barbata can be considered adjuvant herbs with side functions.

In the antibacterial activity test, in the same condition, R1 decoction showed better antibacterial capacity than R2 decoction against B. subtilis (11.33 mm and 7.40 mm, respectively). B. subtilis grows in the digestive tract of humans and many animals, especially ruminants. It's beneficial to humans, so they are also called probiotic bacteria [19]. Which means this decoction is safe for users' digestive tracts if they use it in moderation; if not, some unexpected digestive problems may appear. According to the formulation of oriental medicine, the remedy needs one or more additional herbs in the recipe to support the digestive tract.

In comparison between R1 and R2 decoction's HepG2 cytotoxicity activity, R1 decoction has an IC50 value lower than R2 decoction (0.47% < 0.66%). This means that the cytotoxic capacity of R1 decoction on HepG2 cells was better than that of R2. This can be explained by the higher ratio of E. asperula in recipe 1. E. asperula was demonstrated to have strong cytotoxicity capacity on HepG2 cells, with an IC50 value of n-hexane extract of 28.30 |g/mL through the SRB assay [9]. The concentration of H. diffusa inhibiting 50% cell growth (IC50) was estimated to be 4.62 mg/mL. This led to the R2 decoction (which has a higher rate of HD in the recipe) not being as effective as R1. However, the water extract of this herb was proven to have the capacity to enhance the antitumor activities of cytokine-induced killer cells (natural anti-cancer cells) [6] as well as normal liver cells protection ability [4]. These are reasons to consider H. diffusa as an adjuvant herb.

There is a close relationship between ox-idative stress and liver cancer. When free radical content increases, cancer can form [20]. This remedy has strong free radical scavenging activity, so it can be used as a cancer prevention remedy. In addition, the ability to cause cytotoxicity in liver cancer cells was also strong. Thus, the remedy in this study can be used as a preventive, curable liver cancer treatment. Moreover, further studies need to be car-

ried out to demonstrate the molecular mechanism of hepatocellular cytotoxicity as well as its non-toxicity on normal cells to be able to apply it in medicine.

Conclusion. Based on the data and results obtained above, the remedy decoction combining Scutellaria barbata, Hedyotis diffusa, and Ehretia asperula (two different recipes in weight ratio), in which E. asperula accounts for the most proportion, showed outstanding biological activities such as antioxi-dant activity, cytotoxicity activity against Hep-G2, and antibacterial activity against B. subtilis. The R1 decoction (the ratio of SA:HD:EA was 2:2:5, w/w/w) has better biological activities than the R2 decoction (the ratio of SA:HD:EA was 2:3:4, w/w/w).

Financial support

No financial support has been provided for this work.

Conflict of interests

The authors have no conflict of interest to declare.

References

1. Gao JY, Yang X, Liu P, et al. Optimization of ultrasonic-assisted extraction of polysaccharides from Scutellaria barbata and determination of their anticancer and antioxidant activities. International Journal of Pharmacology. 2016;12(7):754-759. DOI: https://doi.org/10.3923/ijp.2016.754.759

2. Liu HL, Kao TH, Shiau CY, et al. Functional components in Scutellaria barbata D. Don with anti-inflammatory activity on RAW 264.7 cells. Journal of Food and Drug Analysis. 2017;26(1):31-40. DOI: https://doi.org/10.1016/] .jfda.2016.11.022

3. Wang M, Ma C, Chen Y, et al. Cyto-toxic neo-clerodane diterpenoids from Scutellaria barbata D.Don. Chemistry and Biodiversity. 2019;16(2):e1800499. DOI: https://doi.org/10.1002/cbdv.201800499

4. Gao X, Li C, Tang YL, et al. Effect of Hedyotis diffusa water extract on protecting human hepatocyte cells (LO2) from H2O2 - induced cytotoxicity. Pharmaceutical Biology. 2015;54(7): 1148-1155. DOI: https://doi.org/10.3109/13880209.2015.1056310

5. Wang C, Zhou X, Wang Y, et al. The antitumor constituents from Hedyotis diffusa Willd. Molecules. 2017;22(12):2101. DOI: https://doi.org/10.3390/molecules22122101

6. Ma C, Wei Y, Liu Q, et al. Polysaccharides from Hedyotis diffusa enhance the antitumor activities of cytokine-induced killer cells. Biomedicine and Pharmacotherapy. 2019; 117:109167. DOI: https://doi.org/10.1016/j.bio-pha.2019.109167

7. Ma TT, Zhang GL, Dai CF, et al. Scu-tellaria barbata and Hedyotis diffusa herb pair for breast cancer treatment: Potential mechanism based on network pharmacology. Journal of Eth-nopharmacology. 2020;259:112929. DOI: https://doi.org/ 10.1016/j.jep.2020.112929

8. Ly NT. Separation process of rosma-rinic acid and their derivatives from Celestrus hindsii Benth leaves. Vietnam Journal of Science and Technology. 2018;54(2C):380-387. DOI: https://doi.org/10.15625/2525-2518/54/2C/11865

