INTRODUCTION:

The Euphorbiaceae family is one of the largest and most diverse plant families, ranking fourth among the 305 higher plant families with vascular systems, comprising 322 genera and 8,910 species. The Euphorbiaceae family is mainly distributed in tropical and subtropical regions¹. The genus Mallotus is primarily found in tropical regions of Asia, Australia, and the Pacific. It is also present in some tropical areas of Africa and Madagascar. The specific distribution of Mallotus species includes countries such as Thailand, Vietnam, India, and Taiwan and throughout Southeast Asia, Malesia, East Australia, and New Guinea. This genus thrives in various habitats, including old-growth forests, roadside areas, riverbanks, and swampy regions. It can be found at altitudes ranging from sea level up to 800 meters².

Studies have reported various natural compounds in the Mallotus genus, such as diterpenoids, triterpenoids, steroids, flavonoids, coumarin, phloroglucinol derivatives, and benzopyrans. Secondary metabolites isolated from Mallotus species exhibit diverse biological activities, including antioxidant effects^3-5, α-glucosidase inhibition⁶, antiviral and antibacterial properties, anti-inflammatory, anticancer effects^7,8 liver protection⁹, and cytotoxicity¹⁰.

In Vietnam, the diversity of the Mallotus genus was documented in 1999 by Pham Hoang Ho, who confirmed 33 Mallotus species in the country. These species are concentrated in mountainous regions with altitudes below 1,000 meters, although some species can grow up to 2,000 meters^11,12. The biological diversity of Mallotus species is a concern, even among scholars in Vietnam. Mallotus barbatus Muell.-Arg has been detailed in branches, leaves, flowers, fruits, and other characteristics. Traditional medicine uses different plant parts, such as roots, stem bark, and leaves, to treat digestive issues, oedema, and headaches¹³.

Globally, Mallotus barbatus Muell.-Arg has been studied for its morphology¹⁴ and chemical composition, including phenolic compounds, flavonoids^15,16, and essential oils¹⁷. However, research on the database of Mallotus barbatus Muell.-Arg in Vietnam still needs to be completed, as there is a lack of in-depth studies on plant anatomy.

This study presents a fundamental aspect of the internal structure of Mallotus barbatus Muell.-Arg and accurately identifies its genetic source. Additionally, it analyzes and evaluates several biological activities. This initial report contributes to developing raw materials for the pharmaceutical industry.

MATERIALS AND METHODS:

Materials:

Leaves of Mallotus barbatus Muell.-Arg were collected in June 2022 from Lam Dong Province, Vietnam. Leaves were dried, ground into powder, and stored at room temperature.

a) Cell line:

The human breast cancer cell line MDA-MB-231 was sourced from the ATCC (American Type Culture Collection, USA). The cells were stored, activated, and cultured at the Pasteur Institute in Ho Chi Minh City.

b) Experimental microorganisms:

The experimental microorganisms, including MSSA (ATCC 25923), MRSA (ATCC 43300), Streptococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25922), Aspergillus niger (ATCC 16404), Penicillium sp., Mucor sp., Rhizopus sp., and Candida albicans, were activated and cultured at the Faculty of Pharmacy, Nguyen Tat Thanh University.

c) Solvents and chemicals:

Methanol, n-hexane, chloroform, ethyl acetate, and dimethyl sulfoxide (DMSO) were obtained from Xilong (China). Isopropanol and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were sourced from Merk (Germany); 2,2-diphenyl-1-picrylhydrazyl (DPPH), (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and trypan blue were provided by Sigma (USA). Ascorbic acid was supplied from VWR (FUSA) and resazurin from Thermo Scientific (USA).

Morphological and histological characteristics:

Description of visual characteristics of the plant parts, including the stem and leaves, in terms of shape, colour, and size. Fresh samples were cut into thin slices and underwent bleaching with a 10% bleach solution. Subsequently, staining was performed using 1% methylene blue and 2% carmine red. After the staining process, samples were placed on slides for observation under a microscope with different magnifications, including 4X, 10X, and 40X, and images were captured for documentation¹⁸.

rbcL and ITS genes sequencing:

DNA extraction and amplification were performed to obtain the target genes from plant samples, specifically the rbcL (ribulose-bisphosphate carboxylase)^19,20 and ITS (internal transcribed spacer)^21,22 genes. The obtained sequences were compared with reference sequences from the Nucleotide Collection Database through BLAST analysis on GenBank (NCBI).

