Cytotoxic Activity of Secondary Metabolite Compounds from Myanmar Medicinal Plants

1. Introduction

Cancer is defined as the abnormal proliferation of cells in our bodies, which can result in death. As a result, there is currently no effective cancer therapy available, and the disease has the potential to spread globally. Plants are a rich source of novel bioactive compounds. This is because of their innate biological capabilities, which have the potential to be used in medicine and other disciplines of human health promotion. There is a significant need for new anticancer medications that are more effective and less damaging to healthy cells, such as plant-derived substances. The preliminary strategy for identifying active chemicals in plants is cytotoxic screening [1, 2, 3].

Chromolaena odorata is a tropical and subtropical flowering shrub widely distributed in various parts of the world. Fresh leaves or a decoction of C. odorata have been used for folk medicine in Vietnam. In other traditional medicine, it is useful for burn wounds, leech bites, skin infections, soft tissue wounds, and dento-alveolitis. In Myanmar, local people are used for stomachache, cancer, and urinary tract infection. Chemical components of this plant have been discovered as chalcones, flavonoids, glycosides, anthraquinones, triterpenoids, fatty acids, coumarino lignoids, and phenolic compounds [4, 5, 6, 7, 8, 9, 10, 11, 12].

Croton oblongifolius is a medium-size tree and is widely distributed throughout Asia and Myanmar. In folk medicine, this plant is useful for dysmenorrhea, purgative, dyspepsia, and dysentery. In Myanmar, it is used to treat diarrhea (seeds) and liver disorders (barks). Phytochemical studies of C. oblongifolius have been reported in several different types of research and many clerodane diterpenes, cembranoid diterpenes, halimane-type diterpenoids, labdane diterpenoids, furanocembranoids, and megastigmane glycosides have been isolated from the stem barks, leaves, and roots [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21].

Tinospora cordifolia is a huge evergreen and annual climbing plant distributed throughout the world. The plant is used to treat skin diseases, allergic conditions, jaundice, urinary disorders, anaemia, rheumatism, inflammation, and diabetes in both traditional and homeopathic medicine. In Myanmar, this plant is used for diabetes and hypertension. An important phytoconstituents reported from this plant were clerodane furano diterpene glucosides, phenylpropene disaccharides, phenylpropanoids, clerodane diterpenoid, steroids, terpenoids, and alkaloids [22, 23, 24, 25, 26, 27, 28, 29, 30, 31].

Melastoma malabathricum is an evergreen shrub that belongs to the family Melastomataceae. In Malaysia, India, and Indonesia, this plant has traditionally been used to treat a variety of symptoms and disorders. In Myanmar, this plant is used to treat various kinds of cancer, toothache, diabetes, asthma, and lung disorders. Chemical constituents reported from M. malabathricum were flavonoids, triterpenes, alkaloids, steroids, lipids, phenolic compounds, and tannins [32, 33, 34, 35, 36, 37].

Dioscorea bulbifera is a perennial vine widely distributed throughout tropical and temperate areas. It is commonly known as air potato. This herb is used in the treatment of leprosy and cancer by triple people in Bangladesh. In China, this herb is utilized for cancer and thyroid diseases. In Myanmar, this plant is used for various cancer diseases such as breast cancer, cervix cancer, etc. Phytochemical studies revealed the presence of alkaloids, anthraquinones, norclerodane diterpenoids, clerodane diterpenoids, flavonoids, phenolic compounds, steroidal saponins, steroidal sapogenins, and glycosides [38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49].

Our main focus for this chapter is secondary metabolites derived from the plant’s kingdom, i.e., C. odorata, C. oblongifolius, Tinospora cordifolia, M. malabathricum, and D. bulbifera, and cytotoxic compounds obtained from them (Table 1).

2. Secondary metabolites

Secondary metabolites can be found in abundance in plants. They are the focus of several research since they have a wide range of biological actions. Scientific reports have demonstrated the medicinal value of various parts of plant species (C. odorata, C. oblongifolius, T. cordifolia, M. malabathricum, and D. bulbifera) against a wide range of human ailments. Only the cytotoxicity of various secondary metabolites from different plant species will be discussed in this study.

