Thanaka (H. Crenulata, N. Crenulata, L. Acidissima L.): A Systematic Review Of Its Chemical, Biological Properties And Cosmeceutical Applications Part 2

4. Biological Properties of Thanaka 

4.1. Antioxidant Activity

Antioxidants, or free-radical scavengers, are substances that prevent or slow down the cell damage caused by free radicals, which is an unstable molecule produced by the body reacting to environmental stress, thus protecting human health [17]. Sources of antioxidants can be natural or artificial; however, natural sources of antioxidants from plants are generally preferred by people as they are believed to be safer. Polyphenols that can be found commonly in most plants are considered a highly effective antioxidant,  as the structural chemistry of polyphenols derived from plants is ideal for scavenging free radicals, which have been shown to possess more effective antioxidants in vitro than vitamins E and C [17]. Antioxidants are also able to preserve food.

Glycoside of cistanche can also increase the activity of SOD in heart and liver tissues, and significantly reduce the content of lipofuscin and MDA in each tissue, effectively scavenging various reactive oxygen radicals (OH-, H₂O₂, etc.) and protecting against DNA damage caused by OH-radicals. Cistanche phenylethanoid glycosides have a strong scavenging ability of free radicals, a higher reducing ability than vitamin C, improve the activity of SOD in sperm suspension, reduce the content of MDA, and have a certain protective effect on sperm membrane function. Cistanche polysaccharides can enhance the activity of SOD and GSH-Px in erythrocytes and lung tissues of experimentally senescent mice caused by D-galactose, as well as reduce the content of MDA and collagen in lung and plasma, and increase the content of elastin, have a good scavenging effect on DPPH, prolong the time of hypoxia in senescent mice, improve the activity of SOD in serum, and delay the physiological degeneration of lung in experimentally senescent mice With cellular morphological degeneration, experiments have shown that Cistanche has the good antioxidant ability and has the potential to be a drug to prevent and treat skin aging diseases. At the same time, echinacoside in Cistanche has a significant ability to scavenge DPPH free radicals and can scavenge reactive oxygen species, prevent free radical-induced collagen degradation, and also has a good repair effect on thymine free radical anion damage.

Click on rou cong rong benefits

【For more info:george.deng@wecistanche.com / WhatApp:86 13632399501】

Total phenolic content (TPC) and diphenyl-picrylhydrazyl (DPPH) are commonly employed to determine the antioxidant activities of plant extracts using a spectrophotometer. The amount of phenolic content of the plant extract is evaluated using the Folin-Ciocalteu colorimetric method. The commonly used standards are gallic acid, pyrocatechol, and tannic acid. The DPPH assay is widely used for antioxidant activities by measuring the total radical scavenging capacity of antioxidants toward the stable free radical, which subsequently reacts with hydrogen donor compounds.

Antioxidant activities of Thanaka extracts were evaluated by several scientists as mentioned in Table 3. In particular, Wangthong et al. in 2010 evaluated various extracts of Thanaka stem bark using different solvents including hexane, methanol, ethyl acetate, 85% aqueous ethanol, and water. Through the DPPH antioxidant assay, they found that the 85% ethanol extracts of Thanaka stem bark possessed the highest antioxidant activity while hexane had the lowest activity [18]. They also evaluated the TPC in the Thanaka stem bark extracts where the methanol extracts possessed the highest amount of TPC while hexane extract possessed the lowest TPC amount. In 2012, Shermin et al. also performed a DPPH  antioxidant test on Thanaka stem bark extracts of different solvents including chloroform,  petroleum ether, and methanol. The results showed that the chloroform extract possessed the highest antioxidant activity followed by petroleum ether, then methanol [19]. In 2017, Sonawane and Arya evaluated the antioxidant activity of the protein hydrolysates obtained from Thanaka seeds through DPPH, 2,20 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP) and metal chelating ability assay. They found that the DPPH assay was not suitable for protein hydrolysates, while in the FRAP  assay, the absorbance reading did increase as the concentration of protein hydrolysates increased; however, the results are lower compared to the Trolox standard. In the ABTS  assay and metal chelating ability assay, the relationship between the concentration of protein hydrolysates and antioxidant activity was linear and the antioxidant activity was observed to be higher than the Trolox standard [20]. Later in 2020, Sonawane et al. tested Thanaka seed protein hydrolysates in the storage and color stability of anthocyanins. They reported that the storage stability of anthocyanin slightly increased by 10 h and 34 min at 0.12% protein hydrolysate concentration but decreased at a higher concentration by 2%, while the color stability of anthocyanin increased as the concentration of Thanaka seed protein hydrolysates increased [21]. In 2018, Jantarat et al. formulated four herbs including Thanaka bark powder into an herbal ball and tested for their antioxidant activity through a DPPH assay resulting in antioxidant activity lower than the gallic acid standard by 40-fold [22].