Chemical composition of M. barbatus extract:

Preliminary identification of chemical components in the extract from Mallotus barbatus leaves revealed whether the chemical groups, including flavonoids, steroids, terpenes, carotenoids, and iridoid glycosides, are present in the extract.

Extraction and fractionation of M. barbatus extracts:

Extraction of herbal materials was proceeded by the ultrasonic method using methanol as the solvent in a 1:10 ratio of material to solvent. The extraction process was carried out in triples, combining the extracts. The rotary evaporator was used under reduced pressure at 45 °C to remove the solvent. Subsequently, the fractionation was carried out after dissolving the total extract in 150ml of distilled water. In the liquid-liquid partitioning technique, 500ml of each sequentially increasing polar solvent was added, including n-hexane, chloroform, and ethyl acetate. Each fraction was extracted three times, and the resulting extracts were then combined. The remaining portion was the aqueous extract, which was not used in the subsequent steps. Finally, solvent removal was continued by reduced-pressure evaporation.

Antimicrobial activity:

The antimicrobial activity was assessed using the agar well diffusion method on Muller-Hinton agar medium or Muller-Hinton agar medium supplemented with 2% glucose and methylene blue for C. albicans. 60µl of the extract was added to sterile wells with a diameter of 6 mm. Subsequently, the samples were incubated at a temperature of 35-37°C for a period ranging from 18 to 48 hours. The results were recorded by measuring the diameter of the inhibition zone^23,24.

The dilution broth method was used according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) to find the minimum inhibitory concentration (MIC)²⁵. 30µl of the resazurin test reagent (0.015%) was added to the wells to indicate growth²⁶. Results were read after 16-48 hours of incubation. Two control groups were used during the test for comparison with the test sample: the negative control using 1% DMSO and the positive control including amikacin and gentamycin, amphotericin B, and fluconazole^27,28.

Antioxidant assays:

a) DPPH method:

The mixture containing 10µl of the extract from Mallotus barbatus with 190µl of 0.06mg/ml DPPH solution was incubated at room temperature for approximately 30 minutes. Subsequently, the optical absorption of the sample was measured triple at a wavelength of 517nm using a Biotek reader (USA), and vitamin C was used as a control sample. The calculated absorption results determine the percentage of DPPH free radical inhibition and the IC₅₀ value, showing the antioxidant capacity of the samples^29-32.

b) ABTS method:

A 7mM ABTS solution and a 2.45mM K₂S₂O₈ solution were prepared. Subsequently, these two solutions were mixed and incubated in the dark for 12-16hours to generate the ABTS^*+ radical solution. The ABTS^*+ radical solution was then diluted by combining 2.8 ml of the solution with 65ml of acetic acid buffer solution (pH = 4.5). Each well consisted 10µl of the test sample and 190µl of ABTS^*+ solution. The reaction mixture was incubated at room temperature for approximately 30 minutes. The optical absorption of the sample at a wavelength of 734nm was measured using a Biotek reader (USA), along with the Trolox as a control sample. The absorption results were then calculated to determine the percentage of ABTS free radical inhibition. The antioxidant capacity of the samples was assessed by the IC₅₀ value^33-35.

Cytotoxic activity:

The extract was dissolved in a 10% DMSO solvent to achieve the test concentrations of 300, 200, and 100 µg/ml. Samples at the appropriate concentration were introduced into each well on the activated cell plate. A negative control with 1% DMSO and a positive control containing cultivation medium without the test sample were included. The test plate was positioned in a 5% CO₂ incubator for 72hours at 37°C. Then, the current medium was replaced by a non-serum medium, and MTT solution (5mg/ml) was added to each well. The plate was shaken for 5 minutes at a speed of 900rpm and further incubated for 4-6hours at 37°C in a 5% CO₂ incubator. 150µl of acidified isopropanol is added to each well, mixed thoroughly, and allowed to stand for 10minutes before measuring optical absorption at a wavelength of 570nm. The percentage of viable cells was calculated using the formula: cell viability (%) = (average absorption of the test sample × 100)/(average absorption of the negative control sample). The experiment was repeated three times to calculate the average value^36,37.