3. Cytotoxic activity of asteraceae

In 2013, Kouamé and his coworkers isolated 5-hydroxy-7,4′-dimethoxyflavanone, 2′-hydroxy-4,4′,5′,6′-tetramethoxychalcone, and cadalene from the hexane soluble fraction of C. odorata. These were screened for their cytotoxicity and anticancer properties. A chalcone, 2′-hydroxy-4,4′,5′,6′-tetramethoxychalcone has both cytotoxic and anticlonogenic actions against a wide range of cell lines (Cal51, MCF7, and MDAMB-468). Also, it enhances apoptosis in Cal51 breast cancer cells when combined with the Bcl2 inhibitor ABT737 [50]. Another research group reported that genkwanin 4′-O-[α-L-rhamnopyranosyl(1 → 2)-β-D-glucopyranoside] has cytotoxic effects on LLC (IC₅₀: 28.2 μM) and HL-60 (IC₅₀: 11.6 μM) cancer cell line. Similarly, sakuranetin 4′-O-[β-D-glucopyranosyl(1 → 2)-β-D-glucopyranoside] displayed potential cytotoxic activity in HL-60 (IC₅₀: 10.8 μM) cancer cell line [11]. Likewise, Suksamrarm and colleagues document that acacetin (IC₅₀: 24.6 μM) and luteolin (IC₅₀: 19.2 μM) possessed moderate cytotoxic activity in human small cell lung cancer cells (NCI-H187). Luteolin (IC₅₀: 38.4 μM) showed weak activity on human breast cancer cells (BC) [51]. A recent study proved that the EtOH leaves extract from C. odorata has anticancer and antiproliferative activity [52]. Another finding reported that EtOAc soluble fraction from EtOH leaves extract from C. odorata had cytotoxic and antiproliferative actions on HeLa cells [53]. In other reports, both the aqueous and EtOH extract of C. odorata demonstrated cytotoxic effects, with LC₅₀ values of 324 and 392 μg/mL, respectively [54]. The chemical structures of some cytotoxic compounds are shown in Figure 1.