4.2. Antimicrobial Activity

It is worth mentioning that traditionally Thanaka is used as an acne treatment remedy as well as an antifungal. This resulted in scientists’ enthusiasm to evaluate the antimicrobial activity of Thanaka. Wangthong et al. (2010) did both minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assay by treating various solvent extracts of Thanaka stem bark onto Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) and by using clindamycin as the standard. The results showed that all extracts possessed a slight antibacterial activity that is 10 to 20-fold lower against E. coli and 300-fold lower against S. aureus when compared with the clindamycin standard [18]. Interestingly, the herbal ball comprised of four herbs, i.e., Andrographis paniculata, Centella Asiatica, Benchalokawichian, and Thanaka bark powder was prepared by Jantarat et al., 2018, showed antibacterial activity against Propionibacterium acnes (P. acnes) at a concentration of 31.25 µg/mL in both MIC and MBC assay [22].

4.3. Cytotoxicity and Cell Viability

Since it is a traditional remedy that is used on the skin, it is important to know the cytotoxicity of Thanaka to determine the safety of its use. Wangthong et al. in 2010 evaluated both cytotoxicity and genotoxicity of Thanaka stem bark extracts on the human melanoma A-375 cell line. According to their observation, the original bark powder did not show any signs of cytotoxicity towards the A-375 cells, while the water, methanol, and 85%  ethanol extracts showed very low cell cytotoxicity. However, ethyl acetate, hexane, and dichloromethane showed slightly higher cytotoxicity against A375 cells but overall, much lower than the doxorubicin standard. As for genotoxicity, Wangthong et al., 2010 claimed that all extracts and original bark powder exhibited no genotoxicity [18]. Ma et al. (2020)  evaluated the cytotoxicity of Thanaka leaf extract mediated tin (IV) oxide nanoparticles (SnO2 NPs) against human cervical cancer (SiHa) cell line and resulted in the cell viability of SiHa cells reduced as the concentration of Thanaka leaf extract mediated SnO2 NPs increased. They also assessed the morphology of the SiHa cell to observe cell apoptosis. SiHa cells displayed the existence of necrotic and apoptotic cell morphology after treating the SiHa cells with the Thanaka leaf SnO2 NPs for 24 h [23].

Additionally, toxicity models such as Artemia salina (A. salina) brine shrimp and Culex quinquefasciatus (C. quinquefasciatus) mosquito larvae were employed in cytotoxicity screening of Thanaka extracts [14,19]. Shemin et al. (2012) evaluated the cytotoxicity of Thanaka stem bark extracts on brine shrimp A. salina and observed that there is slightly higher cytotoxicity in petroleum ether and chloroform extracts, while very low cytotoxicity was observed in methanol extract; however, cytotoxicity of all extracts is much lower than the vincristine sulfate standard [19]. Pratheeba et al. (2019) evaluated the larvicidal effi-  cacy activity of various Thanaka leaf extracts against C. quinquefasciatus. A higher mortality rate was observed in C. Quinn fasciitis larvae at 24 h post-treatment with acetone extract of Thanaka leaf with lethal concentration 50 (LC50) and lethal concentration 90 (LC90)  values as low as 1.02 mg/L and 1.93 mg/L respectively, followed by methanol extract with 1.13 mg/L LC50 and 2.24 mg/L LC90 values, ethyl acetate extract with 1.81 mg/L LC50  and 4.14 mg/L LC90 and hexane extract with 9.74 mg/L LC50 and 2.34 mg/L LC90 [14].