RESULT:

Morphological description:

Shrubs to small trees up to 3.6-4.2m high, evergreen. Stem and young branches are colored light brown, densely stellate trichomes, and are very soft-floccose. Leaves alternate, simple; lamina is elliptical or ovate (20-25cm x 19-23cm), entire or shallowly dentate along the margin, reticulate venation with 3-5 principal veins; base cordate, rounded or obtuse; petiole 10-20cm, hairy; upper surface pinkish red when young, dull green when old, lower surface brownish green. Inflorescences terminal, unisexual; staminate inflorescences panicles, becoming pendulous; staminate flowers: pedicels absent, sepals four connate, stamen 40-90, pale light yellow, pistillode absent. Pistillate flowers: pedicels absent or present; ovary with visible spines or not, with glandular hairs, locules 3–5, 1 ovule per locule; staminodes present or absent. Fruits are large and long, surface spiny and densely hairy.

Figure 1. Morphology of the upper surface (a) and lower surface (b) of the leaf stem

(1)

A) Upper epidermis; B) Palisade parenchyma; C) Lower epidermis; D) Collenchyma; E) Calcium; F) Trichome with unicellular globular head; G) Xylem; H) Phloem; I) Pith parenchyma; K) Lower epidermis

(2)

A) Epidermis; B) Collenchyma; C) Primary phloem; D) Primary xylem; E) Trichome with unicellular globular heads; F) Calcium oxalate; G) Parenchyma; H) Secondary phloem-xylem bundle

(3)

A) Trichomes with unicellular globular heads; B) Collenchyma; C) Epidermis; D) Parenchyma; E) Collenchyma with cellulose walls; F) Primary phloem; G) Secondary phloem; H) Sclerenchyma; I) Medullary rays; K) Secondary xylem parenchyma; L) Xylem vessels; M) Primary xylem parenchyma; N) Primary xylem vessels; O) Medullary parenchyma; P) Pith parenchyma

Figure 2. Anatomy and cellular composition of leaf veins (1), petiole (2), and stem (3)

The main vein (midrib) is convex on both sides, and the abaxial side is more convex than the adaxial side of the leaf. Both upper and lower epidermises are uniseriate with rectangular cells covered by a cuticle. Angular collenchyma under the epidermis both above and below the leaf, 9 - 12 cell layer polygonal, irregular, disorderly arrangement. There are druse calcium oxalate crystals scattered throughout the parenchyma. Short-stalked, multicellular, stellate trichomes appear abundantly on the leaf surface. Vascular tissue is arranged in an arc, and the xylem and phloem are arranged into a central and secondary vascular bundle. Xylem vessels are nearly round, lignin-impregnated walls arranged in rows in vascular bundles interspersed with xylem parenchyma. Phloem consists of a group of polygonal cells. Parenchyma consists of many layers of polygonal cells, irregular in size and disorderly arrangement. Figure 2 shows leaf vein anatomy.

Petiole and lamina: Both upper and lower epidermises are uniseriate with rectangular cells covered by a cuticle. Short-stalked, multicellular, stellate trichomes appear abundantly on the leaf surface. Chlorenchyma consists of a long polygonal layered cell, walled with cellulose, containing many chloroplasts. The petiole has a nearly round shape; the petiole's epidermis consists of rectangular cells covered by cuticles and cellulose walls, fairly uniform in size. Many multicellular, stellate trichomes appear abundantly on epidermises. Angular collenchyma in petiole 8-10 cell layers has a nearly round polygonal shape, cellulose walls, discontinuous arrangement and irregular size. The parenchyma of the petiole consists of many layers of polygonal cells, irregular in size and disorderly arrangement.

Stem: Epidermise is uniseriate with rectangular cells that are quite uniform in size, tightly packed and covered by cuticle. There are many multicellular, stellate trichomes scattered on the stem surface. Angular collenchyma under the epidermise, 6-8 cell layers with nearly round shape, cellulose walls, uneven size and disorderly arrangement. Cortex parenchyma is located below the angular collenchyma, consisting of 2-3 layers of nearly round cells and cellulose walls that are uneven in size and haphazardly arranged. The sclerenchyma cylinder consists of 3-5 layers of polygonal cells with thick cell walls and disorderly arrangement. Phloem fibres consist of 3-5 layers of polygonal cells located in the secondary phloem area. The secondary xylem includes xylem vessels and xylem parenchymas. Xylem parenchyma in the secondary xylem region is arranged in rows. The secondary xylem vessels have an oval shape, lignified walls, uneven size and disorderly arrangement. The primary xylem includes primary xylem vessels and primary xylem parenchymas. The primary xylem vessels consist of 3-5 cell layers and lignified walls, which are uneven in size and have disorderly arrangements. The primary xylem parenchymas are 7-10 layers of cells with polygonal shape, uneven size and disorderly arrangement. Between the vascular bundles are narrow rays consisting of 3-4 rows of fairly regular polygonal cells arranged in rows, lignified walls. Pith consists of 3-6 layers of nearly round cells, uneven in size and disorderly arrangement.