4. Cytotoxic activity of euphorbiaceae

Numerous investigations indicated that the C. oblongifolius shows cytotoxic properties. Neocrotocembranal obtained from the stem bark of C. oblongifolius displayed cytotoxic activity toward P-388 cells with an IC₅₀ value of 6.48 μg/mL [13]. C. oblongifolius contain methyl 15,16-epoxy-3,13(16),14-ent-clerodatrien-18,19-olide-17-carboxylate, dimethyl 15,16-epoxy-12-oxo-3,13(16),14-ent-clerodatrien-17,18-dicarboxylate, nasimaluns A and B, levatin, (−)-hardwickiic acid, 15-hydroxy-cis-ent-cleroda-3,13-(E)-diene, and patchoulenone. These diterpenoids exhibited mild cytotoxic activity against HUCCA-1 (human cholangiocarcinoma cancer cells), KB (human epidermoid carcinoma of the mouth), HeLa (cervical adenocarcinoma cells), MDA-MB231 (human breast cells), and T47D (human mammary adenocarcinoma cells) cell lines with the IC₅₀ values ranging from 10 to 50 μg/mL [14]. Additionally, the in vitro cytotoxic activity of 2,3-dihydroxy-labda-8(17),12(E),14-triene has been demonstrated non-specific moderate activities in KATO-3 (IC₅₀ value 2.2 μg/mL), SW620 (IC₅₀ value 2.7 μg/mL), BT474 (IC₅₀ value 4.6 μg/mL), HEP-G2 (IC₅₀ value 3.7 μg/mL), and CHAGO (IC₅₀ value 3.3 μg/mL) [15]. Moreover, crotohalimaneic acid, crotohalimonetic acid, and 12-benzouloxy crotohalimaneic acid from C. oblongifolius were examined for their cytotoxic activity against human breast ductol carcinoma (BT474), lung carcinoma (CHAGO), human liver hepatoblastoma (HEP-G2), human gastric carcinoma (KATO-3), and human colon adenocarcinoma (SW620). The latter compound was inactive and the remaining two compounds displayed non-specific strong cytotoxic activity, and the IC₅₀ values for crotohalimaneic acid were 7.5, 0.1, 0.2, 0.4, 0.2, and for crotohalimonetic acid were 0.1, 0.1, 5.2, 8.2, 0.1 μg/mL, respectively [17]. In another study, novel furanocembranoids, found from the stem bark of C. oblongifolius, have been shown broad cytotoxic effects on BT474, CHAGO, Hep-G2, KATO-3, and SW-620 [18]. Besides, croblongifolin obtained from C. oblongifolius was reported to have high cytotoxic activities against human hepatocarcinoma (HEP-G2) (IC₅₀ 0.35 μM), breast carcinoma (BT474) (IC₅₀ 0.12 μM), colon carcinoma (SW620) (IC50 0.47 μM), lung carcinoma (CHAGO) (IC50 0.24 μM), and gastric carcinoma (KATO 3) (IC₅₀ 0.35 μM) [19]. In a cytotoxic study by Sommit et al., natural labdanes (labda-7,12(E),14-triene-17-al and 17-hydroxylabda-7,12(E)14-triene) and their modified derivatives (15-hydroxylabda-7,13(E)diene-17,12-olide, labda-7,13(E)-diene-17,12-olide, 12,17-dihydroxylabda-7,13(E)-diene) were found to have non-specific moderate cytotoxicity against BT474, CHAGO, HEP-G2, KATO-3, and SW620 [20]. The ethyl acetate extract of C. oblongifolius was found to be cytotoxic toward the A549 lung cancer cell line [55]. In 2020, Poofery et al. studied the cytotoxic activity of ethyl acetate extract from Bridelia ovata and Croton oblongifolius. They reported that both extracts possessed anticancer properties in breast cancer cells and induce apoptosis in these cells via a mitochondrial pathway and oxidative stress [56]. In addition, Chaichantipyuth and coworkers isolated eight new labdane-type diterpenoids and a clerodane from the stem bark of C. oblongifolius. They examined the cytotoxic capability of specific compounds and semisynthesis products against human tumor cell lines (KATO-3, BT474, CHAGO, HEPG-2, and SW620) in vitro MTT assay. Among which isolates, ent-3-oxomanoyl oxide, ent-1,2-dehydro-3-oxomanoyl oxide, ent-3α-hydroxymanoyl oxide, nidorellol, and ent-6α,7β,8α,-trihydroxylabda-13(16),14-diene showed strong cytotoxic activity (<1 μg/mL) against KATO-3, moderated activity against BT474, and undifferentiated against CHAGO, HEPG-2, and SW620. Similarly, hardwickiic acid had a substantial effect (<1 μg/mL) on BT474, while semisynthesis products (ent-3β-hydroxymanoyl oxide and ent-3β-hydroxy-1,2-dehydromanoyl oxide) had a modest effect in all cell lines. The remaining isolates were inert [57]. The authors found, two new cleistanthane-type diterpenoids, 3-hydroxycleistantha-13(17),15-diene and 3,4-seco-cleistantha-4(18),13(17),15-triene-3-oic acid, have cytotoxic activity against various human tumor cell lines. Both were isolated from the stem bark of C. oblongifolius. 3-hydroxycleistantha-13(17),15-diene showed nonspecific strong cytotoxic activity (KATO-3; IC50 value 6.0 μg/mL, BT474; IC₅₀ value 6.1 μg/mL, HEP-G2; IC₅₀ value 0.5 μg/mL, CHAGO; IC₅₀ value 5.5 μg/mL) and 3,4-seco-cleistantha-4(18),13(17),15-triene-3-oic acid showed weak activity (KATO-3; IC₅₀ value 9.6 μg/mL, BT474; IC50 value 10 μg/mL, HEP-G2; IC₅₀ value 8.6 μg/mL) [58]. In another study, Nasimalun A has a cytotoxic effect on the MOLT-3 cell line with an IC₅₀ value of 26.44 μg/mL [59]. The chemical structures of some cytotoxic compounds are shown in Figure 2.