9. Nguyet VT, Dat NT, Ha TTH, et al. Evaluating cytotoxic effect of the extracted compounds from Ehretia asperula Zoll. & Mor. stem on several cancer cell lines. Academia Journal of Biology. 2018;40(2):145-152. DOI: https://doi.org/10.15625/0866-7160/v40n2.12955

10. Bui TTD, Vu MH, Bui TT. Cytotoxicity and antioxidant effects of Celastrus hindsii Benth. leaf extract. VNU Journal of Science: Medical and Pharmaceutical Sciences. 2020;36(1):39-45. DOI: https://doi.org/10.25073/2588-1132/vnumps.4203

11. Oyaizu M. Studies on Products of Browning Reactions: Antioxidative Activities of Product of Browning Reaction Prepared from Glucosamine. Japan Journal of Nutrition. 1986;44(6):307-315. DOI: https://doi.org/10.5264/eiyogakuzashi.44.307

12. Zhou J, Yang Q, Zhu X, et al. Antioxi-dant activities of Clerodendrum cyrtophyllum Turcz leaf extracts and their major components. PLoS ONE. 2020;15(6):e0234435. DOI: https://doi.org/10.1371/journal.pone.0234435

13. Brand-Williams W, Cuvelier ME, Ber-set C. Use of free radical method to evaluate antioxidant activity. LWT. 1995;28(1):25-30. DOI: https://doi.org/10.1016/S0023-6438(95)80008-5

14. Adjimani JP, Asare P. Antioxidant and free radical scavenging activity of iron chelators. Toxicology Reports. 2015;2:721-728. DOI: http s://doi.org/10.1016/j.toxrep.2015.04.005

15. Hadacek F, Greger H. Testing of antifungal natural products: Methodologies, comparability of results and assay choice. Phytochemical

Analysis. 2000;11(3):137-147. DOI:

https://doi.org/10.1002/(SICI)1099-1565(200005/06)11:3<137::AID-PCA514>3.0.CO;2-I

16. Balouiri M, Sadiki M, Ibnsouda SK. Methods for in vitro evaluating antimicrobial activity: A review. Journal of Pharmaceutical Analysis. 2016;6(2):71-79. DOI: https://doi.org/10.1016/jjpha.2015.11.005

17. Monks A, Scudiero D, Skehan P, et al. Feasibility of a High-Flux Anticancer Drug Screen Using a Diverse Panel of Cultured Human Tumor Cell Lines. Journal of the National Cancer Institute. 1991;83(11):757-766. DOI: https://doi.org/10.1093/jnci/83.11.757

18. Nguyen VT, Vu KT, Nguyen XN, et al. Oleanane-type saponins from Glochidion hirsutum and their cytotoxic activities. Chemistry and Biodiversity. 2017;14(5):e1600445. DOI: https://doi.org/10.1002/cbdv.201600445

19. Nguyen LD, Nguyen DQ, Pham VT. Microbiology. Ha Noi: Education Publisher; 2012. Vietnamese.

20. Wang Z, Li Z, Ye Y, Xie L, Li W. Oxidative stress and liver cancer: Etiologi and therapeutic targets. Oxidative Medicine and Cellular Longevity. 2016;2016:7891574. DOI: https://doi.org/10.1155/2016/7891574

Received 3 July 2023 Revised 13 September 2023 Accepted 19 October 2023

Information about the authors

Hang N. Thi, MSc, Laboratory Manager of the

Genetics and Evolution, Biology Department, Ho

Chi Minh City University of Education, Ho Chi

Minh City, Vietnam, E-mail:

[email protected], ORCID:

https://orcid.org/0000-0002-3334-3291.

Tai P. Anh, Student at the Laboratory of Genetics

and Evolution, Biology Department, Ho Chi Minh

City University of Education, Ho Chi Minh City,

Vietnam, E-mail:

[email protected], ORCID:

https://orcid.org/0000-0002-6254-1537.

Dang T.D. Khanh, Student at the Laboratory of

Genetics and Evolution, Biology Department, Ho

Chi Minh City University of Education, Ho Chi

Minh City, Vietnam, E-mail:

[email protected], ORCID:

https://orcid.org/0009-0003-2530-3710.

Tran B.N. Huyen, Student at the Laboratory of

Genetics and Evolution, Biology Department, Ho

Chi Minh City University of Education, Ho Chi Minh City, Vietnam, E-mail:

[email protected], ORCID:

https://orcid.org/0009-0009-1353-9906. Duy D.N. Anh, Student at the Laboratory of Genetics and Evolution, Biology Department, Ho Chi Minh city University of Education, Ho Chi Minh City, Vietnam, E-mail:

[email protected], ORCID: https://orcid.org/0000-0002-3809-0115. Hien N. Thanh, Student at the Laboratory of Genetics and Evolution, Biology Department, Ho Chi Minh City University of Education, Ho Chi Minh City, Vietnam, E-mail:

[email protected], ORCID: https://orcid.org/0000-0003-2695-8922.