Phytochemical investigation and antimicrobial activity from rhizomes of alocasia odora K. Koch

. Alocasia odora is a common plant species in Viet Nam. A phytochemical investigation of the ethyl acetate extract (EtOAc) of A. odora rhizomes collected in Tuyen Quang province led to the isolation and determination of five compounds, including three alkaloids, alocasin A (1), hyrtiosin B (2), hyrtiosulawesine (3), one mono-phenol, p-hydroxycinamic acid (4)


INTRODUCTION
Alocasia, belonging to the Araceae family, is a genus of more than 100 species of perennial, herbaceous, diminutive to extremely large, usually robust herbs with clear-to-milky latex.They are distributed throughout typical subtropical Asia and were used as a common traditional medicine [1].Previous phytochemical investigations have revealed that Alocasia is rich in alkaloids, carbohydrates, phenols, phytosterols, saponins, and tannins with various pharmacological activities such as antimicrobial antioxidant, antitumor and cytotoxic, hepatoprotective, anti-inflammatory, glycemic and lipidemic activities [2 -4].Alocasia odora, a widespread Alocasia, is a common medicinal herb known in Vietnamese as "Ráy dại, Bạc hà, Dã vu".Traditional experience of using the plant was used to treat many diseases such as liver, gout, detoxication, cutaneous diseases, malaria, and rheumatism [5].Despite this multitude of bioactivities, a few phytochemical investigations of A. odora are found in the literature [6 -7].Therefore, this study was undertaken to identify more of the phytoconstituents and antimicrobial activity of A. odora.In this paper, we report the isolation and structural determination of three alkaloids (1)(2)(3), one mono-phenol (4), and one fatty acid (5) from the EtOAc extract of the rhizomes of A. odora.Three alkaloids, alocasin A (1), hyrtiosin B (2), and hyrtiosulawesine (3) from A. odora were reported for the first time.

Materials
The fresh rhizomes of A. odora were collected in Na Hang, Tuyen Quang province in January 2021 and were identified by Dr. Nguyen Van Du, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology.A voucher specimen (AO-2021) was deposited at the Laboratory of Applied Biochemistry, Institute of Chemistry, Vietnam Academy of Science and Technology.

Broth microdilution assays
The antimicrobial activity of compounds was evaluated as previously described [8].Briefly, samples were dissolved in DMSO and ionized water to concentrations of 128.0, 32.0, 8.0, 2.0 and 0.5 µg/mL.Microbacteria were kept at -80 o C. Before assaying, they were activated with culture medium and adjusted to a concentration of 5 × 10 5 CFU/mL.10 µL of sample were combined with 190 µL of microbacterial solution in a 96-well plate which was further incubated within 18 -24 h at 37 o C. The experiment was performed in triplicate.Positive controls were wells with a bacterial suspension in growth medium, and culture medium without bacteria served as a negative control.Ampicillin and cefotaxime (Sigma-Aldrich) were used as positive controls.The results were recorded at 590 nm using a microtiter plate reader and calculated by Raw data software.The MIC value was considered to be the lowest concentration of the compound at which bacterial growth was completely inhibited under the assay conditions.
Compound 5 was obtained as a white powder.Its molecular formula C 14 H 28 O 2 was determined by ESI-MS with a pseudomolecular negative ion peak at m/z 227 [M-H] -.The 1H-NMR spectrum showed proton signals of the hydrocarbon chain at  H 2.35 (t, J = 7.5 Hz, H-2), 1.65 (m, H-3), 1.28 (br s, H-4 → H-13) and the signal of a terminal methyl group at  H 0.91 (t, J = 6.0 Hz, H-14).Compound 5 was identified as myristic acid by comparing the spectral data with those published in the literature [13].
Five compounds (1-5) were evaluated for antimicrobial activity against pathogenic microorganisms using the broth microdilution method (Table 1).The results showed that alkaloids 1-3 exhibited antimicrobial activity against bacteria strains.µg/mL, respectively, while it was inactive against P. aeruginosa.Compounds 4 and 5 did not inhibit microbial effect (MIC > 128 µg/mL).For ampicillin and cefotaxime, positive controls showed MICs from 0.125 µg/mL to 32 µg/mL.This is the first time A. odora antimicrobial capacity has been studied.The results showed the potential use of this plant in the treatment of infections and further models of research are needed to provide a scientific basis for the direction of the use of this plant.In another study, the extract from A. odora stems was shown to possess proliferation of skin fibroblasts and antioxidant activities [6].So far, seven Alocasia species have been studied for their antimicrobial and antifungal activities.The results showed that A. indica and A. macrorrhizos exhibited significant antimicrobial effects.Meanwhile, A. fornicata and A. decipiens showed moderate-to-good antimicrobial activities.In contrast, A. sanderiana, A. denudata, and A. brisbanensis extracts did not exhibit antimicrobial activities.Thus, it was believed that different extracts of the Alocasia species might contain different bioactive molecules that were responsible for their antimicrobial and antifungal activity [14].

CONCLUSIONS
From the EtOAc extract of A. odora rhizomes, five compounds were isolated.They were elucidated to be alocasin A (1), hyrtiosin B (2), hyrtiosulawesine (3), p-hydroxycinamic acid (4), and myristic acid (5).The antimicrobial activity of the compounds has been evaluated.Alkaloids 1-3 exhibited antimicrobial activity against reference strains with MIC values ranging from 8 µg/mL to 128 µg/mL.This is the first time Alocasia odora has been studied for antimicrobial activity.

Table 1 .
Antimicrobial activity of the isolated compounds from Alocasia odora.Compound 1 exhibited a strong antimicrobial effect on strains S. aureus, B. subtilis, E. coli and P. aeruginosa with MIC values ranging from 8 µg/mL to 32 µg/mL.Compound 2 displayed antimicrobial activity against four strains with MIC values ranging from 32 to 128 µg/mL.Compound 3 significantly inhibited three bacteria strains S. aureus, B. subtilis and E. coli with MIC values of 32, 128 and 128