5. Cytotoxic activity of menispermaceae

In 2010, Uddin and his coworkers tested the cytotoxicity of various fractions (pet ether, CCl₄, CHCl₃, and aqueous soluble fraction) of T. cordifolia methanolic crude extract by brine shrimp lethality bioassay. They recorded that CCl₄ and CHCl₃ soluble fractions showed highly cytotoxic in Artemia salina with LC₅₀ values of 0.402 and 0.691 μg/mL [60]. Likewise, in 2021, Modi and coworkers documented that ethanol stem and leaves extract of T. cordifolia had a cytotoxic effect in A. salina with LC₅₀ values of 462.38 and 676.08 μg/mL [61]. Furthermore, Jagetia and Rao studied the cytotoxic effects of DCM extract of T. cordifolia on cultured HeLa cells. They suggested that the cytotoxic effect on DCM extract of T. cordifolia may be due to lipid peroxidation and release of lactase dehydrogenase (LDH) and decline in glutathione-S-transferase (GST) [62]. In another study, the cytotoxic activity of aqueous and hexane extract of T. cordifolia was evaluated against six cancer lines, including prostate (PC-3), colon (Colo-205), HCT-116), lung (A-549, NCI-H322), and breast cancer (T47D). Results showed that aqueous extract has potent cytotoxic activity (67–99%) in prostate, lung, and colon cancer lines [63]. Also, the in vitro cytotoxic activity of methanolic stem extract from T. cordifolia against human breast cancer cell line MDA-MB-231 and normal Vero epithelial cell line. It was revealed that methanolic stem extract displayed cytotoxic activity toward human breast cancer cells with an IC₅₀ value of 59 ± 4.05 μg/mL [64]. In addition to the anti-proliferative activity, various fractions (ethanol, pet ether, DCM, butanol, and aqueous) from T. cordifolia were evaluated against cervical carcinoma (HeLa) cell lines by MTT and SRB methods. It was noted that both DCM (MTT; IC₅₀: 54.23 ± 0.94 μg/mL, and SRB; 48.91 ± 0.33 μg/mL) and ethanol extract (MTT; IC₅₀: 101.26 ± 1.42 μg/mL, and SRB; 87.93 ± 0.85 μg/mL) displayed significant cytotoxic effect in HeLa cell with their respective assay [65]. Moreover, Bala and his coworkers also reported the anti-cancer and immunomodulatory activities of different extracts, fractions, and isolated compounds from the stem of T. cordifolia. They were screened against four different human cancer cell lines, KB (human oral squamous carcinoma), CHOK-1 (hamster ovary), HT-29 (human colon cancer), SiHa (human cervical cancer), and murine primary cells. All extracts and fractions showed promising activity against KB and CHOK-1 cells while the isolated palmatine (KB; IC₅₀: 46.1 μM, HT-29; IC₅₀: 49.1 μM), tinocordiside (KB; IC₅₀: 45.5 μM, CHOK-1; IC₅₀: 44.9 μM), and yangambin (KB; IC₅₀: 43.8 μM) also showed promising activity with their respective cell lines [25]. The chemical structures of some cytotoxic compounds are shown in Figure 3.