Genetic identification:

The BLAST software demonstrated the similarity between the rbcL and ITS gene sequences with reference sequences from the NCBI database. The rbcL gene sequence, approximately 467 bps long, showed the highest similarity with species belonging to the Mallotus genus. Simultaneously, the ITS gene sequence, about 691 base pairs long, matched Mallotus barbatus well (MN715380.1). The results of the plant classification are presented in Figures 4 and 5.

(a)

(b)

Figure 3. Phylogenetic tree results based on (a) rbcL and (b) ITS gene sequence similarities

Extraction efficiency:

Initially, the extraction process involved using methanol to extract 346g of medicinal powder, resulting in a methanol extract weighing 30.30g and exhibiting an extraction efficiency of 8.76%. Subsequently, fractions, including n-hexane, chloroform, ethyl acetate, and water, were derived from the methanol extract. The outcomes indicated that the n-hexane fraction yielded 7.00g with an efficiency of 23.10%, the chloroform fraction produced 7.20g with an efficiency of 23.76%, the ethyl acetate fraction contributed 6.00g with an efficiency of 19.80%, and finally, the water fraction furnished 8.84g with an efficiency of 29.17%. The cumulative mass extracted from all fractions amounted to 29.04g. An efficiency of approximately 8.40% was determined after calculating the overall extraction efficiency.

Antimicrobial activity:

Experimental findings revealed that all extracts inhibited bacterial growth, exhibiting a more prominent antibacterial activity than the antifungal effect. The total extract demonstrated moderate antibacterial activity (inhibition zone from 10mm to 20mm). Notably, n-hexane and chloroform extracts exhibited enhanced antibacterial effects against MRSA, MSSA, and S. faecalis (11mm to 19mm). The ethyl acetate (EA) extract displayed robust antibacterial and antifungal properties, with 18, 19 and 21mm inhibition diameters for MRSA, MSSA, and S. faecalis, respectively. EA also showed antifungal activity against various test microorganisms (30mm). In contrast, the water extract exhibited weak antibacterial activity, limited to MRSA and MSSA, with zone diameters of 16mm and 13mm, respectively. Detailed results are presented in Table 1.

The ethyl acetate fraction stands out with the lowest MIC values against most bacteria and fungi. This indicates the strong antibacterial and antifungal capabilities of EA. For Methicillin-sensitive Staphylococcus aureus (MSSA), EA showed the most vigorous activity at a MIC level of 4µg/ml, showing that it has the potential to resist the MSSA growth effectively. Regarding fungi such as Rhizopus sp. and Mucor sp., the MIC values ranged from 8 µg/ml to 64 µg/ml, showcasing their antifungal efficacy.

The n-hexane and chloroform fractions also displayed low MIC values against various microbial strains. They exhibited good antibacterial and antifungal properties, although not as pronounced as EA. For MRSA, the MIC values of n-hexane and chloroform were 128µg/ml and 64µg/ml, respectively. Concerning the fungus Rhizopus sp., n-hexane and chloroform fractions showed the best MIC values at 32µg/ml, slightly lower than EA.

However, the total extract and water extract often exhibited significantly higher MIC values against most tested bacteria and fungi. They did not demonstrate substantial antibacterial or antifungal activity and may not be favourable choices for developing antibacterial and antifungal pharmaceutical products. Table 2 provides detailed results.

Table 1. Antimicrobial diameters of extract fractions

Strains	Diameter of antimicrobial (mm)
Strains	Methanol	n-hexane	Chloroform	Ethyl acetate	Water
MRSA	10	12	11	18	16
MSSA	10	11	12	19	13
S. faecalis	20	15	19	21	-
P. aeruginosa	-	-	-	13	-
E. coli	-	-	-	10	-
C. albicans	-	-	-	28	-
A. niger	-	-	-	-	-
Penicillium sp.	-	-	-	30	-
Mucor sp.	16	-	-	28	-
Rhizopus sp.	-	20	18	26	-

Note: (-) No activity

Table 2. MIC of extract fraction

Strains	MIC (µg/ml)
Strains	Methanol	n-hexane	Chloroform	Ethyl acetate	Water
MRSA	128	128	128	32	256
MSSA	128	64	64	4	256
S. faecalis	256	128	128	32	-
P. aeruginosa	-	-	-	256	-
E. coli	-	-	-	-	-
C. albicans	-	-	-	256	-
A. niger	-	-	-	-	-
Penicillium sp.	-	-	-	128	-
Mucor sp.	32	64	64	16	32
Rhizopus sp.	32	32	32	8	-