4.4. Other Biological Properties

Since Thanaka bark powder has been traditionally used for UV protection and skin conditioning by the Burmese, it encouraged scientists to evaluate its tyrosinase inhibition activity. Tyrosinase is a copper-containing enzyme that is recognized for melanogenesis and pigmentation activity [25]. Therefore, tyrosinase inhibition activity is important to prevent forming of pigments on the skin. Wangthong et al. (2010) evaluated the tyrosinase inhibition activity of the Thanaka stem bark extracts. The overall tyrosinase inhibition  activity of Thanaka stem bark extracts and pure powder is mild when compared with  the Kojic acid standard; however, when compared among the various Thanaka extracts,

The anti-inflammatory activity of the Thanaka stem bark extracts was also evaluated by Wangthong et al. (2010). Inflammation is a response pattern of the body towards injury or allergens that involves the accumulation of cells and exudates in irritated tissues to prevent and protect from further damage. However, inflammatory reactions cause pain,  redness, swelling, and heat to the body; hence, the role of anti-inflammatory activity is important in relieving these symptoms. The anti-inflammatory activity of various Thanaka stem bark extracts was performed in the murine macrophage-like cell line RAW 264.7  using the stem bark extracts and they observed that all extracts possessed an 80–90% high anti-inflammatory activity at non-toxic dosages (80% cell viability) ranked in the order: Hexane > dichloromethane > ethyl acetate > 85% ethanol > methanol > water. Hence, it is proven that the stem bark extracts of Thanaka possess high anti-inflammatory activity and a mild level of tyrosinase inhibition activity [18].

Vasant and Narasimahcharya (2013) experimented with the ability of petroleum ether extract from Thanaka fruit powder the regulation fluoride-induced hyperglycemia and hyperlipidemia in colony-bred male albino rats. Hyperglycaemia is the condition of high glucose level circulating in the blood and when persistently high, may cause diabetes while hyperlipidemia is the condition of elevation of cholesterol or triglycerides in blood circulation, both conditions are menacing to health. In rat groups fed with Thanaka fruit powder (2.5 g/kg in feed, 5 g/kg in feed, 10 g/kg infeed), it showed dose-dependent significant results in the decrease of plasma glucose levels, G-6-Pase activity, plasma total lipid (TL), total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), very-low-density lipoprotein cholesterol (VLDL-C), apolipoprotein (AI) content and hepatic lipid profiles, while an increase in plasma high-density lipoprotein cholesterol (HDL-C) content is also observed [15]. Therefore, the petroleum ether extract of Thanaka fruit extract can regulate hyperglycemia and hyperlipidemia conditions.

The wound healing activity of Thanaka leaf biomolecules coating silver nanoparticles (AgNPs) was also evaluated by Bhuvaneswari et al. (2014) on Wistar male albino rats and compared with the standard drug Betadine [24]. Silver nanoparticles are antimicrobial agents that have been used in skin ointments and creams to deliver extensive applications to prevent infection of burns and open wounds [26]. The results showed that the Thanaka leaf AgNPs had higher wound healing activity than the standard drug betadine [24]. However,  the authors did not compare the Thanaka leaf AgNPs healing effect with only Thanaka leaf extract; therefore, no conclusions could be made as to whether the wound healing is mainly because of the silver nanoparticles or if the Thanaka extract boosted the healing effect of AgNPs or both.