6. Cytotoxic activity of Melastomaceae

The cytotoxic effect of the ethyl acetate fraction from Melastoma malabathricum in human breast and lung cancer cell lines (MCF-7 and A549) was studied by using an MTT assay. Results showed that MCF-7 and A549 cells undergo secondary necrosis 24 hours post-treatment with M. malabathricum extract [66]. Another observation showed that after 72 h of treatment, the M. malabathricum leaves extract exhibited important anticancer activity in the MCF-7 (IC₅₀: 7.14 μg/mL) cell line, although the methanol and chloroform extract from the flowers had modest activities in MCF-7 (IC₅₀: 33.63 μg/mL and 45.76 μg/mL) cell line [67]. In addition, Zakaria et al. studied the in vitro antiproliferative activity of various extracts (aqueous, chloroform, and methanol extracts) from M. malabathricum. The aqueous extract was found to be potent inhibitor of Caov-3 (IC₅₀: 58 μg/mL), HL-60 (IC₅₀: 58 μg/mL) cell lines, whereas CHCl₃ extract showed antiproliferative activity toward Caov-3 (IC₅₀: 34 μg/mL), HL-60 (IC₅₀: 96 μg/mL), and CEM-SS (IC₅₀: 22 μg/mL) cell lines. The methanol extract established antiproliferative activity in more cell lines, such as MCF-7 (IC₅₀: 87 μg/mL), HeLa (IC₅₀: 88 μg/mL), Caov-3 (IC50: 41 μg/mL), HL-60 (IC50: 13 μg/mL), CEM-SS (IC₅₀: 30 μg/mL), and MDA-MB-231 (IC₅₀: 59 μg/mL) cancer cell lines [68]. In 2019, Isnaini et al. studied the quercetin and kaempferol level in M. malabathricum ethanolic fruit extract. The analysis was done by HPLC MS/MS. In comparison to kaempferol (43.52 μg/g), quercetin (67.78 μg/g) had a greater concentration in the test sample. They also compared the extract to quercetin in terms of antioxidant and cytotoxic properties. The authors concluded that the extract exhibits antioxidant (IC₅₀: 16.82 ± 0.24 ppm) and cytotoxic properties (LC₅₀: 313.44 ppm) which may be attributed to the presence of quercetin [69]. The compounds such as flavonoids, naringenin, and kaempferol-3-O-(2′′,6′′-di-O-p-trans-coumaroyl)glucoside, isolated from the flower of M. malabathricum, have been proved to be the potent inhibitor of MCF7 with IC₅₀ values of 0.28 μM and 1.3 μM [70]. In another cytotoxic study by Hamid et al., the M. malabathricum leaves extract showed inhibitory effects on the MDA-MB-231 cell line after 48 and 72 h treatments [71]. The chemical structures of some cytotoxic compounds are shown in Figure 4.