Note: (-) No activity

Antioxidative activity:

These results indicate that the ethyl acetate extract possesses the most potent antioxidant activity among all the studied samples, followed by the n-hexane and chloroform extracts. The n-hexane fraction did not exhibit antioxidant activity. The ethyl acetate fraction demonstrated the most prominent antioxidant activity, with IC₅₀ values of 14.40µg/ml and 20.08µg/ml in DPPH and ABTS assays. The total extract, water extract, and chloroform extract also showed antioxidant activity, but their potency was lower than the ethyl acetate fraction. Their IC₅₀ values were 55.10µg/ml, 101.44µg/ml, and 167.45µg/ml for the DPPH method, respectively; 51.07µg/ml, 80.50µg/ml, and 88.45µg/ml for ABTS method, respectively (Table 3).

Table 3. IC₅₀ results of the extracted fraction

No.	Fractions	DPPH IC₅₀ (µg/ml)	ABTS IC₅₀ (µg/ml)
1	Methanol	55.10 ± 1.52	51.07 ± 1.94
2	n-hexane	-	-
3	Cloroform	167.45 ± 5.28	88.45 ± 1.16
4	Ethyl acetate	14.40 ± 0.69	20.08 ± 0.26
5	Water	101.44 ± 3.62	80.50 ± 2.67
6	Vitamin C	8.04 ± 0.10
7	Trolox		3.50 ± 0.14

Note: (-) No activity

These results are consistent with the research of Van Hong, Rivière et al. 2011¹³ on the antioxidant activity of Mallotus barbatus MA29, equivalent to the activity of tocopherol and antioxidant compounds extracted with ethyl acetate solvent. Mallotus barbatus in this study can be considered as a potential alternative source of antioxidants.

Cytotoxic activity:

Based on the IC₅₀ values, it can be observed that n-hexane extract has the lowest IC₅₀ (42.81±0.72µg/ml), followed by chloroform extract (52.91±0.93µg/ml), and total extract (156.20±2.30µg/ml). This indicates that the n-hexane and chloroform fractions contain compounds with stronger anticancer activity compared to the original total extract. At the same time, ethyl acetate and water extracts could also inhibit cancer cells at a concentration of 300µg/ml, but not as well as n-hexane and chloroform extracts. The rates of killing cells were between 50.93% and 69.04% for ethyl acetate and between 15.81% and 35.45% for water extract. Table 4 presents the results of the tested anticancer activity.

Table 4. The results inhibited the growth of cancer cells with the extract

No	Fractions	(%) Inhibition at a concentration of 300 µg/ml	IC₅₀ (µg/ml)
1	Methanol	84.27 ± 0.54	156.20 ± 2.30
2	n-hexane	94.34 ± 0.86	42.81 ± 0.72
3	Cloroform	93.82 ± 0.20	52.91 ± 0.93
4	Ethyl acetate	69.04 ± 0.70	-
5	Fraction of water	35.45 ± 0.14	-

Note: (-) No activity

Studies on the anticancer potential of Mallotus barbatus species are still limited. However, some Mallotus species, such as Mallotus apelta and Mallotus philippinensis, have shown promising biological activities against cancer cells¹³. Less polar fractions like n-hexane and chloroform are thought to contain compounds that are more effective at fighting cancer. The makeup of these secondary compounds may change depending on the soil, the region, and the harvesting time for Mallotus barbatus.

CONCLUSION:

Mallotus barbatus has been thoroughly described, providing accurate morphological and anatomical characteristics and genetic information on the rbcL and ITS gene sequences. The study assessed the biological activity of various extracts from Mallotus barbatus. The results revealed that certain extracts, particularly the ethyl acetate fraction, inhibited the growth of Gram-positive bacteria and moulds. Additionally, these extracts displayed antioxidant capabilities, with the ethyl acetate fraction exhibiting more vigorous activity than standard compounds such as vitamin C and trolox. The results of the anticancer activity test indicated that the n-hexane and chloroform fractions have significant potential to inhibit the growth of breast cancer cells MDA-MB-231.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 12.04.2024 Revised on 10.09.2024

Accepted on 02.12.2024 Published on 28.01.2025

Available online from February 27, 2025

Research J. Pharmacy and Technology. 2025;18(2):744-750.

DOI: 10.52711/0974-360X.2025.00110

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