5. Cosmeceutical Products Containing Thanaka in the Southeast Asia Market

In Table 4, we tabulated the Thanaka cosmetic products that are manufactured and sold in Southeast Asian countries such as Myanmar, Thailand, and Malaysia. Notable brands,  namely, Shwe Pyi Nann [27] and Truly Thanaka [28] from Myanmar, Suppaporn [29] and De Leaf [30] from Thailand, Thanaka Malaysia [31], and Bio Essence [32] from Malaysia are commonly found over the counter in shopping malls and pharmacies as well as online. Local brands from Myanmar (snake brand, Pann Chit Thu, Myat Bhoon Pwint), and Malaysia (Taté Skincare Malaysia [33]) are also producers of Thanaka cosmeceutical products.

Shwe Pyi Nann Co. Ltd. is the leading manufacturer and exporter of Thanaka to Thailand, Malaysia, Singapore, and the Philippines, which lead to the production of Thanaka products in Thailand and Malaysia. Companies manufacturing Thanaka products are mainly found in Southeast Asia since Thanaka bark thrives in the hot weather of countries located near the equator, where sunlight is more intense as compared to other parts of the Earth. Thus, to protect our skin from harmful UV rays, the demand for sunblock products and after-sun treatment is essential in Southeast Asian countries.

The Burmese apply Thanaka powder directly onto their skin as sunscreen. However,  the yellow patches left on the cheek (Figure 1) are not widely accepted by other countries except Myanmar. Hence, to benefit more people with natural sunscreen, Thanaka skincare products such as soap, loose powder, foundation powder, face scrub, body lotion, and face scrub are produced. To meet the consumers and market demand, Thanaka is also formulated into cleansers, serums, moisturizers, acne spot treatment creams, and tone-up creams. Most manufacturers add active ingredients such as vitamins, collagen, and hyaluronic acid to increase the synergic effect and provide treatment for various skin conditions. Some of the products of Shwe Pyi Nannare are enhanced with the scent of flowers and herbs to make the products appear more attractive to consumers. In general, a scent is added to the beauty product to neutralize the unpleasant odor of its ingredients. Brands such as Truly Thanaka, de Leaf, and ThanakaMalaysia produce Thanaka products containing vitamins A, C, and E, collagens, 24 k gold, hyaluronic acid, aloe vera, turmeric, glutathione, jasmine rice, and pomegranate powder that beneficial to the skin, such as protect our skin from environmental damage such as pollution, improve our skin condition and help to fight the effects of aging such as wrinkles and pigmentation. Furthermore, quality ingredients such as bamboo charcoal were added as an exfoliator, meanwhile, kaffir lime and honey were added to enhance the cleansing effect and body brightening.

6. Conclusions and Perspectives

Thanaka has been used as the traditional skincare by the people of Myanmar for over 2000 years due to the belief in its anti-aging, acne-clearing, and sun-protecting benefits. The people in Southeast Asia also use it as a traditional remedy for various purposes such as insect repellent and wound healing. Among its renowned benefits, its antioxidant, antibacterial, and cytotoxicity are the most studied properties using various Thanaka extracts. The phytochemical analysis is among the favorite method by scientists to discover the natural bioactive compounds in Thanaka.

Although the extensive chromatography analysis of Thanaka was performed on extracts of various solvents, the authors did not further discuss the biological functions of each compound in most of the publications reviewed in Table 2. Conversely, most of the biological assays were performed using extracts of Thanaka without further isolation of pure bioactive compounds from the extracts (Table 3). This gap could be improved by collaboration between chemists and biologists in the discovery of bioactive compounds in natural products. Moreover, most authors utilized organic solvents such as hexane, chloroform, ethyl acetate, ethanol, and methanol (Table 3) to perform the extraction. Wangthong et al. (2010) mentioned that the solubility of extracts and toxicity of organic solvents may affect the accuracy of results as most of the biological assays use polar buffer solutions [18]. Thus, the use of green solvents (such as glycerol) in extracting bioactive ingredients may be a good alternative to organic solvents in the extraction of natural products, particularly, in the development of skincare products. The development of green skincare products will significantly prevent users from experiencing any allergic skin reactions. Furthermore, green solvents require minimum waste management. It is hoped that this review may serve as a reference that will lead to new scientific discoveries.