7. Cytotoxic activity of dioscoreaceae

Several studies have been performed to examine the cytotoxic activity of constituents in Dioscorea bulbifera. Many studies have observed cytotoxic compounds, largely found as steroidal saponins. Isolated pennogenin-3-O-α-L-rhamnopyranosyl-(1 → 3)-[α-L-rhamnopyranosyl-(1 → 2)]-β-D-glucopyranoside and penogenin-3-O-α-L-rhamnopyranosyl-(1 → 4)-[α-L-rhamnopyranosyl-(1 → 2)]-β-D-glucopyrano-side demonstrated cytotoxic activity in two human hepatocellular carcinoma cell lines (Bel-7402; 99.1% and 92.6% inhibition, SMMC7721; IC₅₀ value 4.54 μM and 4.85 μM) [47]. Diosgenin-3-O-[α-L-rhamnopyranosyl-(1 → 2)-[α-L-rhamnopyranosyl-(1 → 3)]-β-D-glucopyrano- side and diosgenin-3-O-[α-L-rhamnopyranosyl-(1 → 2)-[α-L-rhamnopyranosyl-(1 → 4)]-β-D-glucopyranoside were obtained from the rhizomes of D. bulbifera and displayed moderate cytotoxic activities against two human hepatocellular carcinoma cell lines (Bel-7402; IC₅₀ values 10.87 and 19.10 μM, and SMMC7721; IC₅₀ values 3.89 and 7.47 μM) [48]. In another study, spiroconazol A, penogenin-3-O-α-L-rhamnopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 4)-[α-L-rhamnopyranosyl-(1 → 2)]-β-D-glucopyranoside, and 26-O-β-D-glucopyranosyl-(25R)-5-en-furost-3β,17α,22α,26-tetraol-3-O-α-L- rhamno-pyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 4)-[α-L-rhamnopyranosyl-(1 → 2)]-β-D-glucopyrano-side were reported to have moderate activity against human urinary bladder carcinoma cell (ECV-304) [72]. In 2018, Wang and colleagues evaluated the cytotoxic activity of three new norclerodane diterpenoids (diosbulbin E acetate, diosbulbin R, and diosbulbin S) in the SMMC-7721 cancer cell line. These compounds were isolated from the ethanol extract of D. bulbifera. Interestingly, the isolates showed no inhibitory effects on SMMC-7721 (IC₅₀ > 40 μM) [73]. Chloroform and methanol soluble fractions from D. bulbifera were tested for their cytotoxic activity in T47D breast cancer cells by Rinto Muhammad Nur and Laurentius Hartanto Nugroho. The results revealed that the chloroform fraction displayed the highest activity with an IC₅₀ value of 115.63 ± 86.01 μg/mL. They also screened the combined leaf chloroform fractions and the data showed that fractions F-5 (IC₅₀: 14.55 ± 8.62 μg/mL) and F-6 (IC50: 14.55 ± 8.62 μg/mL) were found to be the most toxic fractions. Nevertheless, both fractions (F-5 and F-6) were very weak when compared with the cancer drug, Doxorubicin (IC₅₀: 0.04 ± 0.02 μg/mL) [74]. Mainasara et al. reported that D. bulbifera has cytotoxic effects on MAD-MB-231 and MCF-7 breast cancer cell lines. The IC₅₀ values for MAD-MB-231 were found to be 41.17, 15.71, and 11.53 μg/mL, at 24, 48, and 72 h of incubation time. For MCF-7 cell lines, the IC₅₀ values were 4.29, 1.86, and 1.23 μg/mL, respectively, after 24, 48, and 72 h of incubation time. They also studied cycle analysis and apoptosis by using flow cytometry analysis. It was found that D. bulbifera induced apoptosis at various phases, with a considerable drop in viable cells after 24 h and significant improvements after 48 and 72 h of treatment [75]. Wang et al. observed the antitumor activity of various extracts and compound diosbulbin B from D. bulbifera in mice model [76]. In another study, two new phenolic derivatives (diosbulbiol A and diosbulbiol B) from the tubers of D. bulbifera were tested for their cytotoxic activity against SMMC7721, MCF-7, K562 and A549 cancer cell lines. Interestingly, these two compounds showed no inhibitory effect on all selected cell lines (IC₅₀ > 40 μM) [77]. The chemical structures of some cytotoxic compounds are shown in Figure 5.

8. Conclusions

Several investigations have been conducted on the bioactivities of Myanmar medicinal herbs. Novel anticancer medicines and lead compounds are still abundant in higher plants. The key benefits of anticancer natural compounds are their low toxicity, low cost, and wide range of modes of action. In this chapter, we examined and studied the potential of Myanmar medicinal plants as a source of cytotoxic compounds. Although several of these plants’ metabolites exhibited potential bioactivities, some of them were determined to be inert or to have weak activities in present investigations. This chapter will surely assist academics and practitioners working with these specific plant species in determining the best application for them. Because the majority of cytotoxic effects are evaluated in vitro, their therapeutic advantages may not be adequately demonstrated. Animal models and human trials should be used in a future study to assess how effective they are at providing a platform for enhancing anticancer therapeutic approaches.

Acknowledgments

The authors are grateful to: (1) Daw Ni Ni Aung at the Department of Botany, Lashio University for her valuable support, and (2) Universitas Airlangga for the funding support.

Conflict of interest

The authors declare no conflict of interest.