Author Contributions: Conceptualization, M.W.L., L.T.G. and M.K.A.; methodology, L.T.G.; original  draft preparation of the manuscript, M.W.L. and L.T.G.; review and editing the manuscript, L.T.G. and M.K.A.; supervision, L.T.G.; funding acquisition, L.T.G. and M.K.A. All authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by Fundamental Research Grant Scheme (FRGS) (Grant no.: FRGS/1/2020/STG01/SYUC/02/1) awarded by the Ministry of Education Malaysia (MOE).

Institutional Review Board Statement: Not applicable. 

Informed Consent Statement: Not applicable. 

Data Availability Statement: Not applicable.

Acknowledgments: The authors would like to acknowledge Sunway Postgraduate Research Scholarship (PGSUREC2020/006) which was granted to MW Lim by Sunway University, Malaysia.

Conflicts of Interest: The authors declared no conflict of interest. 

References

1. Yeni. Beauty That’s More Than Skin Deep. 2011.

2. Nayar, M.N.S.; Sutar, C.V.; Bhan, M.K. Alkaloids of the stem bark of Hesperethusa crenulata. Phytochemistry 1971, 10, 2843–2844. [CrossRef] 

3. Nayar, M.; Bhan, M.K. Coumarins and other constituents of Hesperethusa crenulata. Phytochemistry 1972, 11, 3331–3333. [CrossRef] 

4. Joo, S.H.; Lee, S.C.; Kim, S.K. UV absorbent, parmesan, from the bark of Thanakha, Hesperethusa crenulate L. J. Plant Biol. 2004, 47, 163–165. [CrossRef] 

5. Kim, K.H.; Yang, M.C.; Lee, K.H.; Lee, I.K.; Ha, S.K.; Choi, P.; Bae, W.; Kim, S.Y.; Lee, K.R. Three new tyramine and two new phenolic constituents from Limonia acidissima. Planta Med. 2008, 74, PB116. [CrossRef] 

6. Aoyama, Y.; Katayama, T.; Yamamoto, M.; Tanaka, H.; Kon, K. A new antitumor antibiotic product, demethylchartreusin isolation, and biological activities. J. Antibiot. 1992, 45, 875–878. [CrossRef] 

7. Matos, M.J.; Santana, L.; Uriarte, E.; Abreu, O.A.; Molina, E.; Yordi, E.G. Coumarins—An important class of phytochemicals. Phytochem. Isol. Characterisation Role Hum. Health 2015, 25, 533–538. 

8. Stefanachi, A.; Leonetti, F.; Pisani, L.; Catto, M.; Carotti, A. Coumarin: A natural, privileged and versatile scaffold for bioactive compounds. Molecules 2018, 23, 250. [CrossRef] [PubMed] 

9. Liberati, A.; Altman, D.G.; Tetzlaff, J.; Mulrow, C.; Gøtzsche, P.C.; Ioannidis, J.P.; Clarke, M.; Devereaux, P.J.; Kleijnen, J.; Moher, D. The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluate health care interventions: Explanation and elaboration. J. Clin. Epidemiol. 2009, 62, e1–e34. [CrossRef] 

10. Niu, X.M.; Li, S.H.; Peng, L.Y.; Lin, Z.W.; Rao, G.X.; Sun, H.D. Constituents from Limonia crenulata. J. Asian Nat. Prod. Res. 2001, 3, 299–311. [CrossRef] [PubMed] 

11. Sarada, K.; Margret, R.J.; Mohan, V. GC-MS Determination of bioactive components of Naringi crenulata (Roxb) Nicolson. Int. J. ChemTech Res. 2011, 3, 1548–1555. 

12. Sampathkumar, S.; Ramakrishnan, N. GC-MS analysis of methanolic extract of Naringi crenulata (Roxb.) Nicols. stem. J. Pharm. Res. 2012, 5, 1102–1104. 

13. Sampathkumar, S.; Ramakrishnan, N. Pharmacognostical analysis of Naringi crenulata leaves. Asian Pac. J. Trop. Biomed. 2012, 2, S627–S631. [CrossRef] 

14. Pratheeba, T.; Vivekanandhan, P.; Faeza, A.N.; Natarajan, D. Chemical constituents and larvicidal efficacy of Naringi crenulata (Rutaceae) plant extracts and bioassay-guided fractions against Culex quinquefasciatus mosquito (Diptera: Culicidae). Biocatal. Agric. Biotechnol. 2019, 19, 101137. [CrossRef] 

15. Vasant, R.A.; Narasimhacharya, A.V. Limonia fruit as a food supplement to regulate fluoride-induced hyperglycemia and hyperlipidemia. J. Sci. Food Agric. 2013, 93, 422–426. [CrossRef] [PubMed] 

16. Pandavadra, M.; Chanda, S. Development of quality control parameters for the standardization of Limonia acidissima L. leaf and stem. Asian Pac. J. Trop. Med. 2014, 7, S244–S248. [CrossRef] 

17. Rice-Evans, C.; Miller, N.; Paganga, G. Antioxidant properties of phenolic compounds. Trends Plant Sci. 1997, 2, 152–159. [CrossRef] 

18. Wangthong, S.; Palaga, T.; Rengpipat, S.; Wanichwecharungruang, S.P.; Chanchaisak, P.; Heinrich, M. Biological activities and safety of Thanaka (Hesperethusa crenulata) stem bark. J. Ethnopharmacol. 2010, 132, 466–472. [CrossRef] 

19. Shermin, S.; Aktar, F.; Ahsan, M.; Hasan, C.M. Antioxidant and Cytotoxic Activitiy of Limonia acidissima L. Dhaka Univ. J. Pharm. Sci. 2012, 11, 75–77. [CrossRef] 

20. Sonawane, S.K.; Arya, S.S. Bioactive L acidissima protein hydrolysates using Box-Behnken design. 3 Biotech 2017, 7, 1–11. [CrossRef] 

21. Sonawane, S.K.; Patil, S.; Arya, S.S. Effect of protein hydrolysates from Limonia (L.) acidissima and Citrullus (C.) lanatus on anthocyanin degradation. Int. J. Fruit Sci. 2020, 20 (Suppl. 2), S231–S239. [CrossRef] 

22. Jantarat, C.; Sirathanarun, P.; Chuchue, T.; Konpian, A.; Sukkua, G.; Wongprasert, P. In vitro antimicrobial activity of gel containing the herbal ball extract against Propionibacterium acnes. Sci. Pharm. 2018, 86, 8. [CrossRef]

23. Ma, J.; Zhao, M.; Zhang, C.; Wu, X.; Yang, G. Synthesis of L. Acidissima mediated tin oxide nanoparticles for cervical carcinoma treatment in nursing care. J. Drug Deliv. Sci. Technol. 2020, 57, 101745. [CrossRef] 

24. Bhuvaneswari, T.; Thiyagarajan, M.; Geetha, N.; Venkatachalam, P. Bioactive compound loaded stable silver nanoparticle synthesis from microwave irradiated aqueous extracellular leaf extracts of Naringi crenulata and its wound healing activity in an experimental rat model. Acta Trop. 2014, 135, 55–61. [CrossRef] [PubMed] 

25. Merimsky, O.; Shoenfeld, Y.; Fishman, P. A focus on anti-tyrosinase antibodies in melanoma and vitiligo. Decade Autoimmun. 1999, 261, 267. 

26. Durán, N.; Marcato, P.D.; Alves, O.L.; De Souza, G.I.; Esposito, E. Mechanistic aspects of biosynthesis of silver nanoparticles by several Fusarium oxysporum strains. J. Nanobiotechnol. 2005, 3, 1–7. [CrossRef] [PubMed] 

27. Thanaka Shop.

28. Truly Thanaka. 

29. Supaporn Group. 

30. De Leaf. 

31. Thanaka Malaysia. 

32. Bio essence. 

33. Taté Skincare Malaysia. 

【For more info:george.deng@wecistanche.com / WhatApp:86 13632399501】